阅读说明：本技术 试验装置 (Testing device ) 是由 凯瑟琳·安格普拉帕考恩 史蒂芬·卡利斯勒 巴比尔·拉伊 于 2020-04-09 设计创作，主要内容包括：本发明提供用于检测样本中分析物的存在的试验装置和方法。本发明的装置包括试剂区、一个或更多个捕获区和检测区。在样本中存在阴性标志物和/或样本中不存在阳性标志物的情况下,捕获区可以减少到达检测区的经标记偶合物的量,促进高灵敏度和改进的特异性测试。(The present invention provides assay devices and methods for detecting the presence of an analyte in a sample. The device of the invention comprises a reagent zone, one or more capture zones and a detection zone. In the presence of a negative marker in the sample and/or the absence of a positive marker in the sample, the capture zone may reduce the amount of labeled conjugate reaching the detection zone, facilitating a high sensitivity and improved specificity test.)

1. A test device for detecting the presence of an analyte in a sample, the device comprising a test flow path, the flow path comprising:

(a) a reagent zone comprising a mobilizable labeled conjugate comprising a detectable label directly or indirectly attached to a detectable label

(i) Means for associating said labeled conjugate with said analyte and means for associating said labeled conjugate with a first negative marker, said first negative marker being likely to be present in said sample, and/or

(ii) Means for associating the labeled conjugate with the analyte, wherein the labeled conjugate further comprises a positive marker or a positive marker conjugate, the positive marker portion of the positive marker conjugate being available for binding, and/or wherein a mobilizable positive marker or positive marker conjugate is provided separately in the reagent zone and is configured to associate with the labeled conjugate upon mobilization such that the positive marker portion of the conjugate is available for binding, and/or the labeled conjugate associates with the positive marker or positive marker conjugate

(iii) Means for associating the labeled conjugate with the analyte and means for associating the labeled conjugate with a positive marker or a positive marker conjugate in the device or a positive marker that may be present in the sample, optionally and wherein the reagent zone further comprises a mobilizable positive marker or positive marker conjugate for immobilization downstream of the reagent zone,

(b) one or more capture zones, wherein,

(i) in the case where the reagent zone comprises option (i),

at least one capture zone comprising an immobilized capture substance configured to capture a complex comprising the labeled conjugate and the first negative marker, wherein capturing the complex is preferably achieved by a specific binding reaction with the first negative marker,

(ii) in the case where the reagent zone comprises option (ii),

at least one capture zone comprises an immobilized capture substance configured to capture a labeled conjugate comprising the positive marker or positive marker conjugate or a labeled conjugate associated with the positive marker or positive marker conjugate or the positive marker in the case of the presence of a positive marker in the sample, wherein capturing the labeled conjugate is preferably achieved by a specific binding reaction with a positive marker or positive marker conjugate,

(iii) in the case where the reagent zone comprises option (iii),

at least one capture zone comprises an immobilized capture substance configured to capture labeled conjugates comprising means for associating the labeled conjugates with a positive marker or positive marker conjugate, wherein the immobilized capture substance comprises or consists of an immobilized positive marker or positive marker conjugate, and/or in case the reagent zone comprises a mobilizable positive marker or positive marker conjugate, at least one capture zone comprises an immobilized capture substance for localizing the mobilizable positive marker or positive marker conjugate at the capture zone, thereby making the positive marker portion of the conjugate available for binding, wherein capture of the labeled conjugates is preferably achieved by a specific binding reaction with the means for associating the labeled conjugate with a positive marker or positive marker conjugate ,

(c) a detection zone comprising an immobilised binding reagent for capturing a complex comprising the labelled conjugate and the analyte, wherein capture of the complex is preferably achieved by a specific binding reaction with the analyte,

wherein the or each capture zone is downstream of the reagent zone and the detection zone is downstream of the or each capture zone.

2. An assay device according to claim 1, wherein the device is arranged such that the or each capture zone is not visible to a user in use.

3. An assay device as claimed in claim 1 or 2, wherein the or each capture zone is not readable by the assay reading means.

4. An assay device as claimed in any preceding claim, wherein the means for associating the labelled conjugate with the analyte comprises or consists of a binding reagent, for example an antibody which binds to an epitope of the analyte.

5. An assay device as claimed in any preceding claim, wherein the means for associating the labelled conjugate with a first negative marker comprises or consists of a binding reagent, for example an antibody which binds to an epitope of the first negative marker.

6. The test device of any one of the preceding claims, wherein the means for associating the labeled conjugate with the analyte and the means for associating the labeled conjugate with the first negative marker are (a) the same or (b) different.

7. An assay device as claimed in any preceding claim, wherein the means for associating the labelled conjugate with the analyte comprises or consists of a binding reagent, for example an antibody that binds to an epitope shared by the analyte and the first negative marker.

8. An assay device as claimed in any preceding claim, wherein the means for associating the labelled conjugate with a positive marker comprises or consists of a binding reagent, for example an antibody which specifically binds to the positive marker or positive marker conjugate.

9. The test device of any one of the preceding claims, wherein the one or more capture zones comprise an immobilized capture substance that specifically binds to the first negative marker.

10. The test device of any one of the preceding claims, wherein the one or more capture zones comprise an immobilized capture substance that specifically binds to the positive marker or positive marker conjugate.

11. An assay device as claimed in any preceding claim, wherein the immobilised capture species in the capture zone binds to an epitope of the first negative marker which is not present in the analyte.

12. A test device according to any preceding claim, wherein the reagent zone comprises a mobilizable first negative marker binding reagent specific for the first negative marker, the mobilizable first negative marker binding reagent linked to a first binding partner, and the capture zone comprises an immobilized second binding partner for the first binding partner.

13. A test device according to any preceding claim, wherein the reagent zone comprises a mobilizable positive marker binding reagent specific for the positive marker, which mobilizable positive marker binding reagent is linked to a first binding partner, and the capture zone comprises an immobilized second binding partner to the first binding partner, preferably, wherein the labeled conjugate comprises a positive marker or a positive marker conjugate and/or, wherein a mobilizable positive marker or positive marker conjugate is provided separately in the reagent zone and is configured to associate with the labeled conjugate upon mobilization, such that in the event that the labeled conjugate is associated with the positive marker or the positive marker conjugate, the positive marker or the positive marker portion of the conjugate is available for binding.

14. A test device according to any preceding claim, wherein the reagent zone comprises a mobilizable positive marker or positive marker conjugate linked to a first binding partner and the capture zone comprises an immobilized second binding partner for the first binding partner, preferably wherein the labelled conjugate comprises means for associating the labelled conjugate with a positive marker or positive marker conjugate.

15. An assay device as claimed in any preceding claim, wherein at least one capture zone comprises a first line or region of immobilised capture species and one or more additional lines or regions of immobilised capture species located downstream of the first line or region of immobilised capture species.

16. A test device according to any preceding claim, wherein the device comprises a housing which obscures the view of the or each capture zone from the user.

17. An assay device as claimed in any preceding claim, wherein the detection zone comprises an immobilised binding reagent that binds specifically to the analyte.

18. The test device of any one of the preceding claims, wherein the immobilized binding reagent in the detection zone does not bind to a positive marker or a negative marker.

19. An assay device as claimed in any preceding claim, wherein the immobilised binding reagent in the detection zone comprises or consists of an antibody.

20. An assay device as claimed in any preceding claim, wherein the immobilised binding reagent in the detection zone binds to an epitope of the analyte which is not present in the first negative marker.

21. An assay device as claimed in any preceding claim, wherein the reagent zone comprises a mobilisable binding reagent which binds specifically to the analyte, the mobilisable binding reagent being linked to a first binding partner, and the detection zone comprises an immobilised second binding partner to the first binding partner.

22. A test device according to any preceding claim, wherein the analyte is indicative of the presence of a first condition and optionally a second condition, and the first negative marker is indicative of the absence of the first condition.

23. A test device according to any preceding claim, wherein the positive marker indicates the presence of a first condition.

24. A test device according to claim 22 or 23, wherein the first condition is pregnancy.

25. An assay device as claimed in any preceding claim, wherein the first negative marker and the analyte share a common epitope.

26. An assay device as claimed in any preceding claim, wherein the first negative marker and the analyte share a common structural feature, such as a subunit of the analyte.

27. An assay device according to any preceding claim, wherein the analyte is hCG and/or wherein the first negative marker is FSH and/or the positive marker is a progesterone metabolite, such as P3G.

28. An assay device as claimed in any preceding claim, wherein the mobilisable labelled conjugate comprises an anti-a-hCG antibody.

29. An assay device as claimed in any preceding claim, wherein the capture region comprises an anti- β -FSH antibody.

30. The test device of any one of the preceding claims, wherein the mobilizable, labeled conjugate comprises an anti-P3G antibody and the capture zone comprises an immobilized P3G conjugate.

31. The test device of any one of the preceding claims, wherein the mobilizable, labeled conjugate comprises a P3G conjugate and the capture zone comprises an immobilized anti-P3G antibody.

32. An assay device according to any one of the preceding claims, wherein the detection zone comprises an anti- β -hCG antibody.

33. An assay device as claimed in any preceding claim, wherein the mobilisable labelled conjugate comprises an anti-a-hCG antibody coupled to P3G and the capture zone comprises an immobilised anti-P3G antibody.

34. A test device according to any preceding claim, wherein the label is a gold sol or latex.

35. The test device of any one of the preceding claims, adapted to reduce the amount of labeled conjugate reaching the detection zone in the presence of one or more additional negative markers.

36. The test device of any one of the preceding claims, adapted to reduce the amount of labeled conjugate reaching the detection zone in the absence of one or more additional positive markers.

37. The test device of any one of the preceding claims, wherein the positive marker moiety of option (ii) is different from the positive marker of option (iii) in the presence of the reagent zone of option (ii) and the reagent zone of option (iii).

38. A test device for detecting the presence of an analyte in a sample, the device comprising a test flow path, the flow path comprising:

(a) a reagent zone comprising a mobilizable labeled conjugate comprising a detectable label directly or indirectly linked to a binding agent that binds to the first negative marker and a binding agent that binds to the analyte, optionally wherein the binding agent is an anti-a-hCG antibody,

(b) one or more capture zones, wherein at least one capture zone comprises an immobilized capture substance that specifically binds to the first negative marker, optionally wherein the immobilized capture substance is anti-beta-FSH,

(c) a detection zone comprising an immobilized binding reagent for capturing complexes comprising the labeled conjugate and the analyte, wherein capture of the complexes is preferably achieved by a specific binding reaction with the analyte, optionally wherein the immobilized binding reagent is anti-beta-hCG,

wherein the or each capture zone is downstream of the reagent zone and the detection zone is downstream of the or each capture zone.

39. A test device for detecting the presence of an analyte in a sample, the device comprising a test flow path, the flow path comprising:

(a) a reagent zone comprising a mobilizable, labeled conjugate configured to associate with the analyte, wherein the mobilizable, labeled conjugate is further provided

i. Is configured to associate with the first negative marker, and/or

is configured to associate with a positive marker, and/or

Comprising or configured to associate with a positive marker or positive marker conjugate such that the positive marker or positive marker portion of the conjugate is available for binding,

(b) one or more capture zones containing an immobilized capture substance for capturing the labeled conjugate if the sample contains a negative marker and/or an immobilized capture substance for capturing the labeled conjugate if the sample does not contain a positive marker,

(c) a detection zone for capturing the labeled conjugate if the sample contains the analyte and is not captured by the capture zone,

wherein the one or more capture zones are downstream of the reagent zone and the detection zone is downstream of the one or more capture zones.

40. The test device of claim 39, wherein, where the reagent zone comprises option (ii) and option (iii), the positive marker of option (ii) is different from the positive marker or positive marker conjugate of option (iii) in its positive marker portion.

41. The test device of any one of the preceding claims, wherein, in the presence of the negative marker in the sample and/or the absence of the positive marker in the sample, the capture zone reduces the amount of the labeled conjugate reaching the detection zone and, in the presence of the analyte, forms a detectable signal in the detection zone.

42. A test device according to any one of the preceding claims, wherein detection of a signal in the detection zone is indicative of the presence of the analyte in the sample and of the presence of a first condition in a subject from which the sample is derived.

Technical Field

The present invention provides a test device for detecting the presence of an analyte in a sample.

Background

Referring for example to EP291194, simple lateral flow immunoassay devices for detecting analytes in liquid samples have been developed and commercialized. Such devices typically comprise a porous carrier comprising a dried, mobile (mobilisable) labelled binding reagent capable of binding to the analyte in question, and an immobilised binding reagent also capable of binding to the analyte provided at a detection zone downstream of the labelled binding reagent. The mobilisable labelled binding reagent is mobilisable within the porous carrier in the wet state. The immobilized binding reagent is permanently immobilized in the detection zone and therefore is not mobile in the wet state. The presence of the captured labeled binding reagent at the detection zone indicates the presence of analyte in the sample.

Alternatively, where the analyte of interest is a hapten, the immunoassay device may employ a competition reaction in which the labeled analyte or analyte analog competes with analyte present in the sample for binding to the immobilized binding reagent at the detection zone. Alternatively, the assay device may employ an inhibition reaction to provide an immobilised analyte or analyte analogue at the detection zone, the assay device further comprising a mobilisable labelled binding reagent for the analyte.

The test device is capable of detecting the presence and/or amount of more than one analyte. For example, in the case of an assay that detects the presence of a drug of abuse, the device is capable of determining the entire drug group. Such lateral flow immunoassay devices are typically provided with more detection zones provided on a single or more lateral flow carriers within the test device.

Digital devices have been developed which comprise optical detection means for identifying the results of the test and display means for displaying the results of the test. A digital test reader used in combination with a test strip to determine the concentration and/or amount of an analyte in a liquid sample is referred to as an assay device containing an integrated digital test reader. An embodiment of such a device is disclosed in EP 1484601.

Light from a light source, such as a Light Emitting Diode (LED), is directed onto a portion of the porous support, and reflected or transmitted light is detected by a photodetector. Typically, the reader has more than one LED to illuminate various areas of the carrier, and a respective light detector is provided for each of the more LEDs. EP1484601 discloses an optical path arrangement for a lateral flow test strip digital reading apparatus comprising a baffle arrangement which reduces the likelihood of the amount of light detectors in the apparatus.

The above-described types of test techniques have been applied to "self-test" pregnancy test devices. Typically, these devices are used by women who are suspected of being potentially pregnant themselves. Therefore, they must be designed in a manner that is easy to use (without any medical or technical training) and generally disposable after a single use. The device is typically a lateral flow immunoassay device and the test is typically initiated by contacting the sampling portion of the lateral flow test stick with a urine sample. The sampling portion of the test stick may be dipped into the urine sample in the container or, more generally, the user may urinate directly on the sampling portion. The test is then carried out without the user having to perform any further steps, the results are displayed and read with the naked eye, or, in a digital device, the results are identified by test result reading means and presented to the user via a display such as a Liquid Crystal Display (LCD) or the like.

Such conventional pregnancy tests measure human chorionic gonadotropin (hCG) in a sample. hCG is produced by developing embryos and hCG concentrations in a sample above a certain threshold will result in a positive (i.e., "pregnant") result.

There is a need for improvements in pregnancy tests, particularly self-test pregnancy tests: many women want to know whether or not they are pregnant as soon as possible. Early confirmation of pregnancy is beneficial because positive pregnancy tests can cause important positive behavioral changes that promote maternal and fetal health. For example, pregnant women are prompted to adjust their diet, begin taking pregnancy supplements and avoid the risks to developing fetuses caused by the use of drugs, alcohol and occupational exposure to mutagens/teratogens. Recently, Prior et al (Obstetrics and Gynaecology,2017,129(4,727-. They concluded that improving early pregnancy awareness is more effective than promoting abstinence of alcohol among concentent persons, which may be applied to other important health information related to pregnancy behavior.

This has led to the need for highly sensitive tests that can detect very low concentrations of hCG in samples such as urine at the early stages of pregnancy. However, one problem arises with the use of a high sensitivity test for measuring hCG, which is sometimes present in urine at relatively low concentrations for non-pregnant reasons, and therefore may give false positive results. For example, low levels of hCG may be produced in the pituitary gland of peri-and menopausal women.

According to the world health organization, the accepted definitions of "menopause" and "peri-menopause" are as follows:

menopause (natural menopause) -is defined as the permanent menopause resulting from the loss of viability of the follicles. Continuous amenorrhea for 12 months without other obvious pathological or physiological causes is considered natural menopause. Menopause occurs in the last menstrual period (FMP), which can only be determined after one year or more.

Perimenopause, includes the perimenopausal period (where endocrine, biological, and clinical features begin to approach menopause) and the first year after menopause.

Thus, perimenopausal women are currently defined as women in perimenopause according to the WHO definition above. Postmenopausal women are defined as women WHO have undergone menopause according to the WHO definition above.

Women build families later in the united states and most developed countries. From 2000 to 2014, the average age of the first time to become mom in the United states increased by 1.4 years, from 24.9 to 26.3 years (Mathews and Hamilton, National Center for Health Statistics, Data Brief 2016; No 232). Between 2000 and 2012, the first time fertility rate of women aged 35-39 and 40-44 years increased by 24% and 35%, respectively (Mathews and Hamilton, National Center for Health Statistics, Data Brief 2014; No 152). Recently, data of 2017 by the department of Health and public service in the united states (Brady et al, National Center for Health statistics. report No 004.May 2018) showed that the fertility rate of women in almost all age groups under 40 years old decreased, but the fertility rate of women in the early stage of 40 years old increased. The fertility rate of 40-44 year old women in 2017 is 11.6 per 1000 women, which is 2% higher than 2016. Fertility rates in this age group have generally risen since 1982. According to one study, up to 10% of over-the-counter pregnancy tests are sold by women over the age of 40 (Leavitt SA 2006, "A private title recommendation: the home prediction test in American Culture". Bulletin of the History of Medicine 200680,317-45).

Perimenopause can last as long as 10 years from the age of women more than 30 (American College of ostricias and gynocology 2015, FAQ047), which can lead to changes in hormone levels and can affect ovulation, leading to irregular menstrual cycles. Menopause, occurring on average in the age of 51 years, but 1% of women occur before the age of 40 years (Haller-Kikkatalo et al, Human Reproduction 201530(5),1229-1238). Many women have little knowledge of their fertility and are unaware that they are undergoing peri-menopause/menopause. Interruptions in the menstrual cycle during perimenopause, or the disappearance of menses during menopause, can be mistaken for menopause, leading to pregnancy tests.

Published literature reports elevated perimenopausal hCG levels, Snyder et al (Clinical Chemistry 2005)51,1830-1835) found to have serum hCG levels above 5mIU/ml in 1.3% of the population they examined.

During menopause, increased pituitary hCG production has been demonstrated (Odell and Griffin, New England Journal of Medicine 1987317,1688–91)，(Stenman et al,Journal of Clinical Endocrinology&Metabolism 1987 64,730-6). As women approach menopause, the number of follicles available for recruitment per cycle decreases, requiring more Follicle Stimulating Hormone (FSH) to stimulate the remaining follicles. Thus, increased FSH levels may also persist until menopause for a proportion of women. An increase in FSH content in perimenopausal and postmenopausal women leads to co-production of small amounts of hCG in the pituitary.

Such as Snyder et al (Clinical Chemistry 200551, 1830-han et al (Clinical Chemistry 1992)38,1981-1987) had recorded the production of hCG in postmenopausal women. Serum levels have been reported to be as high as 13mIU/ml, with 6.7% of women having hCG concentrations higher than 5mIU/ml (Snyder et al, 2005).

In order to be able to detect pregnancy as early as the first six days of menopause, a highly sensitive hCG detection is required. However, a highly sensitive pregnancy test that detects only low levels of hCG (i.e. a test that detects both pregnancy and pituitary-derived hCG) is at risk in that it is also more likely to detect low levels of pituitary hCG and therefore would give a false positive result.

In a different context, almost all female patients of child bearing age undergo routine serum hCG testing in many countries before any medical intervention that may harm the developing fetus is performed. The problem of elevated serum hCG levels in perimenopausal and postmenopausal women due to "pituitary" hCG is well recognized. Snyder et al (Clinical Chemistry 2005)511830-1835) the non-pregnant women were examined for changes in serum hCG concentration with age and investigated using serum FSH measurements as an aid to explain higher than expected hCG results. They suggest that combining serum hCG measurements, subject age and serum FSH measurements may reduce or avoid "false positive" pregnancy outcomes. However, these researchers are not concerned with self-test pregnancy tests, particularly with pregnancy detection at very early stages.

Attempts have been made to reduce the occurrence of false positive pregnancy results resulting from the detection of non-pregnancy associated hCG present in urine samples. For example, WO2016156981 discloses a test device for detecting pregnancy comprising: a test means for measuring the absolute or relative amount of hCG in a sample from a subject; a test means for measuring the absolute or relative amount of FSH in a sample from the subject; a test member for measuring the absolute or relative amount of one or more progesterone metabolites in a sample from a subject. This concept is based on the following observations. First, after conception, no further ovulation cycles are required as the woman is already pregnant. Thus, after conception, a decrease in FSH inhibits folliculogenesis. Therefore, the expected hCG levels are high and FSH levels are low in pregnant women. Second, during the first 10-12 weeks of pregnancy, progesterone produced by the corpus luteum supports the endometrium, thus allowing pregnancy to continue. Progesterone is elevated during part of the menstrual cycle, but if there is no pregnancy, the level will fall back to the baseline level (i.e. in the case of menstruation). However, if pregnant, the level of progesterone (and its urinary metabolites) will remain elevated and will continue to rise throughout pregnancy, so progesterone (and its urinary metabolites) can be used as an adjunct to hCG for additional confirmation of pregnancy. WO2016156981 therefore attempts to solve the problem of false positive pregnancy outcomes by proposing a test device comprising, in addition to the assay for hCG, an assay for measuring FSH and one or more progesterone metabolites. The results of the FSH and progesterone metabolite assay are used to determine whether pregnancy is indicated by a positive hCG result.

Reading and interpreting many test results to determine whether a positive result can be declared is laborious and leaves room for error generation, particularly for typical users of such devices (who have not received training to perform or interpret biochemical tests). WO2016156981 proposes the use of a digital assay reader to reduce the burden on the user. However, one disadvantage of this approach is that it increases the cost and complexity of the test apparatus.

Disclosure of Invention

The present invention provides a test device for detecting the presence of an analyte in a sample. The device includes a test flow path, the flow path including:

(a) a reagent zone comprising a mobilizable labeled conjugate configured to associate with the analyte, wherein the mobilizable labeled conjugate is also

i. Is configured to associate a first negative marker and/or

is configured to correlate positive markers, and/or

Comprising or configured to associate a positive marker or positive marker conjugate such that the positive marker or positive marker portion of the conjugate is available for binding,

(b) one or more capture zones comprising an immobilized capture substance for capturing the labeled conjugate if the sample contains a negative marker and/or an immobilized capture substance for capturing the labeled conjugate if the sample does not contain a positive marker,

(c) a detection zone for capturing the labeled conjugate if the sample contains the analyte and is not captured by the capture zone,

wherein the one or more capture zones are downstream of the reagent zone and the detection zone is downstream of the one or more capture zones.

Where the reagent zone comprises option (ii) and option (iii), the positive marker of option (ii) is different from the positive marker or positive marker moiety of the positive marker conjugate of option (iii).

There is also provided a test device for detecting the presence of an analyte in a sample, the test device comprising a test flow path comprising:

(a) a reagent zone comprising a mobilizable labeled conjugate comprising a detectable label directly or indirectly attached to a detectable label

(i) Means for associating said labeled conjugate with said analyte and means for associating said labeled conjugate with a first negative marker, said first negative marker being likely to be present in said sample, and/or

(ii) Means for associating the labeled conjugate with the analyte, wherein the labeled conjugate further comprises a positive marker or a positive marker conjugate, the positive marker portion of the positive marker conjugate being available for binding, and/or wherein a mobile positive marker or positive marker conjugate is provided separately in the reagent zone and is configured to associate with the labeled conjugate upon movement such that the positive marker or positive marker portion of the conjugate is available for binding, and/or the labeled conjugate associates with the positive marker or positive marker conjugate

(iii) Means for associating the labeled conjugate with the analyte and means for associating the labeled conjugate with a positive marker or positive marker conjugate in the device or a positive marker that may be present in the sample, optionally and wherein the reagent zone further comprises a mobilizable positive marker or positive marker conjugate for immobilization downstream of the reagent zone,

(b) one or more capture zones, wherein,

(i) in the case where the reagent zone comprises option (i),

at least one capture zone comprising an immobilized capture substance configured to capture a complex comprising the labeled conjugate and the first negative marker, wherein capturing the complex is preferably achieved by a specific binding reaction with the first negative marker,

(ii) in the case where the reagent zone comprises option (ii),

at least one capture zone comprises an immobilized capture substance configured to capture a labeled conjugate comprising the positive marker or positive marker conjugate or a labeled conjugate associated with the positive marker or positive marker conjugate or the positive marker in the case of the presence of a positive marker in the sample, wherein capturing the labeled conjugate is preferably achieved by a specific binding reaction with a positive marker or positive marker conjugate,

(iii) in the case where the reagent zone comprises option (iii),

at least one capture zone comprises an immobilized capture substance configured to capture a labeled conjugate comprising means for associating the labeled conjugate with a positive marker or positive marker conjugate, wherein the immobilized capture substance comprises or consists of an immobilized positive marker or positive marker conjugate, and/or in case the reagent zone comprises a mobilizable positive marker or positive marker conjugate, at least one capture zone comprises an immobilized capture substance for localizing the mobilizable positive marker or positive marker conjugate at the capture zone, thereby rendering a positive marker moiety of the positive marker or conjugate available for binding, wherein the capture of the labeled conjugate is preferably by means of a specific binding to the means for associating the labeled conjugate with a positive marker or positive marker conjugate The anisotropic combination reaction is realized,

(c) a detection zone comprising an immobilised binding reagent for capturing a complex comprising the labelled conjugate and the analyte, wherein capture of the complex is preferably achieved by a specific binding reaction with the analyte,

wherein the or each capture zone is downstream of the reagent zone and the detection zone is downstream of the or each capture zone.

Embodiments of the present invention include assay devices that include, but are not limited to, the following combinations of reagent zone and capture zone options:

additional embodiments of the invention are set forth in the dependent claims and in the following description. In one embodiment, the test device is adapted to reduce the amount of labeled conjugate reaching the detection zone in the presence of one or more additional negative markers. In one embodiment, the test device is adapted to reduce the amount of labeled conjugate reaching the detection zone in the absence of one or more additional positive markers.

As discussed in further detail below, where the test device of the present invention includes an immobilized capture material for reducing the amount of labeled conjugate that reaches the detection zone in the presence of the first negative marker, the test device may further include any suitable reagent disclosed herein and an immobilized capture material to reduce the amount of labeled conjugate that reaches the detection zone in the presence of the second negative marker. This concept can be extended to reduce the amount of labeled conjugate reaching the detection zone in the presence of more negative markers.

In embodiments where both reagent zone option (ii) and reagent zone option (iii) are present, preferably the positive marker of option (ii) is different from the positive marker of option (iii). For example, the positive marker of option (ii) may be a first positive marker, and the positive marker of option (iii) may be a second positive marker. This means that the positive marker conjugate of option (ii) or the positive marker of the device will be different from the positive marker conjugate of option (iii) or the positive marker of the device when both are present in the test device of the present invention.

Where the test device of the present invention comprises an immobilised capture material for reducing the amount of labelled conjugate reaching the detection zone in the absence of the first positive marker, the test device may further comprise any suitable reagent disclosed herein and an immobilised capture material for reducing the amount of labelled conjugate reaching the detection zone in the absence of the second positive marker. This concept can be extended to reduce the amount of labeled conjugate reaching the detection zone in the absence of more positive markers.

In one embodiment, the device does not comprise any means for reducing the amount of labeled conjugate reaching the detection zone in the absence of a positive marker. In one embodiment, the device does not comprise any means for reducing the amount of labeled conjugate reaching the detection zone in the presence of a negative marker.

The invention also provides a method of testing using the testing apparatus of the invention, the method comprising:

(i) the sample is applied to the test device and,

(ii) the assay result at the detection zone is determined without reference to any capture zone present in the device.

Detailed Description

The present invention is based, in part, on the recognition that there is a need to improve the specificity of analyte testing. In conventional test devices, a positive test result indicating the presence of the first condition in the subject can be based on identifying the presence of the analyte in a sample from the subject (e.g., by observing the accumulation of a detectable label associated with the analyte in a detection zone of the test device). However, where the analyte is associated with more than one state, then the presence of the analyte does not necessarily imply a risk of the presence of the first state in the subject. The present invention addresses this problem by utilizing the presence or absence of additional markers in a sample from the subject to modulate the ability of the test device to generate a detectable signal in the detection zone in response to the presence of analyte in the sample. The present invention contemplates using the presence of one or more negative markers in the sample to reduce the amount of labeled conjugate associated with the analyte that reaches the detection zone and generates a signal. The present invention also contemplates using the absence of one or more positive markers in the sample to reduce the amount of labeled conjugate associated with the analyte that reaches the detection zone and generates a signal. Both strategies, alone or in combination, reduce the risk of generating a signal (i.e., observing a positive test result) in the detection zone in the absence of the first state in the subject.

One non-limiting example of this principle is in the context of pregnancy testing. In this embodiment, the analyte indicative of pregnancy (but may also be present in other states such as menopause or perimenopause) is hCG. According to the present invention, pregnancy negative markers such as FSH (elevated peri-or menopause) may be used to reduce the amount of labeled conjugate associated with hCG which reaches the detection zone and generates a signal. In such embodiments, the reagent zone may comprise a mobile labelled conjugate capable of associating with both hCG and FSH (e.g. may comprise an anti-hCG antibody and an anti-FSH antibody). An FSH capture zone (invisible to the user, or visible but not interpretable by the user, or indeed unmeasured by the device) may be provided downstream of the reagent zone and upstream of the detection zone. In the case where the sample contains hCG but no FSH, the mobilisable labelled conjugate associates with hCG, passes through the capture zone and binds at the detection zone, thereby allowing a signal to be observed at the detection zone indicative of pregnancy in the subject. In the case where the sample contains hCG and FSH (e.g. because the subject is perimenopause), the mobilizable, labeled conjugate can associate with hCG and FSH to form a complex. The complex is captured at the FSH capture zone and therefore cannot move to the detection zone. Thus, although the sample contains hCG, the amount of labelled conjugate which reaches the detection zone and produces an observable signal is reduced relative to that in the presence of hCG and in the absence of FSH.

Pregnancy positive markers such as P3G, according to the present invention, can be used to modulate the amount of labeled conjugate associated with the analyte that reaches the detection zone and generates a signal. For example, the reagent zone may comprise a mobile labelled conjugate (e.g. may comprise an anti-hCG antibody and an anti-P3G antibody) which is capable of associating with both hCG and P3G. A capture zone (invisible to the user, or visible but not interpretable by the user, or indeed unmeasured by the device) may be provided downstream of the reagent zone and upstream of the detection zone and may include a P3G conjugate. The detection zone may contain, for example, anti-hCG antibodies. In the case of a sample containing hCG and P3G, the mobile labelled conjugate will associate with hCG and P3G to form a complex, preventing the P3G conjugate in the capture zone from capturing the complex. The complex passes through the capture zone and binds to the detection zone by hCG in the sample, thereby allowing a signal to be observed at the detection zone indicating pregnancy in the subject. In the case of samples containing hCG but not P3G, the mobilizable, labeled conjugate will associate with hCG to form a complex. The P3G conjugate captures the labeled reagent in the capture zone, preventing it from continuing into the detection zone. Thus, although the sample contains hCG, the amount of labelled conjugate reaching the detection zone and producing an observable signal is reduced. In an alternative arrangement, the P3G conjugate can instead be provided on a mobile conjugate, and an immobilized capture substance (e.g. anti-P3G antibody) configured to capture the P3G or P3G conjugate is provided in the capture zone. In this embodiment, where the sample contains P3G, it will be captured by the capture zone, meaning that the labelled conjugate containing P3G conjugate will not be captured in the capture zone and will be able to continue to enter the detection zone where it will react with hCG in the sample and generate a signal. In the presence of hCG but in the absence of P3G in the sample, the mobile conjugate will be captured by the immobilised capture species at the capture zone and will not continue into the detection zone. Thus, although the sample contains hCG, the amount of labelled conjugate entering the detection zone and producing an observable signal is reduced relative to that in the presence of both hCG and P3G.

One application of the present invention is to provide an improved pregnancy test of high sensitivity, with improved specificity, allowing earlier determination of pregnancy. Such an improved pregnancy test is particularly useful for peri-or post-menopausal individuals because, in the event that these individuals are not pregnant, the risk of obtaining a positive result due to the small amount of pituitary-derived hCG in the test sample is reduced. It will be appreciated that the principles may be applied to other analytes and markers, including those associated with other states. For example, one can envision assays that test for disease X based on identifying analyte a in the sample. Analyte a is also present in state Y. Negative marker N is present at a relatively high level in state Y, but absent or present at a relatively (significantly) low level in disease X. The test device of the present invention may comprise a mobilisable labelled conjugate comprising a binding reagent for analyte a and a binding reagent for negative marker N, a capture zone for an immobilised capture species comprising the binding reagent for negative marker N and a detection zone for an immobilised binding reagent for analyte a. In the presence of negative marker N and analyte a in the sample applied to the device (because the subject from which the sample is derived is in state Y and not disease X), N binds to the labelled conjugate to form a complex bound at the capture zone. Thus, in the presence of the negative-form marker N in the sample, the capture zone reduces the amount of labeled conjugate that reaches the detection zone and produces an observable signal in the presence of analyte a. Therefore, especially in the case of testing a sample containing analyte a in the presence of state Y, the risk of a positive result indicating the presence of disease X is reduced and the specificity of the test is improved.

In another embodiment, the assay for testing for disease X can be based on the identification of analyte a in the sample. Analyte a is also present in state Y. Positive marker P is present at a relatively high level in disease X, but absent or present at a relatively (significantly) low level in state Y. The test device of the present invention may comprise a mobilisable labelled conjugate comprising a binding reagent for analyte a and a binding reagent for positive marker P, a capture zone comprising an immobilised positive marker or positive marker conjugate and a detection zone comprising an immobilised binding reagent for analyte a. In the presence of the positive marker P and analyte a in the sample, the binding reagent for the positive marker P will bind to the positive marker P in the sample, preventing capture of the labeled conjugate by the immobilized positive marker or positive marker conjugate at the capture zone. The labeled conjugate will continue to enter the detection zone where it is captured by the immobilized binding reagent for analyte a, thus generating a signal at the detection zone indicative of the presence of disease X. In the presence of analyte a and in the absence of positive marker P in the sample, the binding reagent for positive marker P of the labeled conjugate will bind to the immobilized positive marker or positive marker conjugate in the capture zone, preventing the labeled conjugate from continuing into the detection zone. Thus, in the presence of analyte in the sample and in the absence of the positive marker P, the capture zone reduces the amount of labeled conjugate that reaches the detection zone and produces an observable signal.

The device of the invention may be adapted to include a mobilizable reagent and an immobilized capture substance to reduce the amount of labeled conjugate reaching the detection zone in the presence of any amount of additional negative marker and/or in the absence of any amount of additional positive marker.

Test flow path

In one embodiment, the assay device of the invention comprises an assay flow path comprising a reagent zone, one or more capture zones and a detection zone. The or each capture zone is downstream of the reagent zone and the detection zone is downstream of the or each capture zone.

"upstream" and "downstream" are used to describe the location of components of the assay device with reference to the direction of flow of a sample applied to the device. If component a is located upstream of component B and/or component B is located downstream of component a, the sample applied to the device (at the predetermined sample application location) will reach component a first and then component B.

The dried binding reagent is provided in the flow path of the microfluidic device, or in the case of a lateral flow device, on a porous carrier material provided upstream of the porous carrier material containing the detection zone. The upstream porous support material may be macroporous. The macroporous support material should be low or non-protein binding, or should be easily blocked with a reagent such as BSA or PVA to minimize non-specific binding and to promote free movement of the labeled reagent after the macroporous support is wetted with the liquid sample. Where desired, the macroporous support material may be pretreated with a surfactant or solvent to make it more hydrophilic and to promote rapid absorption of the liquid sample. In addition, one or more sugars (e.g., sucrose, trehalose) can be used to stabilize and aid in the movement of the labeled reagent. These may conveniently be applied to the flow paths and/or the porous support material as part of a solution in which the labelled reagent is applied to the porous support. Suitable materials for the macroporous support include plastic materials (e.g. polyethylene and polypropylene) or other materials (e.g. paper or glass fibre). In the case where the detectable particles are labeled with a labeled binding reagent, the pore size of the macroporous support may be at least ten times the maximum particle size of the labeled particles. Larger pore sizes may better release the labeled reagent.

The test flow path may comprise a lateral flow porous support. The porous carrier material is typically a material with a high protein binding capacity that is blocked with a reagent such as BSA or PVA after the reagent is applied to form the immobilised capture and detection zone to minimise non-specific binding and to promote free movement of the labelled conjugate after wetting of the macroporous carrier by the liquid sample. Suitable materials that can be used as porous carriers include nitrocellulose, acetate fiber, cellulose or cellulose derivatives, polyesters, polyamides, polyolefins or glass fibers. The porous carrier may comprise nitrocellulose. Its advantage is no need of chemical treatment to fix the binding reagent firmly. If the porous solid phase material comprises paper, for example, immobilization of the antibody may be performed by chemical coupling using, for example, CNBr, carbonyldiimidazole or trifluoroethanesulfonyl chloride. The test device may include a sample application zone. It is a region of porous (usually absorbent) material to which an aqueous sample (e.g. urine) can be applied.

An alternative to the lateral flow type test flow path is the "flow through" test device. In such devices, the sample flows substantially vertically rather than laterally. Preferably, the device comprises a sample addition zone; one or more membranes comprising one or more capture zones as described herein, disposed vertically or substantially vertically below the sample addition zone; and a membrane comprising a detection zone as described herein disposed vertically or substantially vertically below the one or more capture zone membranes. The sample is applied to the application zone and flows down through the or each capture zone membrane to reach the detection zone membrane. The assay result is determined, for example, by viewing or reading the detection zone from the bottom surface of the flow-through device. The flow-through assay device may or may not include a reagent zone as previously described. If provided, the reagent zone is located in a further membrane upstream (in a substantially vertical direction) of the capture zone membrane. Such a reagent zone may include any of the reagents described herein (e.g., labeled conjugates) in a dry state. Alternatively, or in addition, a reagent such as a labeled conjugate described herein may be provided in a dry state adjacent to the capture zone membrane. Alternatively, the labeled conjugate may be provided separately and applied to the device either before or after application of the sample, or applied to the device with the sample. For example, such reagents may be mixed with the sample prior to application to the device. The invention includes kits comprising a flow-through assay device as described herein, a labeled reagent as described herein, and optionally any other reagent described herein (e.g., a reagent provided in a reagent zone).

Typically, the sample is applied by the user urinating directly in the sample application zone. Alternatively, the sample application zone can be immersed in a container containing the sample. Where the sample is blood, serum or plasma, the sample may be applied to the device using a pipette or other instrument, and then an elution buffer may be added to move the sample components along the assay flow path.

The sample application zone is preferably upstream of the reagent zone.

An absorption "sink" may be provided at the distal, downstream end of the test flow path. The absorption sink may preferably comprise a highly absorbent material, e.g. CF7 wadman paper, and should provide sufficient absorption capacity to remove any unbound labels from the vicinity of the detection zone. A solid phase porous material having a length extending beyond the detection zone may be used as a substitute for such a sink. An advantage of providing a superabsorbent sink is that it removes or substantially removes excess labeled binding reagent from the assay flow path, minimizing the amount of unbound labeled binding reagent near each zone. Removal or reduction of unbound labeled reagent near the detection zone (and control zone, if any) helps to determine the correct test result.

Reagent zone

The reagent zone of the test device of the present invention comprises a mobile labeled conjugate which may contain a detectable label.

A "mobile" substance refers to a substance that is associated with a matrix (e.g., a component of a test flow path) in a dry state, and that is mobile and capable of flowing along the matrix upon contact with a fluid.

"label" in the context of a labeled conjugate refers to any substance capable of producing a signal that can be detected by visual or instrumental means. Various labels suitable for use in the present invention include labels that generate a signal by chemical or physical means (e.g., optically detectable). Such labels include enzymes and substrates, chromogens, catalysts, fluorescent compounds, chemiluminescent compounds, electroactive species, dye molecules, radioactive labels, and particle labels. The particle labels may comprise magnetic or charged labels, which may be detected by magnetic or electrochemical means. The label may be covalently linked to the binding agent. The label may be optically detectable. Preferred optically detectable labels include colloidal metal particle labels and dye particles, hereinafter.

Labels may include colloidal metal particles (e.g., gold, silver, platinum, silver-enhanced gold sol, carbon sol, or carbon nanoparticles), colloidal metalloid or non-metal particles (e.g., tellurium or selenium), or dyed or colored particles (e.g., polymer particles incorporating a dye or dye sol). The dye may be any suitable color such as blue. The dye may be fluorescent or comprise quantum dots. Suitable fluorescent materials are well known to those skilled in the art. Dye sols can be prepared from commercially available hydrophobic dyes (e.g., Foron Blue SRP (Sandoz) and Resolin Blue BBLS (Bayer)). Suitable polymer labels may be selected from a range of synthetic polymers (e.g. polystyrene, polyvinyltoluene, polystyrene-acrylic acid and polyacrolein). The monomers used are generally water-insoluble and are emulsified in an aqueous surfactant so that monomer micelles are formed, and then polymerization is induced by adding an initiator to the emulsion, resulting in nearly spherical polymer particles. The desired size range for such polymer particles is from about 0.05 μm to about 0.5. mu.m. Larger polymer particles can also be used coupled to appropriately sized porous membranes. According to an exemplary embodiment, the label is a gold colloid, preferably with a particle mean diameter in the range of 0.02 μm to 0.25 μm. In one embodiment, the label is a blue latex. In one embodiment, the label is a gold sol. In one embodiment, the label is a 40nm gold sol.

Currently, the term "binding agent" refers to one of a binding pair (i.e., two different molecules, wherein one molecule chemically and/or physically binds to the second molecule). The two molecules are related in the sense that they bind to each other such that they can distinguish their binding partners from other assay components having similar characteristics. Members of a binding pair may be referred to as a ligand and receptor (anti-ligand), a binding pair member and a binding pair partner, and the like. The molecule may also be a binding pair member of an aggregate of molecules; for example, an antibody and its corresponding antigen of an immune complex for a secondary antibody may be considered a binding pair member of the immune complex. The binding reagent is typically a specific binding reagent. For example, the binding agent may specifically bind to the alpha subunit of hCG and the alpha subunit on FSH, the alpha subunit on Luteinizing Hormone (LH) or the alpha subunit on Thyroid Stimulating Hormone (TSH). The specific binding reagent may be one which binds to the β subunit of hCG.

The binding agent may consist of or comprise an antibody. As used herein, the term "antibody" includes whole antibodies and antigen-binding fragments thereof. Antigen-binding fragments can be produced by conventional techniques. Examples of such fragments include, but are not limited to, Fab, F (ab')2, and Fv fragments. The binding agent may consist of or comprise an aptamer. The binding agent may consist of or comprise an affimer. Binding reagents such as antibodies for use in the present invention are available from various suppliers (e.g., Medix Biochemica Espoo, Finland).

In addition to antigen and antibody binding pair members, other binding pairs include, but are not limited to, biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, peptide sequences and antibodies specific for the sequences or the entire protein, polymeric acids and bases, dyes and protein binding agents, peptides and specific protein binding agents (e.g., ribonuclease, S-peptide, and ribonuclease S-protein), and the like. In addition, specific binding pairs may include analogs of the original specific binding reagent.

In some embodiments, the mobilizable labeled conjugate comprises means for associating the labeled conjugate with an analyte and means for associating the labeled conjugate with a first negative marker that may be present in the sample.

The means for associating the labeled conjugate with the analyte may comprise or consist of a binding reagent. The binding reagent may bind the analyte directly or indirectly. Where the binding reagent indirectly binds the analyte, the mobilizable analyte binding reagent linked to the first binding partner can be provided separately from the labeled conjugate (e.g., in the reagent zone). In these embodiments, the labeled conjugate can be provided with a second binding partner that is directed to the first binding partner. In this way, the binding reagent for the analyte can be localized to the labeled conjugate without being directly linked to the labeled conjugate. The first and second binding partners may be any suitable binding partners (e.g., antibody-antigen) that are specific for each other. Examples include binding reagent pairs such as biotin and streptavidin, biotin and anti-biotin antibodies, fluorescein and anti-fluorescein antibodies.

Either one of the binding pairs may be linked to the labeled conjugate while the other is provided elsewhere, e.g., in the reagent zone or along the flow path but upstream of the capture zone. This concept can be adapted to include more intermediate binding partners. This concept can be adapted to locate other binding members disclosed herein at desired locations on the assay flow path.

The associating means for associating the labeled conjugate with the analyte may comprise or consist of an antibody (e.g., an antibody that binds an epitope of the analyte).

The means for associating the labeled conjugate with the first negative marker may comprise or consist of a binding reagent. The binding reagent may bind directly or indirectly to the first negative marker. Where the binding reagent binds indirectly to the first negative marker, the mobilizable first negative marker binding reagent linked to the first binding partner can be provided separately from the labeled conjugate (e.g., in the reagent zone). In these embodiments, the labeled conjugate can be provided with a second binding partner that is directed to the first binding partner. In this way, the binding reagent for the first negative marker may be located at the labeled conjugate, rather than being directly linked to the labeled conjugate. The first and second binding partners may be any suitable binding partners (e.g., antibody-antigen) that are specific for each other. Examples include binding reagent pairs (e.g., biotin and streptavidin, biotin and anti-biotin antibodies, fluorescein and anti-fluorescein antibodies).

Either one of the binding pairs may be linked to the labelled conjugate while the other is provided elsewhere, for example in the reagent zone or along the flow path but upstream of the capture zone. This concept can be adapted to include more intermediate binding partners. This concept can be adapted to locate other binding members disclosed herein at desired locations on the assay flow path.

The means for associating the labeled conjugate with the first negative marker may comprise or consist of an antibody (e.g., an antibody that binds to an epitope of the first negative marker).

The means for associating the labeled conjugate with the analyte and the means for associating the labeled conjugate with the first negative marker may be the same. For example, the labeled conjugate may be directly or indirectly linked to an antibody that binds the analyte or the first negative marker. The antibody may bind to an epitope common to the analyte and the first negative marker. The antibody may bind to a domain common to the analyte and the first negative marker. Where the analyte is hCG and the first negative marker is FSH, the mobilizable, labeled conjugate can comprise an antibody that binds to the α subunit of hCG. Both FSH and hCG are heterodimeric molecules containing α and β subunits. The alpha subunits of FSH and hCG are substantially identical, so antibodies for the alpha subunits can bind both FSH and hCG. In some embodiments, the means for associating the labeled conjugate with the analyte and the means for associating the labeled conjugate with the first negative marker are different. For example, the labeled conjugate can be directly or indirectly linked to an antibody that binds to the analyte but not to the first negative marker, and also directly or indirectly linked to an antibody that binds to the first negative marker but not to the analyte.

In some embodiments, the mobilizable labeled conjugate comprises a positive marker or a positive marker conjugate, wherein the positive marker portion of the positive marker conjugate is available for binding. The positive marker or positive marker conjugate may be linked or otherwise associated with a means for associating the labeled conjugate with an analyte. Where the positive marker is a hapten, it is preferably coupled to another moiety such as a carrier to form a positive marker conjugate. Larger positive markers may or may not be coupled to another moiety. The larger (non-hapten) positive marker may be immobilized directly on the mobile labeled conjugate by methods such as physical absorption or chemical coupling. However, in cases where direct immobilization has an adverse effect on the structure of the positive marker or its ability to participate in a binding reaction, it may be preferable to link the non-hapten positive marker to another moiety. The positive marker may be a progesterone metabolite such as P3G. The means for associating the labeled conjugate with an analyte can be any of the means disclosed herein. The positive marker/positive marker conjugate may be coupled to a binding reagent for the analyte and linked, directly or indirectly, to the labeled conjugate in the manner discussed herein. For example, where the analyte is hCG and the positive marker is a progesterone metabolite such as P3G, the progesterone metabolite such as P3G can be coupled with an anti-hCG antibody (e.g., an anti- α -hCG antibody). Alternatively or additionally, a mobilizable positive marker or positive marker conjugate can be provided separately in the reagent zone. In this case, the mobilizable positive marker or positive marker conjugate is capable of associating with the labeled conjugate upon mobilization, such that the positive marker or positive marker portion of the conjugate is available for binding where the labeled conjugate associates with the positive marker or positive marker conjugate. To facilitate such association, the labeled conjugate and the positive marker/positive marker conjugate may each comprise a member of a binding pair such that upon contact of the members of the binding pair with each other, the labeled conjugate associates with the positive marker/positive marker. The first and second binding partners may be any suitable binding partners (e.g., antibody-antigen) that are specific for each other. Where the liquid contacts the positive marker or positive marker conjugate, the positive marker or positive marker conjugate is mobile and can be associated with a labeled conjugate. Examples of binding pairs include biotin and streptavidin, biotin and anti-biotin antibodies, fluorescein and anti-fluorescein antibodies. Either partner may be linked to the labeled conjugate and the other partner (linked to the positive marker or positive marker conjugate) is provided, for example, in the reagent zone or elsewhere along the flow path upstream of the capture zone. This concept can be adapted to include more intermediate binding partners.

The positive marker conjugate comprises a positive marker moiety. The positive marker moiety may be bound by a binding agent such as an antibody. The positive marker conjugate may comprise a progesterone metabolite such as P3G or a portion thereof. The positive marker conjugate may comprise a protein carrier (e.g., BSA, ovalbumin, or mouse antibody).

In some embodiments, the mobilizable labeled conjugate comprises a means for associating the labeled conjugate with a positive marker or a positive marker conjugate. The means for associating the labeled conjugate with the positive marker/positive marker conjugate may comprise or consist of a binding reagent. The binding reagent may bind directly or indirectly to the positive marker or positive marker conjugate. Where the binding reagent indirectly binds to the positive marker or positive marker conjugate, the mobile binding reagent for the positive marker/positive marker conjugate linked to the first binding partner may be provided separately from the labeled conjugate, e.g., in the reagent zone, or elsewhere along the flow path but upstream of the capture zone. In these embodiments, the labeled conjugate can be provided with a second binding partner that is directed to the first binding partner. In this way, the binding reagent for the positive marker/positive marker conjugate can be located at the labeled conjugate without being directly linked to the labeled conjugate. The first and second binding partners may be any suitable binding partners (e.g., antibody-antigen) that are specific for each other. Examples include biotin and streptavidin, biotin and anti-biotin antibodies, fluorescein and anti-fluorescein antibodies. Either one of the partners is linked to the labeled conjugate, while the other partner (linked to a binding reagent for the positive marker/positive marker conjugate) is provided, e.g., in the reagent zone or elsewhere along the flow path but upstream of the capture zone. This concept can be adapted to include more intermediate binding partners.

The means for associating the labeled conjugate with the positive marker or the positive marker conjugate may comprise or consist of an antibody (e.g., an antibody that binds to an epitope of the positive marker). For example, where the positive marker is P3G, the means for associating the labeled conjugate with the positive marker or positive marker conjugate may comprise or consist of an anti-P3G antibody.

In some embodiments, the reagent zone further comprises a mobilizable positive marker or positive marker conjugate for immobilization at a capture zone, such as downstream of the reagent zone. Such mobilizable positive marker/positive marker conjugates can comprise or be linked to one member of a binding pair. The other member of the binding pair is provided downstream of the reagent zone, e.g., the capture zone. The mobile positive marker/positive marker conjugate may thus be immobilised at the capture zone such that it can be associated with means for associating the labelled conjugate with the positive marker/positive marker conjugate. Examples of binding pairs include biotin and streptavidin, biotin and anti-biotin antibodies, fluorescein and anti-fluorescein antibodies. Either of the partners may be linked to a mobile positive marker/positive marker conjugate, while the other partner is provided, for example in the capture zone. This concept can be adapted to include more intermediate binding partners.

Capture zone

The device of the invention comprises one or more capture zones located downstream of the reagent zone and upstream of the detection zone. The function of the one or more capture zones is to ensure that detection of the labeled conjugate at the detection zone reliably indicates the presence of the first condition in the subject from which the sample was derived. This functionality is achieved by: (i) in the case where the sample contains one or more negative markers (preferably, markers indicative of the absence of the first state), capturing the labeled conjugate at the capture zone such that the amount of labeled conjugate reaching the detection zone is reduced, and/or (ii) capturing the labeled conjugate at the capture zone unless the sample contains one or more positive markers (preferably, markers indicative of the presence of the first state) such that the amount of labeled reagent reaching the detection zone is reduced. Thus, in the presence of a negative marker and/or in the absence of a positive marker, the capture zone will reduce the amount of labeled conjugate reaching the detection zone, thereby reducing the likelihood that a detectable signal will be generated at the detection zone even in the presence of small amounts of analyte. This means that the likelihood of false positives (i.e. the presence of a first state is indicated in the absence of a first state) is reduced, improving the specificity of the test. Advantageously, not only can the capture zone reduce the amount of labelled conjugate reaching the detection zone, but the capture zone can also reduce the amount of analyte reaching the detection zone, as the complex captured at the capture zone can also contain analyte. In the case where the test sample is from a subject not having the first condition, this dual effect significantly reduces the risk of detecting a signal at the detection zone. Preferably, the device does not comprise a capture zone comprising an immobilised capture species capable of binding the analyte.

The capture zone may be designed to take advantage of the presence of one or more negative markers in the sample to reduce the amount of labeled conjugate (which may be associated with the analyte) that reaches the detection zone.

The capture zone may be designed such that the presence of one or more negative markers in the sample reduces the amount of labeled conjugate (which may be associated with the analyte) that reaches the detection zone. The capture zone can comprise an immobilized capture material configured to capture a complex comprising the labeled conjugate and the first negative marker. The capture of the complex is preferably achieved by a specific binding reaction with the first negative marker. Preferably, the binding reaction occurs at an epitope of the first negative marker that is different from the epitope bound by the means used to associate the labeled conjugate with the first negative marker of the mobilizable labeled conjugate, thereby forming a "sandwich". In the case where an analyte is present in the sample, the captured complex may further comprise the analyte. Preferably, the immobilized capture substance for the negative marker is configured not to capture a complex not comprising the first negative marker.

The one or more capture zones may comprise an immobilized capture substance specific for the first negative marker. In some embodiments, upon application of the sample comprising the first negative marker to the test device, the first negative marker is associated with the labeled conjugate by means for associating the labeled conjugate with the first negative marker, forming a first negative marker-labeled conjugate complex. In case the analyte is also present in the sample, then the complex may further comprise the analyte, since the labeled conjugate is capable of binding both the analyte and the negative marker. Where the complex reaches a capture zone containing an immobilized capture material configured to capture a complex comprising the labeled conjugate and the first negative marker, the complex is captured and unable to flow to the detection zone. Thus, the presence of the first negative marker in the sample prevents or reduces the total amount of labeled conjugate that reaches the detection zone and produces a detectable signal, even if the analyte is present in the sample. This also effectively reduces the amount of analyte reaching the detection zone, further reducing the signal from the detection zone in samples containing both analyte and negative marker.

The immobilized capture substance can bind directly to the first negative marker. The immobilized capture substance can bind to an epitope of the first negative marker that is not present in the analyte. The immobilized capture substance may comprise or consist of an antibody. The antibody may bind to an epitope of the first negative marker that is not present on the analyte. Where the first negative marker is FSH, the capture zone may comprise an immobilized anti-FSH specific antibody (e.g. an anti- β -FSH antibody).

The capture zone may comprise an immobilized capture material that indirectly binds to the first negative marker. For example, in some embodiments, a mobilizable first negative marker binding reagent specific for the first negative marker linked to the first binding partner can be provided, e.g., in the reagent zone or elsewhere along the flow path but upstream of the capture zone. The mobilizable first negative marker binding reagent can bind to an epitope of the first negative marker that is not present in the analyte. The or each capture area may provide an immobilised second binding partner to the first binding partner. In this way, the mobilizable first negative marker binding reagent can be located at the capture zone without being directly immobilized to the flow path. The first and second binding partners may be any suitable binding partners (e.g., antibody-antigen) that are specific for each other. Examples include binding reagent pairs such as biotin and streptavidin, biotin and anti-biotin antibodies, fluorescein and anti-fluorescein antibodies. Either member of the binding reagent pair may be immobilized at the capture zone, while the other member (linked to the binding reagent for the negative marker) is provided, for example, in the reagent zone or elsewhere along the flow path but upstream of the capture zone. This concept can be adapted to include more intermediate binding partners. This concept can be adapted to locate other binding members disclosed herein at desired locations on the assay flow path. In one embodiment, the reagent zone comprises a mobilizable anti- β -FSH antibody labeled with a first member of a binding pair (e.g., biotin), and the capture zone comprises a second member of an immobilized binding pair (e.g., anti-biotin antibody or streptavidin). Thus, the anti- β -FSH antibody may be located at the capture zone without being immobilized directly at the capture zone. This concept can be adapted to capture zones for other negative markers.

Alternatively, or in addition, the capture zone may be designed to take advantage of the absence of one or more positive markers in the sample to reduce the amount of labeled conjugate (which may be associated with the analyte) that reaches the detection zone.

The presence of a positive marker in the sample reduces the likelihood that the labeled conjugate will be captured in the capture zone (e.g., by occupying the capture zone or the positive marker binding site of the labeled conjugate).

In some embodiments, at least one capture zone comprises an immobilized capture material configured to capture a positive marker or a labeled conjugate comprising or associated with a positive marker/positive marker conjugate. Capture of the labeled conjugate is preferably achieved by a specific binding reaction with the positive marker/positive marker conjugate. The positive marker present in the sample is bound by (and therefore may occupy) the binding site of the immobilised capture material, thereby rendering the binding site unavailable for capture of a labeled conjugate comprising or associated with the positive marker/positive marker conjugate. The labeled conjugate comprising or associated with the positive marker/positive marker conjugate may thus continue to enter the detection zone (unless prevented by other capture members disclosed herein).

The positive marker in the sample may compete with the positive marker/positive marker conjugate of the device for binding at the capture zone. In the absence of a positive marker in the sample, the immobilized capture material captures the positive marker/positive marker conjugate containing the device or a labeled conjugate associated with the positive marker/positive marker conjugate of the device, thereby preventing the labeled conjugate from continuing to enter the detection zone. The immobilized capture material can directly bind to the positive marker or positive marker conjugate. The immobilized capture substance may comprise or consist of an antibody. Where the positive marker is P3G, the capture zone may comprise an immobilized anti-P3G antibody.

The capture zone may comprise an immobilized capture substance that indirectly captures the positive marker or positive marker conjugate. For example, in some embodiments, a mobilizable binding reagent linked to a first binding partner for a positive marker/positive marker conjugate is provided elsewhere, such as in the reagent zone or along the flow path but upstream of the capture zone. In such embodiments, the or each capture area may be provided with an immobilised second binding partner to the first binding partner. In this way, mobile binding reagents for the positive marker/positive marker conjugate can be located at the capture zone without being directly immobilized to the flow path. The first and second binding partners may be any suitable binding partners that are specific for each other. Examples include binding reagent pairs such as biotin and streptavidin, biotin and anti-biotin antibodies, fluorescein and anti-fluorescein antibodies. Either member of the binding reagent pair may be immobilized at the capture zone while the other member is provided in, for example, the reagent zone. This concept can be adapted to include more intermediate binding partners.

In one embodiment, the reagent zone comprises a mobilizable anti-P3G antibody labeled with a first member of a binding pair (e.g., biotin), and the capture zone comprises an immobilized second member of the binding pair (e.g., anti-biotin antibody or streptavidin). Thus, the anti-P3G antibody may be located at the capture zone without being immobilized directly at the capture zone. This concept can be adapted to capture zones for other positive markers.

In some embodiments, at least one capture zone comprises an immobilized capture species configured to capture a labeled conjugate comprising means for associating the labeled conjugate with a positive marker or a positive marker conjugate. Capture of the labeled conjugate is preferably achieved by a specific binding reaction with a means for associating the labeled conjugate with the positive marker or positive marker conjugate. The immobilized capture material may comprise or consist of an immobilized positive marker or positive marker conjugate. Where the positive marker is a hapten, it is preferably coupled to another moiety (e.g. a carrier) to form a positive marker conjugate. Larger positive markers may or may not be coupled to another moiety. Larger (non-hapten) positive markers can be immobilized directly on the flow path. However, for example, in cases where direct immobilization has an adverse effect on the structure of the positive marker or its ability to participate in a binding reaction, it may be preferable to link the non-hapten positive marker to another moiety. Alternatively or additionally, the reagent zone may comprise a mobilizable positive marker or positive marker conjugate. In this case, at least one capture zone comprises an immobilized capture material for localizing the mobile positive marker or positive marker conjugate to the capture zone, thereby enabling the positive marker or positive marker portion of the conjugate to be available for binding. In this way, the positive marker conjugate can be located at the capture zone without being directly immobilized to the flow path. Localization of the positive marker or positive marker conjugate to the capture zone can be achieved using any of the binding reagent pairs disclosed herein. Where the positive marker or positive marker conjugate is immobilized (directly or indirectly) at the capture zone, the mobilizable labeled conjugate comprises means for associating the labeled conjugate with the positive marker. In the absence of a positive marker in the sample, the labeled conjugate is captured by the positive marker conjugate or positive marker immobilized or located at the capture zone, preventing the captured labeled conjugate from continuing to the detection zone. In the event that a positive marker is present in the sample, it becomes associated with the labeled conjugate, such that the labeled conjugate is not captured by the positive marker conjugate/positive marker immobilized or located in the capture zone, and may proceed to the detection zone. Where the positive marker is P3G, the device preferably comprises a P3G conjugate. In one embodiment, the reagent zone comprises a mobilizable P3G conjugate labeled with a first member of a binding pair (e.g., biotin), and the capture zone comprises an immobilized second member of the binding pair (e.g., anti-biotin antibody or streptavidin). Thus, the P3G/P3G conjugate may be located at the capture zone without being immobilized directly at the capture zone. This concept can be adapted to capture zones for other positive markers.

In one embodiment, the device is arranged such that the or each capture zone is not visible to a user in use. In one embodiment, the or each capture zone is not readable by the assay reading device. In one embodiment, the capture area in the device is not visible to the user in the case of use of the device. In one embodiment, where one or more capture zones are fully or partially visible to the user, the device is provided as part of a kit, the kit further comprising instructions to the user to ignore any signal generated at the capture zones and/or to determine the result of the assay based on the presence or absence of a signal at the detection zone without reference to any capture zone. In one embodiment, the device is provided as part of a kit comprising an accessory for masking one or more or all of the capture zones present in the device.

In one embodiment, the capture area may be visible to a user, who may be instructed to capture an image or video of the device on a mobile device, such as a mobile phone, which in turn interprets the test results by itself or by some other means connected directly or indirectly to the mobile device. In this case, the processing/analysis to derive the test results does not include reference or actual measurement capture zones.

Preferably, the assay reading member is unable to read the capture zone. In one embodiment, the test device does not comprise a test reading means. In one embodiment, the assay device is incompatible with an external reading means. In one embodiment, the device does not comprise means for reading or analysing the or each capture zone. The one or more capture areas may be shielded by any suitable means. For example, the assay device may comprise a housing which conceals the or each capture zone. Where the detection zone of the assay device is designed to be read by an assay reader and/or the assay device includes an assay reader for reading the detection zone, such a reader will be configured to read the detection zone and preferably not configured to read or analyse any capture zone. For example, only a reading member for reading the detection zone may be provided. In this case it may not be necessary to shield the capture zone, nor can it be read upstream of the detection zone, as the reading member of the reader is typically aligned with the detection zone. Preferably, the test reader for use with the test device comprises a single reading member for reading the detection zone. In one embodiment, the test device does not comprise a test for measuring the amount of a negative marker or a positive marker.

In one embodiment, the assay device is configured to allow only the detection, measurement or observation of a signal at the detection zone. In one embodiment, the assay device is configured to prevent detection, observation and/or measurement of signals at the or each capture zone. In one embodiment, the or each capture zone is not suitable for detecting the presence or measuring the amount of labelled conjugate captured at the capture zone. In one embodiment, the device does not comprise means to enable a user to obtain test results from the capture zone. In one embodiment, the device does not include means to enable a user to detect or analyze the signal at the capture zone. Preferably, the assay device is configured such that the assay result can be obtained by merely detecting or observing the signal at the detection zone (i.e. the zone where the analyte is detected). In the case of use, any capture zone need not be visible to the user or readable by the assay component, as the results of the assay are determined merely by viewing or analyzing the detection zone. There is no need to measure, view or interpret any signal generated at the capture zone to obtain the assay result. Furthermore, it is advantageous to mask the or each capture area so that the user is not confused or distracted by any signal generated at the capture area.

The immobilized capture substance can be disposed on the flow path in a variety of ways. For example, the flow path may include one or more regions containing immobilized capture species. The capture zone may comprise any immobilized capture species or combination of immobilized capture species within the capture zone disclosed herein. For example, an immobilized capture substance for capturing a complex comprising a first negative marker and an immobilized capture substance for capturing a complex comprising a second negative marker may be provided in the same capture zone. For example, binding reagents specific for the first negative marker and the second negative marker may be immobilized in the same capture zone (although they may be provided in different capture zones). An immobilized capture material configured to capture a positive marker or a labeled conjugate comprising or associated with a positive marker/positive marker conjugate may be provided in a capture zone comprising the immobilized capture material for capturing a complex comprising the first negative marker and/or the other negative markers. The immobilized capture material comprising or consisting of the positive marker/positive marker conjugate or for localizing the positive marker/positive marker conjugate at the capture zone and the immobilized capture material for capturing the complex comprising the first negative marker and/or the further negative marker may be provided in the same capture zone. The same capture zone may comprise an immobilized capture species configured to capture labeled conjugates in the absence of the first positive marker and an immobilized capture species configured to capture labeled conjugates in the absence of the second positive marker (or such capture species may be provided in different capture zones). This concept can be adapted to immobilized capture materials used with other negative and positive markers.

The immobilized capture substance can be disposed in one or more capture lines on the assay flow path. The capture zone may comprise 1, 2, 3, 4, 5 or more different capture lines. The capture lines may be arranged in parallel. The amount of labeled conjugate captured by the capture zone can be controlled by adjusting the amount, location, arrangement and orientation of the immobilized capture material. For example, higher capture efficiency can be achieved by placing the immobilized capture species further downstream, increasing the amount of capture line, and/or using a wider capture line. In addition to variations in the size of the labeled reagent, the pore size of the porous support of the flow path may also be used to adjust the capture efficiency and thus alter the sensitivity of the test for the analyte of interest. Generally, decreasing the pore size of the porous support can increase the capture efficiency.

The sensitivity of the assay to the analyte, negative and positive markers can be controlled by methods known to those skilled in the art, including varying the amount of binding reagent and/or the amount of positive marker/positive marker associated with the mobilizable labeled conjugate, and/or varying the amount of reagent at one or more capture zones, the amount of capture zone in fact, and the amount of labeled reagent used. Further modifications may be made by varying the affinity of the binding reagents used in the device. In embodiments involving positive marker conjugates, the relative amounts of positive marker and carrier protein used, for example, at the capture zone, can be modified. The sensitivity of the test to any analyte and marker involved in the assay can thus be modified.

Detection zone

The test device includes a detection zone containing an immobilized binding reagent for capturing a complex comprising the labeled conjugate and the analyte. Capture of the complex at the detection zone is preferably achieved by a specific binding reaction with the analyte. Preferably, the binding reaction occurs on an epitope of the analyte that is different from the epitope bound by the means for associating the labeled conjugate with the analyte, such that a "sandwich" is formed. Preferably, the detection zone is configured for capturing the labeled conjugate only if the labeled conjugate is part of a complex that also contains the analyte.

The detection zone may comprise an immobilised binding reagent that directly binds the analyte. The immobilized binding reagent at the detection zone may consist of or comprise an antibody specific for the analyte. In one embodiment, the immobilized binding reagent in the detection zone comprises or consists of an antibody that binds to an epitope of the analyte that is not present in the first negative marker.

The detection zone may comprise an immobilized binding reagent that indirectly binds the analyte. For example, in some embodiments, a mobilizable binding reagent for the analyte that is linked to the first binding partner can be provided, e.g., in the reagent zone or elsewhere along the flow path but upstream of the detection zone. The mobilizable binding reagent can bind to an epitope of the analyte that is not present in the first negative marker. An immobilized second binding partner can be provided in the detection zone to the first binding partner. In this way, mobile binding reagents for the analyte may be located at the detection zone without being immobilized directly to the flow path. The first and second binding partners may be any suitable binding partners (e.g., antibody-antigen) that are specific for each other. Examples include biotin and streptavidin, biotin and anti-biotin antibodies, fluorescein and anti-fluorescein antibodies. Either of the partners may be immobilised in the detection zone, while the other is provided elsewhere, for example in the reagent zone or along the flow path but upstream of the detection zone. This concept can be adapted to include more intermediate binding partners. In one embodiment, the reagent zone comprises a mobilizable anti- β -hCG antibody labeled with a first member of a labeled binding pair such as biotin, and the detection zone comprises a second member of an immobilized binding pair (e.g., anti-biotin antibody or streptavidin). Thus, the anti- β -hCG antibody may be located at the detection zone without being immobilized directly at the detection zone. This concept can be adapted to other analytes.

In embodiments where the analyte is hCG, the detection zone may comprise an anti-hCG antibody (e.g., an anti- β -hCG antibody).

Capture of a complex comprising the labelled conjugate and analyte at the detection zone will result in a signal being formed at the detection zone. The signal may be detected or examined, for example visually or using a test reading member. Detection of a signal at the detection zone is indicative of the presence of the analyte in the sample and the presence of a first condition in the subject from which the sample was derived. Failure to detect a signal at the detection zone, or detection of a signal level below a certain level when measured by the reading member, may indicate the absence of the first condition from the subject from which the sample originated.

Where a reading member is used to determine the signal intensity at the detection zone, a set threshold may be used as part of the algorithm to determine the test result as positive or negative for the first state or indeed to quantify the signal at the detection zone to indicate the amount of analyte in the sample.

For example, where the analyte is hCG, detection of a signal at the detection zone is indicative of pregnancy in the subject from which the sample was derived. If no signal is detected at the detection zone after the sample is applied to the device, this indicates that the subject from which the sample is derived is not pregnant.

Negative and positive markers

The device of the present invention is useful for situations where the analyte to be detected is associated with a state of interest ("first state") in the subject, but the detection of the analyte by itself is insufficient to confirm the presence of the first state. For example, the analyte may also be associated with the second state. The analyte may indicate the presence of the second state.

In some embodiments, the analyte, negative marker, and positive marker are such that one or more of:

the presence of analyte and positive marker in the sample indicates the presence of the first state.

The presence of a negative marker and/or the absence of analyte in the sample indicates the absence of the first state.

The presence of analyte and negative marker in the sample indicates the absence of the first state.

The presence of analyte in the sample and a negative marker indicates the presence of the second state.

The presence of analyte in the sample and/or the absence of a positive marker indicates the presence of the second state.

A negative marker may be a marker indicating the absence of the first state. A negative marker may be a marker indicating the presence of the second state. A positive marker may be a marker indicating the presence of a first state. A positive marker may be a marker indicating the absence of the second state.

In the case of a pregnancy test, the analyte may be hCG. The sample from the subject may contain hCG or hCG metabolites from the embryo, which may be indicative of pregnancy (first state). Examples of hCG metabolites include nicked (nicked) intact hCG, free β -hCG, nicked free β -hCG, free α -hCG, and β core (betacore) fragments.

However, hCG may also be present at low levels in the sample due to the presence of a second condition (e.g., peri-or menopause). In this case, the hCG is produced by the pituitary of the subject rather than the embryo. Thus, for example, in conventional devices using lateral flow, detection of low levels of hCG does not necessarily indicate that the subject is pregnant.

Negative marker

A negative marker is preferably a marker indicating the absence of the first state in the subject. In the case of a pregnancy test, the first negative marker may be FSH or LH. A negative marker may be a marker indicative of follicular development or other follicular growth or activity in females, such as FSH or a metabolite of such markers. Examples of FSH and its metabolites include free β -FSH and proteolytic degradation products.

In some embodiments, the amount of mobilizable labeled conjugate that reaches the detection zone (where the analyte of interest is detected) is modulated depending on the level of the first negative marker in the sample. In this case, the mobilizable labeled conjugate can be coated with a reagent that binds directly or indirectly to both the analyte to be detected and the first negative marker. In the case where such a test is performed with a sample containing the analyte and the first negative marker, the amount of mobilizable labeled conjugate reaching the detection zone is reduced due to the presence of the negative marker. In such embodiments, the presence of the negative marker causes capture of the labelled conjugate at a capture zone located upstream of the detection zone, the capture zone comprising an immobilised capture material which directly or indirectly captures the labelled conjugate, preferably by interaction with the first negative marker. The amount of mobilizable labeled conjugate captured at the capture site may depend on the level of the first negative marker in the sample. In a sample containing no or low levels of the first negative marker but having a first amount of analyte to be detected, a relatively large amount of the mobilizable, labeled conjugate will reach the detection zone and produce a first visible signal at the detection zone. Conversely, the presence of a level of the first negative marker in the sample and the first amount of analyte to be detected will result in the capture of the mobilizable, labeled conjugate at the capture zone and a decrease in the amount of mobilizable, labeled conjugate reaching the detection zone, thereby decreasing the signal generated at the detection zone. Thus adjusting the sensitivity of the test to the analyte to be detected.

In one embodiment, the analyte to be detected is hCG indicative of the presence of the first condition and the first negative marker is a marker indicative of the absence of the first condition in the subject.

In some embodiments, the first negative marker may be FSH. FSH may be the result of peri-or post-menopause. In such embodiments, testing with a sample from a perimenopausal or postmenopausal woman containing FSH as the first negative marker will result in capture of the mobilizable, labeled conjugate at a capture zone located upstream of the detection zone, thereby reducing the amount of labeled reagent reaching the detection zone and thus reducing or eliminating the signal seen at the detection zone. Even if the sample from a perimenopausal or postmenopausal woman contains pituitary-derived hCG (in addition to FSH), the presence of FSH causes a reduction or elimination of the signal produced at the detection zone, indicating a negative result in the first state.

The analyte and the first negative marker may share a common epitope. The analyte and the first negative marker may share a common structural feature (e.g., a subunit of the analyte). The analyte may be hCG and the first negative marker may be FSH. The first negative marker may be Luteinizing Hormone (LH). In a preferred embodiment, the first negative marker is FSH.

The mobilizable labeled conjugate can comprise a specific binding reagent that binds to a common structural feature on the analyte and the first negative marker. This may be, for example, an antibody directed against the alpha subunit of analyte hCG, which antibody is also capable of binding to the alpha subunit of the first negative marker FSH. Testing with urine samples from perimenopausal or postmenopausal women containing certain levels of FSH may result in some capture of the mobilizable labeled conjugate by the anti-FSH capture zone, thereby reducing or eliminating the signal seen at the detection zone.

In other embodiments, the mobilizable, labeled conjugate can comprise a binding reagent that specifically binds to the analyte and an additional binding reagent that specifically binds to the first negative marker. Specific binding reagents may be permanently immobilized directly to the mobilizable, labeled conjugate by passive adsorption or covalent coupling. In other embodiments, referred to as indirect binding modes, the specific binding reagent may be indirectly bound to the mobilizable, labeled conjugate prior to or while the assay is being performed. This can be achieved, for example, by coating the mobilisable labelled conjugate in a reagent such as avidin, streptavidin or avidin and biotinylating the specific binding reagent. The specific binding reagent is associated with the mobilizable, labeled conjugate either before or after the assay is performed.

Positive marker

A positive marker is preferably a marker that indicates the presence of a first condition in the subject, and is a marker other than the analyte of interest. In the case of a pregnancy test, a positive marker is preferably a marker indicative of a maternal factor secreted or produced in connection with the maintenance of pregnancy in a woman, such as progesterone or a metabolite of such a marker, such as pregnanediol glucuronide (P3G). Other positive markers that may be used in the method according to the invention, such as in pregnancy tests, include subtypes of intact hCG, such as hyperglycosylated hCG, notched hCG; subunits of hCG, such as the hCG β core fragment, β -hCG, α -hCG, unnotched β -hCG; early Pregnancy Factor (EPF), Early Pregnancy Factor (ECF), Follistatin (FST), activin A, inhibin B, Pro-AlphｃA C, and Pregnancy related plasmｃA protein A (PAPP-A).

A positive marker may be a small molecule called a hapten which is too small to elicit an immune response in an attempt to generate specific binding patterns or antibodies in the animal. In the case of specific binding modalities to haptens, if used to generate antibodies in animals, haptens are typically coupled or linked to immunogenic carriers such as proteins like bovine serum albumin or ovalbumin. The resulting antibodies can be used in lateral flow assays to generate competitive or inhibition assays. For example, antibodies for hapten generation may be coated on a mobile labeled conjugate, and the hapten or an analog of the hapten may be coupled to a carrier, typically a protein, to form a hapten conjugate. The hapten conjugate may be immobilized in a region on a porous support. In the case where the test is performed in the absence of hapten in the sample, the mobile labeled conjugate binds to the immobilized hapten conjugate, and performing the test in the presence of hapten reduces the binding of the mobile labeled conjugate to the immobilized hapten conjugate.

Conversely, the mobilisable labelled conjugate may be coated with a hapten linked to a carrier and the antibody for hapten generation may be immobilised to a region on a porous carrier. Haptens present in a sample applied to such a device occupy sites on the antibody immobilized on the porous carrier and reduce binding to mobile labeled conjugates coated in the haptens attached to the carrier.

This negative correlation is typical of competitive or inhibitory assays, which are commonly employed where the analyte of interest is a hapten and/or is too small to bind two different antibodies simultaneously (e.g., as is the case with progesterone metabolites).

Binding members specific for haptens are usually generated in mice and monoclonal antibodies are selected for the assay. The specific binding member may be coated directly onto the surface of the mobilisable labelled conjugate by direct adsorption or chemical coupling. The specific binding member may be coated onto the surface of the mobilizable, labeled conjugate by indirect means, including, for example, avidin-biotin or biotin and avidin as described above. Such indirect binding may be preformed such that the specific binding means is coated on the mobilizable, labeled conjugate prior to conducting the assay, or the specific binding member is bound to the mobilizable, labeled conjugate in the case of conducting the assay. In the latter case, the mobilisable labelled conjugate may be coated in avidin and the biotinylated specific binding member for the hapten may be provided as a separate reagent in the assay.

The hapten-bound protein can be immobilized directly on a porous support or by chemical coupling. The protein coupled to the hapten can be indirectly immobilized on a porous support, provided as a mobile reagent in the assay, in which case it is immobilized on the porous support.

The positive marker conjugate preferably comprises a positive marker moiety. The positive marker moiety is preferably capable of being bound by a binding reagent such as an antibody. The positive marker conjugate may comprise a progesterone metabolite, such as P3G, or a portion thereof.

The mobilizable labeled conjugate can directly bind to the positive marker or the positive marker conjugate. In some embodiments, the mobilizable labeled conjugate can indirectly bind to the positive marker/positive marker conjugate. Here, the reagent zone may comprise means for associating the mobilizable labeled conjugate with a positive marker or a positive marker conjugate. The means for associating the mobilizable labeled conjugate with a positive marker or a positive marker conjugate may comprise a binding reagent. The binding reagent may bind directly or indirectly to the positive marker or positive marker conjugate. The means for associating the mobilizable labeled conjugate with the positive marker or positive marker conjugate may comprise or consist of an antibody that binds to an epitope of the positive marker conjugate (which may be an epitope on the carrier protein). If the binding reagent binds indirectly to the positive marker or positive marker conjugate, it may bind to another binding reagent provided in the reagent zone that binds directly or indirectly to the positive marker or positive marker conjugate.

In some embodiments, the reagent zone further comprises a mobilizable positive marker/positive marker conjugate for immobilization downstream of the reagent zone. Such a mobile positive marker/positive marker conjugate may comprise or be linked to one member of a binding pair. The other member of the binding pair may be immobilized downstream of the reagent zone, e.g., in the capture zone. Thus, the mobilizable positive marker/positive marker conjugate can be immobilized at the capture zone such that it can bind to the means for associating the mobilizable labeled conjugate with the positive marker/positive marker conjugate.

In some embodiments, where the analyte is hCG and the positive marker is P3G, P3G can be coupled with an anti-hCG antibody (e.g., an anti- α -hCG antibody).

One or more capture zones may be located upstream of the detection zone. The mobilizable, labeled conjugate can be coated directly or indirectly onto a specific binding member for an analyte of interest (e.g., an antibody to hCG for use in pregnancy tests). In addition, the mobilizable labeled conjugate can also be coated directly or indirectly in an antibody for a positive marker (e.g., an anti-P3G antibody where the positive marker is a progesterone metabolite). In one embodiment, a porous carrier and directly or indirectly immobilized anti-hCG antibody are prepared at the detection zone, and a signal is provided at the detection zone by a typical sandwich assay in the presence of hCG from a pregnant source. In such an embodiment, the porous carrier may comprise one or more capture zones located upstream of the detection zone, the capture zones comprising an immobilised hapten (P3G) coupled to a protein carrier, such as BSA. Alternatively, the specific binding member of P3G may be immobilized directly or indirectly at the capture zone, and P3G coupled to the support may be coated directly or indirectly onto the mobilizable labeled conjugate.

In samples from pregnant women, the level of the positive marker (P3G) was elevated along with the pregnancy-derived hCG levels. In some embodiments, the presence of P3G reduces the binding of mobile labelled conjugate at the capture zone, thereby allowing more labelled reagent to reach the detection zone where the presence of hCG causes binding of mobile labelled conjugate at the detection zone.

Conversely, samples from non-pregnant individuals contained significantly lower levels of positive marker (P3G). In some embodiments, application of such a sample to the assay device causes the mobile labeled conjugate to bind to the capture zone, reducing the amount of mobile labeled conjugate that reaches the detection zone. Reducing the amount of mobile labeled conjugate reaching the detection zone reduces the signal generated at the detection zone, making a negative result of the pregnancy test more likely.

Analysis of urine samples from pregnant women showed that the level of positive markers (e.g. P3G) increased with increasing levels of hCG from pregnancy. However, in perimenopausal or postmenopausal women, the level of positive markers is relatively low. According to certain embodiments of the present invention, the lower level of positive marker present in urine samples from perimenopausal or postmenopausal women allows more mobile labeled conjugate to bind to the capture zone, effectively reducing the amount of mobile labeled conjugate reaching the detection zone. Since samples taken from perimenopausal or postmenopausal individuals contain pituitary-derived hCG, the signal seen at the detection zone in such embodiments decreases as the amount of mobile labeled conjugate reaching the detection zone is compromised. In this case, it is more likely that a negative result is reported in the detection region.

In some embodiments, where the analyte is hCG and the positive marker is a hapten such as P3G, P3G can be coupled with an anti-hCG antibody such as an anti-a-hCG antibody and a conjugate that associates with a mobile labeled conjugate. Here, anti- α -hCG acts as a specific binding member for hCG (binding of the mobilisable labelled conjugate at the detection zone) and a specific binding member for the first negative marker (e.g. FSH), and in the presence of FSH binds the labelled conjugate at the capture zone containing the immobilised capture species for FSH. In addition, the anti- α -hCG-P3G conjugate on the mobilizable, labeled conjugate is used to capture the mobilizable, labeled conjugate in the capture zone of the immobilized capture material containing P3G in the absence of P3G in the sample.

More than one positive marker may be used to further reduce the amount of labelled reagent reaching the detection zone, wherein a porous carrier containing a binding member for each positive marker is utilised.

Where the device of the invention comprises a positive marker or positive marker conjugate, the positive marker or positive marker portion of the conjugate may be an analogue or variant of the naturally occurring positive marker. Any analog or variant may be used, provided that both the analog/variant and the naturally occurring marker can participate in the positive marker binding reaction described herein and compete for the relevant binding site.

Combination of negative and positive markers and additional negative and/or positive markers

Specific binding members for one or more negative markers and/or one or more positive markers may be provided within a single test device. This format further improves the ability to report negative results in the absence of the first condition and positive results in the presence of the first condition. In the case of pregnancy tests, this format further improves the ability to report negative results for non-pregnant samples and positive results in the case of true pregnancies. In this case, the mobilizable, labelled conjugate may be coated directly or indirectly with a specific binding member such as anti- α -hCG to bind hCG and the negative marker FSH. The mobilizable labeled conjugate can also be coated with a specific binding member (e.g., P3G) that is linked to a positive marker. In such embodiments, the porous carrier preferably comprises a capture zone for a specific binding reagent comprising FSH and one or more regions of a positive marker or positive marker conjugate (e.g. P3G coupled BSA). The capture zone may comprise separate regions for negative and positive markers, or the capture zone may comprise specific binding reagents, co-immobilized at the capture zone by mixing reagents in any ratio.

In such embodiments, testing with samples taken from pregnant individuals having elevated levels of positive markers results in a lower amount of mobilizable labeled conjugate captured at the capture zone, thus allowing more mobilizable labeled conjugate to pass through to the detection zone. Since the sample also contains a lower level of the first negative marker FSH, less of the mobilizable labeled conjugate is captured at the capture zone by interaction with FSH. This allows more of the mobile labelled conjugate to move to the detection zone and increases the likelihood of a signal being generated at the detection zone by the presence of hCG of pregnancy origin in the sample. Thus, a true pregnancy sample is more likely to provide a positive test result after examining the detection zone. Conversely, a truly negative pregnancy sample that is not from a perimenopausal or postmenopausal woman contains a relatively lower level of positive marker, such that more of the mobilizable labeled conjugate is captured at the capture zone, reducing the amount of mobilizable labeled conjugate that reaches the detection zone. Although the level of negative marker may vary in such samples, the amount of negative marker is not important because the hCG level from the source of the pregnancy is low, resulting in a reduced/no signal formation at the detection zone. In the case of testing samples from perimenopausal or postmenopausal women, a relatively high level of the negative marker FSH allows more of the mobilizable labeled conjugate to be captured at the capture zone. In addition, a greater amount of mobilizable labeled conjugate is captured at the capture zone due to the presence of a relatively lower level of positive marker P3G in the sample. In effect, the amount of mobile labeled conjugate reaching the detection zone is reduced, increasing the likelihood of reporting a negative result in the case of a test at the detection zone. Since in certain embodiments the mobilizable labeled conjugate comprises an anti-alpha antibody as specific binding member, it will also bind pituitary-derived hCG in samples from perimenopausal or postmenopausal women. Due to the presence of the negative marker FSH in the sample, the mobile labelled conjugate is captured at the capture zone and some of the pituitary hCG will also be captured on the mobile labelled conjugate and will not move to the detection zone. Reducing the amount of pituitary-derived hCG in the sample that migrates to the detection zone further reduces the likelihood of mobilizable labeled conjugate binding to the detection zone, increasing the likelihood of reporting a negative result at the detection zone in the case of running individual samples from peri-or post-menopause.

The device of the invention may be adapted to include means in the reagent zone and the capture zone to facilitate further capture of the labelled conjugate prior to its reaching the detection zone in the presence of the second negative marker in the sample. Any of the means for capturing a complex comprising a labeled conjugate and a first negative marker described herein may be modified and included in the device of the invention to capture a complex comprising a labeled conjugate and a second negative marker. If the first negative marker is FSH, the second negative marker may be, for example, LH. If the first negative marker is LH, the second negative marker may be, for example, FSH.

Similarly, the device of the invention may be adapted to include means in the reagent zone and the capture zone to facilitate further capture of the mobilizable, labeled conjugate before it reaches the detection zone in the absence of the second positive marker in the sample (e.g., in the case where the second positive marker is a hapten). In the absence of a positive marker, any of the means for capturing a complex described herein may be modified and included in the device of the invention to capture a complex comprising a mobilizable, labeled conjugate in the absence of a second positive marker in the sample. If the first positive marker is P3G, the second positive marker may be another progesterone metabolite. If the first positive marker is a progesterone metabolite other than P3G, the second positive marker may be P3G. The device of the invention may further be adapted to capture the complex in the presence of any amount of more negative markers and/or in the absence of any amount of more positive markers.

Relative position of zones

One or more capture zones are located downstream from the reagent zone and upstream from the detection zone. The detection zone may be located at the downstream end of the assay flow path (e.g. a porous carrier such as nitrocellulose). The capture zone may be located anywhere between the reagent zone and the detection zone. The position may be adjusted as described herein to adjust the capture efficiency. In one example, the detection zone may be located about 10 to 20mm from the upstream end of the porous carrier (e.g., nitrocellulose membrane). The capture zone may be located about 3 to 15mm from the upstream end of the porous support (e.g. nitrocellulose membrane). In one embodiment, the detection zone is located about 15mm from the upstream end of the porous carrier (e.g. nitrocellulose membrane) and the capture zone is located about 8mm from the upstream end of the porous carrier (e.g. nitrocellulose membrane). The reagent zone may be provided on a separate porous support, upstream of the porous support containing the capture and detection zones. There is no inherent limit to the spacing between the capture and detection zones. However, in embodiments where the assay device is designed to be read visually by a user, the capture zone will be spaced far enough from the detection zone so as to be invisible to a user viewing the detection zone. Thus, the size of the viewing window used to view the detection zone and the position of the detection zone below the window may be factors in determining the most appropriate position of the capture zone.

Sample(s)

While urine is preferred as a sample matrix for home pregnancy testing, the sample may be any suitable bodily fluid, such as whole blood, plasma, serum, or urine. Urine samples are highly preferred because such samples are readily available and do not require invasive procedures. In addition, the use of urine samples facilitates self-testing by the user. Other samples may be collected using swabs, sanitary napkins, bandages, and the like and applied directly to the device, followed by an elution buffer to help move sample components along the flow path. Prior to applying the sample to the test device, the sample may be collected and placed in an elution buffer as described above.

Runtime and reader properties

The results of the test may be determined by a user's visual inspection of the detection zone, typically after a set period of time after application of the sample to the device, or on the development and visual appearance of the control wire or other end of the test indicator.

In general, in the case of a digital assay device, the test results will be determined after a certain time (tE) has elapsed (typically, but not necessarily, by reference to the time at which the sample is in contact with the sampling area of the assay device). If a test result reading device is provided, this may include some sort of integral timing means to determine when tE is reached. The timing means may be activated automatically by contacting the sample with the assay device (e.g. by a liquid sample allowing current to flow), or may be triggered by the user (e.g. by pressing a switch or the like) or by any other convenient means. The test reaction may conveniently reach equilibrium at tE, but this is not essential.

The reader may take one or more measurements of the analyte signal at the detection zone prior to tE. In the case where the signal at the detection zone is greater than the first threshold (which indicates that the detection zone signal is above the set threshold for a positive result in the case of tE), an early measurement of the detection zone signal may be used to output the final result to the user before tE. Also, if the signal from the detection zone is below a set threshold at any stage prior to tE, an early negative result may be output to the user prior to tE.

the tE end point may be conveniently determined by the reader with reference to a particular point in time. (i.e., tE may be considered to occur a certain amount of time after the assay has begun, e.g., a certain interval after activation of the reader and/or insertion of the assay device into the reader and/or application of the sample to the assay device).

Outer casing

In a preferred embodiment, the test device comprises a housing which contains most or all of the functional components of the test and the test reagents. The housing is conveniently formed from a water resistant synthetic plastics material and is preferably substantially opaque. Opacifiers may be added to the plastic material to achieve the desired level of opacity. Suitable synthetic plastics materials for forming the housing include in particular polycarbonate, polystyrene, polyolefins, polypropylene, copolymers of polyethylene and acrylonitrile. Desirably, the housing may be formed in two or more parts which are connected together with most (or all) of the remaining trial components contained within or between the assembled parts of the housing. The parts of the housing may be connected and fastened by conventional fastening means, such as a snap-fit action, or by plastic welding or the like.

In a preferred embodiment, the test device comprises a housing (generally having the characteristics described above) and wherein the sample application portion or zone extends beyond the housing to facilitate application of the urine sample to the sample application portion or zone. The sample application portion or area extending beyond the housing may be covered by a removable cap prior to use. The cap may also preferably be formed from a synthetic plastics material, which may be opaque, transparent or translucent. Typically, the cap is replaced once the sample is applied to the sample application zone.

The housing may include a test window over the detection zone to facilitate viewing of the detection zone. The housing may obstruct the user's view of the or each capture area.

In another example, no housing is provided. In such embodiments, the test device components may be mounted on a support material such as mylar to form a commonly known test strip. In this case, the capture area may be otherwise covered by an additional opaque covering, shielding the user from the capture line. In other cases, using such a strip, the capture zone is visible to the user, merely indicating to the user the result of interpreting the result by observing the detection zone. Such a test strip configuration without a housing may also be read by a reading member as described herein.

Other features

The test device may conveniently contain other features known to those skilled in the art and common in conventional self-test or home-test pregnancy test devices, including, but not limited to, a sample sufficiency indicator (e.g. as described in WO 2015/082646), a "flood control" pad (e.g. as disclosed in WO 2012/069610), the use of a sample stream as a test control (e.g. as disclosed in EP1,484,611; US6,194,222); determining a positive or negative result "early" (e.g., as disclosed in EP1,484,613; US5,679,584); automatic "wake-up" of the electronic device (e.g., automatic start-up after wetting of the device by the sample, thereby completing the circuit); a digital test progress meter (e.g. european community design registration number 1367304) or "color changing wick" or the like is used to give a visual indication in the case that a sample has been applied to the test device and/or that a test has commenced (e.g. WO 2003/058245). The test device will advantageously be presented to the consumer in a waterproof package, optionally further comprising a desiccant, such as a silicone sachet.

The test flow path may conveniently be supported on some sort of support or backing layer to provide mechanical strength and a suitable degree of rigidity. The assay plus support may be conveniently referred to and provided as a test strip for use without a housing, or indeed placed in a housing as described herein to form what is commonly referred to as a test stick. The test stick or sticks may be adapted and configured for insertion by a user into the test device, or the test stick or sticks may more preferably form an integral part of the test device, which is sold to a consumer with test sticks pre-inserted or loaded in the test device.

Reading device

In a preferred embodiment, the test results may be read directly by the user in a manner known from conventional "self-test" pregnancy tests, for example by the user examining one or more windows covering the detection zone to determine the presence or absence of a detectable signal. In such embodiments, the test device preferably does not include a test reader. In one embodiment, the assay device of the present invention is not configured to be read by an assay reader. Typically, in such user-read devices, the user will directly check the detection zone of a lateral flow or microfluidic assay. In other forms, the user may determine the result of the detection zone by reference to a color chart or indicator. Conveniently, the test device may be provided with instructions or guidance for reading the test results (if the device does not interpret the test results for the user). For example, a printed color chart may be provided to a user to facilitate interpretation of such directly read visual tests.

Alternatively, the detection zone may be read by a test result reading member, referred to as a test result reader. In this case, the assay result reader is preferably configured to read only the detection zone and not to read or analyse any capture zone present in the assay device. The test result reader may be external to the test device or integrated with the test device. The test result reader may advantageously comprise electronic components, in particular digital electronic components, such as a microprocessor. The detection zone may be determined by an optical means, i.e. measuring the amount of light reflected and/or transmitted by the detection zone, wherein the optically labelled reagent tends to accumulate in a manner directly proportional (proportional or inversely proportional) to the analyte concentration in the sample. Alternatively, the detection zone may be read by, for example, magnetic or electrochemical measurements. Obviously, the manner in which the assay is read may depend on the nature of the label used to label the test agent. The generated data can be processed by an integrated microprocessor, and the test result can be output to the device itself by a liquid crystal screen or the like, or remotely transmitted to an external device such as a mobile phone for in-situ processing or transmitted to a central processing server through network connection. The results may be reported on a mobile phone or other networked device.

The external test result reading means may comprise a dedicated result reading device (e.g. similar to the device described in EP 1066530). Alternatively, the external test result reader may be "non-dedicated", such as a mobile phone or other portable electronic device (e.g., a tablet computer), preferably equipped with a camera, wherein the test results are read by measuring the signal strength generated by the tags at the detection zone.

The test result reader, whether external or forming part of the integrated test device/test result reader, may read and interpret the test data, or may transmit test data containing device operating characteristics, such as flow time and various outputs from the measurement system to a remote device for interpreting the analytical data, and the results obtained. The test data may be transmitted to the remote device in real time. Data may be transmitted over an internet connection, or may be stored on a storage device (e.g., a "flash" drive, etc.) that is physically transmitted to a remote device, or data may be transmitted over a wireless communication means (e.g., bluetooth, near field communication [ NFC ], etc.).

The microprocessor may control the operation of the optical reading or other test reading means and will conveniently be programmed with or have access to the relevant test signal threshold for each analyte, compare the actual test signal value to a predetermined threshold and interpret the test results in order to determine the outcome of the pregnancy test.

The testing device may further comprise a test result display for displaying the test result to a user. Typically, the display will comprise an LCD, but other types of displays are possible (e.g., using "electronic ink"). In those embodiments where the test device (or more particularly, components thereof) requires a power source to operate, then the test device will preferably be provided with an integrated power source, such as a battery. Very small and inexpensive batteries are readily available on the market. The device may also be provided with a switch to connect the integrated power source and activate the device.

A combined pregnancy test stick/test result reading device with a display may be referred to as a "digital pregnancy test," and such digital test devices are commercially available and may be adapted with the benefit of this disclosure to provide a test device according to the present invention.

The microprocessor will ideally be programmed to cause the test result reading means to read the test results; explaining the test result; and displays the conclusion to the user.

The means for reading the test results will preferably comprise at least one light source and at least one light detector. The at least one light source is preferably a Light Emitting Diode (LED). The at least one photodetector is preferably a photodiode or a phototransistor. The light source illuminates a detection zone of the assay, which zone tends to accumulate labeled species during the course of the assay in a manner that depends on the concentration of the analyte of interest in the sample applied to the assay.

The device may generally employ a reference zone, which is part of the microfluidic or lateral flow assay flow path, for reference to readings taken from the detection zone. The use of reference regions is well known to those skilled in the art and helps illustrate unbound labeled reagent that may be located in the flow path and/or background staining of the flow path of the sample being tested.

In one embodiment, the light source emits light at different wavelengths at different times, and the light detector distinguishes between the different wavelengths. Additionally or alternatively, optical baffles (fixed or adjustable) may be used to control the area illuminated by a particular light source. Further details of optical arrangements of the kind that may be used are disclosed in, for example, EP1,484,601, US6,055,060 and US5,889,585. For the avoidance of doubt, the term "light" as used herein is not intended to refer only to radiation in the portion of the electromagnetic spectrum that is visible to a human observer and includes, for example, ultraviolet and infrared radiation. However, components within the visible portion of the spectrum, components sensitive to the visible portion of the spectrum may be preferred and selected according to the labeled reagent used.

The microprocessor or computerized control means may contain one or more stored analyte thresholds against which the test results may be compared to allow the test result reading device to interpret the results and display an appropriate conclusion (e.g. pregnant or not pregnant) to the user. The microprocessor or control means may be programmed with an algorithm to measure the test results, compare them to predetermined thresholds, and display a conclusion.

Control

In some embodiments, it is desirable that the test device also include some sort of control function. This is conventional in self-test devices to provide some indication that the test has run correctly.

Typically, the control function will involve the use of a control zone in which labeled reagent will tend to accumulate if sufficient sample has been applied to the sample application zone of the test device. Typically, the labelled reagent is a labelled antibody or other reagent which is releasably deposited in dry form on the upstream or proximal portion of the test strip, for example in the reagent zone, and which moves on rehydration by the sample and is captured by a capture reagent immobilised at the control zone. The control indicates whether sufficient sample has been applied to the test device and that the test reagents have retained their binding properties to a reasonable extent and that the labelled reagents have been moved to a sufficient extent. The labeled reagent used to form the control zone may be a second set of reagents of the first set of labeled reagents used to detect the analyte. A mixture of a labelled reagent for detecting the analyte and a separate set of labelled reagents for forming the control zone may be used, both sets participating in the formation of the control zone.

The embodiments described in relation to each aspect of the invention are mutatis mutandis to the embodiments of each of the other aspects. The documents cited herein are incorporated by reference to the fullest extent allowed by law. The prior art documents mentioned herein are incorporated to the maximum extent allowed by law.

The various features of the present invention will now be further described by way of exemplary embodiments and with reference to the accompanying drawings in which:

FIG. 1A shows an embodiment of an assay device (100) for detecting the presence of an analyte (101) in a sample. The assay device comprises a sample addition zone (102), a reagent zone (104) downstream of the sample addition zone, a capture zone (110) downstream of the reagent zone, a detection zone (108) downstream of the capture zone, a control zone (109) downstream of the detection zone, and a sink pad (105) downstream of the control zone. In the case where a sample is applied to the sample application zone (102), it flows along the flow path of the assay device in the direction indicated by arrow (103).

The reagent zone (104) contains a test mobilizable labeled conjugate (106) and a control mobilizable labeled conjugate (107). The test mobilizable labeled conjugate (106) comprises a label (114) coupled to a binding reagent (113) (e.g., an antibody), and the test mobilizable labeled conjugate (106) binds to the analyte (101) or the first negative marker (112). The control mobilizable labeled conjugate (107) includes a label (114) coupled to a binding member that is bound by the immobilized binding member (116) at the control zone (109). Test mobilizable labeled conjugates may also be used as control conjugates.

The capture zone (110) comprises an immobilized capture substance (115) (e.g., an immobilized antibody) specific for the first negative marker (112).

The detection zone (108) comprises an immobilized binding reagent (111) (e.g., an immobilized antibody) specific for the analyte (101).

Test windows (117) are provided in the test device housing (not shown) above the detection zone (108) and control zone (109) to enable a user to view or read by a test reader. The capture area (110) is not visible through the test window (117) and is obscured by the test device housing.

FIG. 1B shows how a positive test result is obtained by the test device shown in FIG. 1A. In this case, the analyte (101) is present in the sample, but the first negative marker (112) is not present in the sample. The test mobilizable, labeled conjugate (106) and the control mobilizable, labeled conjugate (107) are mobilized by the sample and flow downstream from the reagent zone (104). The analyte (101) is bound by a binding reagent (113) of the test mobilisable labelled conjugate (106) to form a complex. The complex is not captured at the capture zone (110) because the immobilized capture substance (115) at the capture zone (110) is specific for the first negative marker (112) and the first negative marker is not present in the sample. The immobilized binding reagent (111) in the detection zone (108) binds to the analyte in the complex, thereby capturing the complex. The control mobilizable labeled conjugate (107) is bound at the control zone (109). This results in the formation of a test line signal (118) and a control line signal (119), both of which are visible through the test window (117), indicating a positive result.

FIG. 1C shows how a negative result is obtained by the test device of FIG. 1A in the absence of the analyte (101) and the first negative marker (112) in the sample. The control mobilizable labeled conjugate (107) is mobilized by the sample, flows downstream from the reagent zone (104) and binds at the control zone (109). The test mobile labeled conjugate (106) is moved by the sample but is unable to bind at the detection zone (108) due to the absence of analyte (101). This causes only the control line signal (119) to be generated and the user to be visible at the test window (117), indicating a negative result.

FIG. 1D shows how a negative result is obtained by the test device of FIG. 1A in the presence of the analyte (101) and the first negative marker (112) in the sample. The test mobilizable, labeled conjugate (106) and the control mobilizable, labeled conjugate (107) are mobilized by the sample and flow downstream from the reagent zone (104). The first negative marker (112) and the analyte (101) are bound by a binding reagent (113) of the test mobilizable labeled conjugate (106) to form a complex. The immobilized capture substance (115) in the capture zone binds to the first negative marker (112) of the complex such that the complex is captured at the capture zone (110). Since the capture zone (110) is upstream of the detection zone (108), the test mobile labeled conjugate (106) is isolated before it reaches the detection zone (108). Thus, despite the presence of analyte (101) in the sample, the amount of accumulation of test mobile labeled conjugate (106) at the detection zone (108) is insufficient to give a positive result. The control mobilizable labeled conjugate (107) is mobilized by the sample and binds to the control zone (109). This allows the user to see only the control line (119) at the test window (117), indicating a negative result. The capture area (110) is obscured from view.

Figure 2 shows a simplified schematic of a test device of the type shown in figure 1. The test device comprises a reagent zone (1), a capture zone (5) downstream of the reagent zone (1) and a detection zone (7) downstream of the capture zone (5). The reagent zone (1) comprises a mobilizable labeled conjugate (2) comprising a detectable label (3) and means for associating the labeled conjugate with an analyte (4a) and means for associating the labeled conjugate with a first negative marker (4 b). The capture zone (5) comprises an immobilized capture substance (6) capable of capturing a complex comprising the labeled conjugate (2) and the first negative marker (10). The detection zone (7) comprises an immobilised binding member (8) for capturing a complex comprising the labelled conjugate (2) and the analyte (9). In the presence of the analyte (9) and in the absence of the first negative marker (10) (left panel), the means for associating the labeled conjugate with the analyte (4a) binds to the analyte, forming a complex. The complex is captured at the detection zone (7) as the immobilised binding member (8) at the detection zone (7) binds to the analyte (9) of the complex. In the presence of the analyte (9) and the first negative marker (10) (right panel), the means for associating the labeled conjugate with the analyte (4a) binds to the analyte (9) and the means for associating the labeled conjugate with the first negative marker (4b) binds to the first negative marker (10). It should be noted that in this embodiment, the means for associating the labeled conjugate with the analyte (4a) and the means for associating the labeled conjugate with the first negative marker (4b) are different (e.g., different antibodies). However, they may be the same, e.g. the same antibody that binds both the analyte and the first negative marker. A complex is formed comprising the labeled conjugate, the analyte (9), and the first negative marker (10). The complex is captured at the capture zone (5) as the immobilized capture substance (6) binds to the first negative marker (10) of the complex. In the assay device, the capture zone (5) is obscured from view, while the detection zone (7) is visible to the user or readable by an assay reader.

Figure 3 shows a schematic diagram of a test device of the type shown in figure 2, with the following differences: (i) the mobilizable labeled conjugate (2) comprises a positive marker conjugate (4c) rather than a means for associating the labeled conjugate with a first negative marker (labeled (4b) in fig. 2), and (ii) the capture zone (5) comprises an immobilized capture substance (6a) capable of capturing the first positive marker or a complex comprising the positive marker conjugate. In the presence of analyte (9) and first positive marker (10a) (left panel), first positive marker (10a) is bound by immobilized substance (6a) at capture zone (5) such that labeled conjugate (2) cannot bind at capture zone (5). The analyte (9) is used to bind the labelled conjugate to the analyte associated member (4a) to form a complex. The complex passes through the capture zone (5) and binds at the detection zone (7) as the immobilised binding member (8) at the detection zone (7) binds to the analyte (9) of the complex. In the absence of the first positive marker (10a) and in the presence of the analyte (9) (right panel), the analyte (9) is used to bind the labeled conjugate to the analyte-associated member (4a) to form a complex. The complex binds at the capture zone (5) because the immobilized capture substance (6a) at the capture zone (5) binds to the positive marker conjugate (4c) of the complex.

Figure 4 shows a schematic diagram of a test device of the type shown in figure 3, with the following differences: (i) the mobilizable, labeled conjugate (2) comprises means for associating the mobilizable, labeled conjugate with the first positive marker or first positive marker conjugate (4d), but not the positive marker conjugate (4c in fig. 3), and (ii) the capture zone (5) comprises an immobilized first positive marker conjugate (6b), but not an immobilized capture substance capable of capturing the first positive marker or complex comprising the first positive marker conjugate (labeled as (6a) in fig. 3). In the presence of analyte (9) and first positive marker (10a) (left panel), first positive marker (10a) is used to bind the mobilizable labeled conjugate to the first positive marker or first positive marker conjugate associated member (4d) such that labeled conjugate (2) cannot bind at capture zone (5). The analyte (9) is used to bind the labelled conjugate to a member (4a) associated with the analyte to form a complex. The complex passes through the capture zone (5) and binds at the detection zone (7) as the immobilised binding member (8) of the detection zone (7) binds to the analyte (9) of the complex. In the absence of the first positive marker (10a) and in the presence of the analyte (9) (right panel), the analyte (9) is used to bind the labeled conjugate to the analyte-associated member (4a) to form a complex. The complex binds at the capture zone (5) because the immobilized first positive marker conjugate (6b) binds to the means (4d) of the labeled conjugate (2) for associating the mobilizable labeled conjugate with the first positive marker or first positive marker conjugate. It will be appreciated that the embodiments shown in the figures may be combined such that, for example, both the positive and negative markers affect the amount of labeled conjugate (and analyte present in the sample) reaching the detection zone and/or more negative and/or positive markers affect the labeled conjugate reaching the detection zone. For example, the mobilizable labeled conjugate can comprise binding reagents for the analyte, the first negative marker, and the first positive marker, and the capture zone can comprise an immobilized capture substance for the first negative marker and an immobilized first positive marker conjugate. In another embodiment, the mobilizable labeled conjugate can comprise a binding reagent for the analyte, the first negative marker, and the first positive marker conjugate, and the capture zone can comprise an immobilized capture substance for the first negative marker and an immobilized capture substance for the first positive marker and the first positive marker conjugate.

Fig. 5 shows indirect capture at a detection zone using a modified version of the device shown in fig. 3. In this embodiment, reagent zone (1) further comprises a mobilizable binding reagent (8a) for the analyte linked to the first binding partner. The detection zone (7) comprises an immobilized second binding partner (8b) to the first binding partner. In this way, mobile binding reagent for the analyte may be located at the detection zone (7) without being immobilised directly to the flow path.

In the presence of the analyte (9) and in the absence of the first negative marker (10) (left panel), the means (4a) for associating the labeled conjugate with the analyte binds to the analyte, forming a complex. The mobilisable binding reagent (8a) for the analyte linked to the first binding partner is mobilised and flows to the detection zone (7) where the first binding partner is bound by the second binding partner (8 b). The complex is captured at the detection zone (7) by the mobile binding reagent (8a) for the analyte, which is now located at the detection zone (7). In the presence of the analyte (9) and the first negative marker (10) (right panel), the means for associating the labeled conjugate with the analyte (4a) binds to the analyte (9) and the means for associating the labeled conjugate with the first negative marker (4b) binds to the first negative marker (10). A complex is formed comprising the labeled conjugate, the analyte (9), and the first negative marker (10). The complex is captured at the capture zone (5) as the immobilized capture substance (6) binds to the first negative marker (10) of the complex. The mobile binding reagent (8a) for the analyte, linked to the first binding partner, flows to the detection zone and is bound by the second binding partner (8b), but is prevented from reaching the detection zone (7) because the complex is captured at the capture zone (5).

Fig. 6 shows a modified version of the device shown in fig. 4. In this embodiment, reagent zone (1) further comprises a mobilizable first positive marker conjugate (6c) linked to the first binding partner. The capture zone (5) comprises an immobilized second binding partner (6d) to the first binding partner. In this way, the movable first positive marker conjugate (6c) may be located at the capture zone (5) without being directly immobilized to the flow path.

In the presence of a sample containing analyte (9) and first positive marker (10a) (left panel), the mobilizable first positive marker conjugate (6c) linked to the first binding partner is mobilized and flows to the capture zone (5) where it is bound by the immobilized second binding partner (6 d). The means (4a) of the mobilisable labelled conjugate (2) for associating the labelled conjugate with the analyte binds to the analyte (9) to form a complex. The means (4a) of the mobilisable labelled conjugate (2) for associating the labelled conjugate with the first positive marker or analyte binds to the first positive marker (10a) preventing the complex now located at the capture zone (5) from being captured by the first positive marker conjugate (6 c). The complex passes through the capture zone (5) and is bound at the detection zone (7) in that the immobilised binding member (8) of the detection zone (7) binds the analyte (9) of the complex.

In the presence of analyte (9) and in the absence of first positive marker (10a) (right panel), the mobilizable first positive marker conjugate (6c) linked to the first binding partner is mobilized and flows to the capture zone (5) where it is bound by the immobilized second binding partner (6 d). The means (4a) of the mobilisable labelled conjugate (2) for associating the labelled conjugate with the analyte binds to the analyte (9) to form a complex. The complex is bound at the capture zone (5) because the mobilizable first positive marker conjugate (6c) now located at the capture zone (5) is bound by the means (4d) of the labeled conjugate (2) for associating the mobilizable labeled conjugate with the first positive marker or first positive marker conjugate.

Figure 7 is a graph showing the effect of P3G on the assay signal generated by labeled reagents coated with anti-P3G and anti- α -hCG, tested on a porous support provided with a capture zone for P3G conjugate and an anti- β -hCG detection zone, over a range of hCG concentrations.

It should be noted that the positive marker conjugate shown in the figures may be replaced with a positive marker, for example, if the positive marker is a non-hapten marker that does not require coupling.

Examples

The invention will now be described with reference to the following non-limiting embodiments.

Example 1

This example describes the preparation of a pregnancy strip with an anti-hCG detection zone and an anti-FSH capture zone for clinical urine samples and post-spiked (spiked) urine standards for pregnant women and peri-or post-menopausal women.

Production of detection reagent

Preparation of gold Sol-labeled antibody

Test sols

40mL of a 40 μ g/mL mouse anti- α -hCG antibody solution in 20mM 2- (N-morpholino) ethanesulfonic acid (MES) buffer, ph6.5, was added to 40mL of a 40nm, a520nm ═ OD5.0 gold sol (BBI International) and mixed rapidly with the gold sol on a magnetic stirrer at room temperature for 30 minutes. After 30 minutes 836.5. mu.L of a 52.6mg/mL beta-casein solution in 50mM carbonate buffer (pH10.5) was added to the reaction mixture and mixing was continued at room temperature for a further 30 minutes. The final concentration of beta-casein in the reaction mixture was 0.55 mg/mL. Anti- α -hCG antibodies immobilized on gold sol are capable of binding these hormones via the α subunits of hCG and FSH. The sol solution was poured into a Falcon tube (50mL) and the solution was centrifuged (4000rcf, 10 min, 15 ℃). The supernatant was carefully removed, wash buffer (1mL, 10mM MES, ph6.5, containing 0.1mg/mL beta casein) was added, and the precipitated sol was vortexed and sonicated to resuspend. After resuspension, the solution was centrifuged (5000rcf, 10 min, 15 ℃). The supernatant was removed and the sol was resuspended in a small amount of storage buffer (10mM MES pH6.5, containing 0.1mg/mL beta-casein). The final OD of the sol formulation was determined by measuring the absorbance at 520 nm.

Control Sol

20mL of a 12.5 μ g/mL solution of Rabbit IgG antibody (Dako) in 20mM (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) (HEPES) buffer, pH 8.0, was added to 20mL of a 40nm A520nm ═ OD5.0 gold sol (BBI International) and mixed rapidly with the gold sol on a magnetic stirrer at room temperature for 30 minutes. After 30 minutes, 76. mu.L of a 52.6mg/mL beta-casein solution in 50mM carbonate buffer (pH10.5) was added to the reaction mixture and mixing was continued for another 30 minutes at room temperature. The final concentration of beta-casein in the reaction mixture was 0.1 mg/mL. The sol solution was poured into a Falcon tube (50mL) and the solution was centrifuged (4000rcf, 10 min, 15 ℃). The supernatant was carefully removed and wash buffer (1mL of 10mM MES pH6.5 containing 0.1mg/mL beta-casein) was added to resuspend the sol. The precipitated sol was vortexed and sonicated. After resuspension, the solution was centrifuged (5000rcf, 10 min, 15 ℃). The supernatant was removed and the sol was resuspended in a small amount of storage buffer (10mM MES pH6.5, containing 0.1mg/mL beta-casein). The final OD value of the sol formulation was determined by measuring the absorbance at 520 nm.

Preparation and fixing method of gold sol labeled binding reagent

anti-hCG coated gold sol (test) conjugate and rabbit IgG coated gold sol (control) conjugate were centrifuged in a centrifuge and the supernatant removed. The resulting particles were vortexed and sonicated, and then reconstituted in a gold sol conjugate spray buffer to twice the OD of the desired gold (16 OD/mL for the test sol and 10OD/mL for the control sol in this example). The test sol and the control sol were then mixed at 1:1 to give a spray solution containing 8OD/ml of the test sol and 5OD/ml of the control sol. The gold sol conjugate spray buffer used in the following examples contained 100mM Tris pH 7.4, 20% w/v sucrose (Sigma) and 10% w/v bovine serum albumin (Proliant Biologicals SKU # 68700). Glass fibers (GE Healthcare) were loaded onto a Biodot spray device. A Biodot spray device was provided to impregnate/impregnate the glass fibers with the test and control conjugates at the desired location (6.5 mm from the downstream edge) on the glass fibers. In this example, the glass fiber was sprayed with the OD10 conjugate in a single pass at a 6.5mm distance from the downstream edge of the glass fiber at a plot rate of 1.7 μ L/cm. In addition, a 0.56M aqueous EDTA solution was sprayed at a plot rate of 1.7. mu.l/mm from a distance of 16mm from the downstream edge of the glass fiber. The glass fibers impregnated with gold sol were dried at 55 ℃ and stored in sealed foil bags at room temperature together with the desiccant.

Method for preparing, positioning and fixing specific binding substance

The mouse anti-beta-hCG antibody was diluted to 3mg/mL in Phosphate Buffer (PBSA) containing 0.05% (w/v) sodium azide before being immobilized on nitrocellulose. anti-beta-FSH antibody was diluted to 1.5mg/mL in PBSA prior to immobilization on nitrocellulose. Polyvinyl alcohol (PVA) blocking buffer (pH9) (20mM Tris base (Sigma), 1% w/v PVA (80% hydrolyzed PVA, 9-10K MW Sigma), 0.05% w/v Tween 20(Sigma) and 150mM NaCl (Sigma)) was prepared. PVA blocking solutions were prepared by adding 2% w/v sucrose (Sigma) and 2.5mL ethanol (Sigma) to 47.5mL PVA blocking buffer. White backed nitrocellulose with pre-perforated pilot holes to 4mm (mdi) was cut into 35cm by 40mm strips. A Biodot plotter was provided to plot a single line of anti- β -hCG antibody on nitrocellulose strip to form a detection zone and anti- β FSH antibody to form a capture zone and goat anti-rabbit (Lampire) to form a control zone at the desired location. The anti- β -hCG antibody was deposited at a distance of 15mm from the bottom (upstream end) of the nitrocellulose membrane at a concentration of 3mg/mL and a plotting rate of 1uL/cm, the anti-FSH antibody was deposited at a concentration of 3mg/mL at a plotting rate of 3 μ L/cm at a distance of 8mm from the bottom (upstream end) of the nitrocellulose membrane, and the goat anti-rabbit antibody was deposited at a concentration of 2mg/mL at a plotting rate of 1uL/cm at a distance of 22mm from the bottom (upstream end) of the nitrocellulose membrane. After drawing, the strips were dried at 55 ℃, blocked with PVA lock solution, then dried at 65 ℃ and stored with desiccant in sealed aluminum foil bags at room temperature.

Test paper structure

The assay components are assembled into test strips with the aid of a kinematic universal laminator module assembly unit. A backing laminate (Lohman) was placed on the kinematic card platen and a closed nitrocellulose strip with a fixed capture, detection and control region was secured in a predetermined position on the backing laminate. Anti- α -hCG and rabbit IgG gold sol conjugate infused glass fiber strips were secured to a backing laminate with a 2mm overlap on the nitrocellulose strip. The roller pad ensures that all components of the strip are in good contact with the backing laminate. The tape was then cut into individual strips of 4mm using an internal rotary cutter and stored in foil bags with desiccant until ready for use.

Preparation device

The test paper is assembled into a plastic housing part together with an absorbent sampler impregnated with a buffer to control the pH. The housing member contains a lower half with alignment pins and an upper half with windows for viewing the lines on the nitrocellulose strip and means to hold the absorbency sampler in contact with the test strip upstream of the reagent zone. The window position is such that the test and control lines are visible to the user, but the capture area lines are hidden. The strip was placed in the lower shell section using pins to locate the strip. A4X 12 mm sink pad (GE Healthcare) was placed on the proximal end of the strip. The top cover is used to close the device.

Example 2

This example demonstrates an assay that utilizes a negative pregnancy marker (FSH) in combination with the analyte of interest hCG. The effect of a negative marker on the sensitivity of the hCG assay was demonstrated by locating a capture zone containing anti- β -FSH antibody upstream of the anti- β -hCG zone as the detection zone. The effect of the FSH capture zone on the amount of labelled particles that migrate to the detection zone as a function of FSH in the sample can thus be determined.

Method

The device was prepared as described in example 1. To test the effect of FSH concentration on the sensitivity of the assay, a non-pregnant urine sample was collected from internal volunteers. Urine was removed using anti-a-TSH antibodies to remove endogenous hCG and LH, followed by addition of a range of concentrations of hCG and FSH. The device was operated by applying spiked urine to the absorbent sampler of the test device. The intensity of the line at the detection zone was measured at 5 minutes using an internal camera system and 40 non-professional users also scored the device for pregnancy or non-pregnancy. Non-professional users are non-SPD workers who have never seen this type of pregnancy test before. Each non-professional user visually scores 7 devices, which are presented to them in a random order.

Results

As the FSH concentration in the urine sample increased, both the line intensity at the detection zone measured by the camera and the amount of the device scored as pregnant by non-professional users decreased, indicating the effect of FSH as a negative marker on the hCG detection sensitivity. The results are shown in table 1 below.

Table 1: the average line intensity measured by the internal camera system and the amount of devices that scored positive visually for pregnancy strips with anti-hCG detection zones and anti-FSH capture zones running mixed negative urine with hCG and FSH. NT-not tested.

Example 3

This example demonstrates an assay utilizing the binding of pregnancy negative marker (FSH) to the analyte of interest hCG in clinical samples containing pregnant urine and perimenopausal urine containing pituitary hCG.

Method

The device was prepared as described in example 1. To test the effect of the present invention on the ability of the assay to distinguish between hCG from pregnancy and pituitary hCG, urine samples collected from pregnant and peri/postmenopausal women were used. 32 gestational urine samples were selected, collected on days-5 to-3 relative to the day of the expected menstrual period, with hCG concentrations ranging from 3-22 mIU/mL. Also selected were 50 urine samples from peri-and postmenopausal women, containing 5-10mIU/ml pituitary hCG. The device was operated by applying urine to the absorbent sampler of the test device. The intensity of the line at the examination area was measured at 5 minutes using an internal camera system and the device was also scored as pregnant or not pregnant by three non-professional users (SPD staff who did not have a reading device as part of their work).

Results

As shown in Table 2 below, although the maximum concentration of hCG was as high as 10mIU/mL, none of the 50 peri-menopausal/post-menopausal urine solutions were termed gestation, while 90% of the pregnant urine was termed gestation, including those with hCG concentrations as low as 3 mIU/mL. This demonstrates the effectiveness of using a capture zone of negative markers in generating a sensitive pregnancy test that maintains high specificity for pregnancy in clinical specimens.

	Device for scoring pregnancy
		Peri-menopausal/post-menopausal urine	0/50
Urine of pregnancy	29/32

Table 2: in the case of a peri/post-menopausal urine sample containing 5-10mIU/ml hcg and a pregnant urine sample from day-5 to day-3 (relative to the expected period), the amount of device pregnant is scored by a non-professional user.

Example 4

This example demonstrates an assay that utilizes a positive marker of pregnancy (P3G) in combination with the analyte of interest hCG. The effect of the negative marker on the sensitivity of the hCG assay was demonstrated by locating a capture zone with anti-P3G-conjugate upstream of the anti- β -hCG zone as the detection zone. The effect of the P3G conjugate on the amount of particles moving to the detection zone as a function of P3G in the sample can thus be determined. Blue latex particles were coated with the anti- α -hCG and anti-P3G antibodies by mixing the antibodies together prior to coating onto the latex particles. In the test, the coated latex formulation was applied to porous carriers in various mixtures of P3G (positive marker) and hCG (analyte of interest) to observe the effect of P3G levels on the detection zone signal in the assay.

Preparation of labeled reagent (latex coated with anti-alpha-hCG and anti-P3G)

1ml of latex (400 nm in diameter) at 2% solids was washed by centrifugation (13000rcf, 20 ℃,4 min), the supernatant removed and the pellet resuspended in 500. mu.l of 100mM borate buffer (pH8.5) to give a final concentration of 4% solids latex. The latex was heated at 46 ℃ for about 30 minutes while gently mixing. A coating of a mixture of 1200. mu.g/ml anti-alpha-hCG (mouse monoclonal) and 600. mu.g/ml anti-P3G (Fab, mouse monoclonal) was prepared in 100mM borate buffer (pH 8.5). The antibody mixture was heated at 46 ℃ for 30 minutes while gently mixing.

The heated antibody mixture was added in equal volume to the heated latex and mixed to give 1ml of a 2% solids latex plus 600 μ g/ml anti- α -hCG and 300 μ g/ml anti-P3G. To the mixture was added 100. mu.l of 95% ethanol plus 0.5% sodium acetate and the resulting mixture was incubated for 60 minutes with gentle mixing at 46 ℃.

110 μ l of 200mg/ml BSA (reagent grade) was added to deionized water to block the latex and mixed for 30 minutes at 46 ℃. After blocking in BSA, the latex was washed by three centrifugation steps and the supernatant removed (discarding the supernatant), and the pellet was resuspended in 10mM borate buffer (pH8.5) after each centrifugation step. After the final centrifugation step the supernatant was removed and the latex was resuspended in 800. mu.l of buffer containing 10% BSA and 20% sucrose in 100mM Tris pH8.5 (final suspension buffer), yielding approximately 2% solid latex. The latex was further diluted 1+7 in final resuspension buffer before use.

A porous carrier was prepared containing an immobilization zone (detection zone) against β -hCG and an immobilization zone of P3G-conjugate as a capture zone.

A nitrocellulose membrane (MDI 12 μm) was used as a porous carrier on which a capture zone and a separate detection zone were deposited.

As described in example 1, PBSA containing 3mg/ml of anti- β -hCG was applied as a zone (detection zone) to the nitrocellulose membrane at 10mm from the proximal end of the test strip using a Biodot plotter. P3G-conjugate (prepared by linking P3G to a mouse monoclonal antibody as a carrier protein) with a protein concentration of 3mg/ml was deposited at 5mm from the proximal end of the test strip upstream of the detection zone as a capture zone. After the drying step, the film was sealed in PVA and dried, then stored in foil bags with desiccant. The resulting membrane was cut into 4mm wide strips, each having a capture zone 5mm from the proximal end of the strip and a detection zone 10mm from the proximal end of the strip.

The test consisted of latex particles coated with anti-P3G and anti- α -hCG on a porous carrier prepared with a capture zone for the P3G conjugate and an anti- β -hCG zone as a detection zone.

The test procedure or "assay" involves preparing a mixture (sample) of coated latex plus standard buffer (sample) containing different levels of P3G and hCG (5 μ l diluted latex plus 50 μ l standard buffer) and applying the mixture to a nitrocellulose membrane containing a capture zone and a detection zone. This was achieved by dipping the proximal end of the test strip into a bath or mixture of coated latex and buffered standard solutions containing varying levels of P3G and hCG. A paper sink material was applied as a sink at the distal end of the strip (about 20mm downstream of the detection zone). Once the mixture at the bottom of the strip dried, it was removed by applying a buffer (50 μ l PBSA plus 0.1% ovalbumin) to the bottom of the strip and passing it through a sink that washed the unbound latex to the end of the strip. After washing, the signal generated by the detection zone is measured on an optical reading system (image capture system), the intensity of the signal generated by the detection zone is reported as a number, the higher the intensity of the signal at the detection zone, the larger the number.

To test the effect of the P3G assay on the detection zone reaction, the latex formulations were tested in a buffer containing 0 μ g/ml P3G but varying levels of hCG. This was repeated in standard buffer containing 75ug/ml P3G but also containing different levels of hCG.

Mixing a latex formulation with a mixture comprising0μg/ml P3GMixed with standard buffer solutions of different levels of hCG (0, 2.5 and 10mIU/ml hCG) and a strip with a capture zone for the P3G conjugate and anti- β -hCG as the detection zone, held vertically while immersed in the mixture, allowed the mixture to move through the membrane into a water bath applied at the distal end. The test strip is washed as described above and a reading taken at the detection zone on the imaging system.

The above procedure was repeated, this time mixing the latex formulation with standard buffer containing 75. mu.g/ml P3G and 0, 2.5 and 10mIU/ml hCG. The above procedure is followed and readings of the inspection area are taken on the internal camera system.

Results

The latex tested in the assay with 0 μ g/ml P3G allowed the majority of the labelled reagent to be captured at the capture zone, (anti-P3G on labelled reagent binds to P3G-conjugate at the capture zone). This significantly reduces the amount of labelled reagent that moves to the detection zone, so that the reaction to hCG seen at the detection zone is reduced. The presence of 75 μ g/ml P3G in the assay reduced the capture of labelled reagent by the capture zone (preventing the labelled reagent from binding to the P3G-coupling zone due to the binding of P3G in standard buffer to anti-P3G on the labelled reagent) allowing more labelled reagent to move to the detection zone where a significantly stronger signal in the presence of hCG could be seen (see FIG. 7). It is clear that the level of positive marker in the assay will affect the reaction of the detection zone to hCG, depending on the amount of positive marker (in this case P3G) in the assay.

Example 5

This example demonstrates an assay that utilizes a negative marker of pregnancy (FSH) in combination with the analyte of interest hCG. The effect of negative markers on the sensitivity of hCG assays in clinical urine samples was demonstrated by locating a capture zone containing anti- β -FSH antibody upstream of the anti- β -hCG zone as the detection zone. Perimenopausal and postmenopausal clinical urine samples with physiological levels of hCG and FSH were used to show the effect of the FSH capture zone on the test signal generated in the detection zone and were compared to standards prepared by adding hCG to FSH and hCG-depleted urine pools.

Method

The device was prepared as described in example 1. To test the effect of negative markers on the sensitivity of hCG assays in clinical samples, tests were performed using urine samples collected from peri-menopausal/postmenopausal women. In addition, standards were prepared by adding known concentrations of hCG to FSH and hCG depleted urine pools (FSH and hCG depleted as described in example 2). A total of 200 perimenopausal samples and 200 postmenopausal samples were tested. The hCG levels in these samples were measured by a clinical analyzer (DELFIA). In addition, 25 devices used 5mIU/ml hCG added to hCG and FSH depleted mixed urine. Another 25 devices used mixed urine with 10mIU/ml hCG added to hCG and FSH depleted urine. The device was operated by applying urine to the absorbent sampler of the test device. The intensity of the hCG line in the detection zone was measured 5 minutes after addition of the sample using an internal camera system and the device was scored visually as pregnant or not pregnant by two technicians by observing the results of the hCG line, with a visible line (indicating a positive, and therefore pregnant result) or no line (indicating a negative, and therefore not pregnant result). The order of testing clinical samples and standards was randomized and the technician reading the test was blinded to randomization to eliminate any technician reading bias.

Results

As shown in table 3 below, 92% d of the device using 5mIU/ml hCG added mixed urine and 100% of the device using 10mIU/ml hCG added mixed urine were visually observed by the technician as pregnancy. All 200 peri-menopausal and 200 post-menopausal urine samples were visually non-pregnant by both technical readers, despite inclusion of hCG concentrations up to 8.8mIU/ml hCG. Such hCG levels seen in peri-and post-menopausal urine may increase false positive results in traditional pregnancy tests designed to detect hCG as low as 5 mIU/ml.

Table 3: the hCG concentration range and visual score for the perimenopause, post-menopause urine samples used in the device, and hCG standards prepared by adding FSH and hCG depleted negative urine pools.

Figure 8 shows the signal intensity of the hCG test line in the detection zone, measured by an internal camera system, plotted against hCG concentration. For post-spiking standards in the absence of FSH, the signal intensity increased with increasing hCG concentration. With respect to perimenopausal and postmenopausal samples comprising hCG and FSH, it is clear that the presence of FSH in the sample reduces the amount of marker reaching the hCG test zone as the marker binds at the capture zone. This results in a decrease in the signal at the hCG detection zone and therefore provides a true result at the hCG detection zone since the results for these perimenopausal and postmenopausal samples are pregnancy negative.