阅读说明：本技术 女性黄体功能快速无创监测技术 (The quick non-invasive monitoring technology of women luteal function ) 是由 顾泳川 吴银秋 于 2015-04-17 设计创作，主要内容包括：本发明涉及女性生育能力无创监测技术,具体而言,涉及一种全定量或半定量的快速无创尿检技术来测定女性尿中孕酮代谢物的周期生成量,以此来监测和评估女性黄体功能、判断不同排卵周期类型、确定是否真正排卵及排卵后能否受孕。本发明的第一实施方案,涉及一种用于测定孕二醇葡糖苷酸的蛋白杂交偶联体,包含孕二醇葡糖苷酸和偶联蛋白,其特征在于,还包含连接两者的连接物。本发明的激素蛋白偶联体用不饱和标记抗体检测PdG,其灵敏度可达0.1ng/ml,线性范围可控制在0.1-50ng/ml之间。(The present invention relates to female fertility non-invasive monitoring technologies, specifically, it is related to a kind of complete quick noninvasive urine examination technology quantitatively or semi-quantitatively to measure the period production quantity of progesterone metabolite in female urine, monitors and assess women luteal function with this, judge different ovulatory cycle types, determines whether to become pregnant after really ovulating and ovulating.First embodiment of the invention, is related to a kind of for measuring the protein hybridization couplet of pregnanediol glucuronate, includes pregnanediol glucuronate and coupling protein, which is characterized in that the attachment also comprising both connections.Neurophysin couplet of the invention detects PdG with unsaturated labelled antibody, and sensitivity is can be controlled between 0.1-50ng/ml up to 0.1ng/ml, the range of linearity.)

1. it is a kind of for measuring the protein hybridization couplet of pregnanediol glucuronate, it include pregnanediol glucuronate and coupling protein And the attachment of both connections, which is characterized in that the attachment includes polyethylene glycol.

2. couplet as described in claim 1, which is characterized in that the attachment is to include 1-100 carbon atom, preferably 6- 25 carbon atoms, more preferable 6-20 carbon atom, the linear chain or branched chain group of most preferably 6-18 carbon atom.

3. couplet as claimed in claim 2, which is characterized in that one or more carbon atoms in the attachment are selected from The hetero atom of oxygen, nitrogen and sulphur replaces.

4. couplet as described in claim 1, which is characterized in that the polyethylene glycol is selected from diethylene glycol, triethylene glycol, four Ethylene glycol.

5. such as couplet of any of claims 1-4, which is characterized in that the coupling protein be selected from ovalbumin, Bovine serum albumin(BSA), hemocyanin.

6. being selected from such as couplet of any of claims 1-4:

(1) pregnanediol glucuronate -- 6 atom carbochain -- ovalbumin couplet；

(2) pregnanediol glucuronate -- 13 atom carbochain -- ovalbumin couplet；

(3) pregnanediol glucuronate -- 20 atom carbochain -- ovalbumin couplet；With

(4) pregnanediol glucuronate -- 18 atom carbochain -- ovalbumin couplet.

7. such as couplet of any of claims 1-4, which is characterized in that the pregnanediol glucuronate and attachment It is 1:1 to 100:1 with coupling protein ratio.

8. it is a kind of for detecting the kit of pregnanediol glucuronate, it is miscellaneous comprising such as described in any item albumen of claim 1-7 Hand over couplet, which is characterized in that the also antibody comprising specifically binding with the protein hybridization couplet.

9. kit as claimed in claim 8, which is characterized in that the antibody is monoclonal antibody or polyclonal antibody.

10. kit as claimed in claim 9, which is characterized in that also containing the detectable marker being connect with the antibody.

11. kit as claimed in claim 9, which is characterized in that also anti-containing second in conjunction with the antibody specificity Body.

12. kit as claimed in claim 11, which is characterized in that the ratio of the antibody and secondary antibody is 1:2 to 2: 1。

13. the kit as described in claim 11 or 12, which is characterized in that also contain and examined with what the secondary antibody was connect Survey marker.

14. the kit as described in claim 10 or 13, which is characterized in that the detectable marker is selected from colloid, adds lustre to Group, chemiluminescent groups, fluorogen, isotope or enzyme.

15. a kind of for monitoring the kit of luteal function, which is characterized in that comprising as described in any one of claim 1-14 Couplet or kit.

16. a kind of method for preparing couplet as described in claim 1, the method includes distinguishing pregnanediol glucuronate With 6-aminocaprolc acid, 7- azepine -8- oxo -13- amino-tridecanoic acid, 7,14- diaza -8,15- dioxo -20- amino - The step of arachic acid and 4- oxo -5- azepine -9,12, the reaction of 15- trioxa -18- amino-octadecanoid acid, and then with The step of ovalbumin, bovine serum albumin(BSA) or hemocyanin react.

Technical field

The present invention relates to female fertility non-invasive monitoring technology, in particular to it is a kind of it is complete quantitatively or semi-quantitatively Quick noninvasive urine examination technology measures the period production quantity of progesterone metabolite in female urine, monitors and assess women corpus luteum with this Can function judge different ovulatory cycle types, determine whether really to ovulate and become pregnant after ovulating.

Background technique

According to " the infertile Current Situation Investigation report of China " display in 2012, the infertile rate of China was up to 12.5%, the wife's side Reason accounts for 50% or more.Number is more than 50,000,000.The patient of infertile treatment failure accounts for 66%, wherein 98.9% is because not having Promptly and accurately detect and caused by.Therefore, the easy-to-use women luteal function monitoring of noninvasive, the inexpensive, hospital of one kind and family is invented Technology, the needs especially suitable for Chinese prenatal and postnatal care.

When the menstrual cycle starts, about 15-20 ovum starts maturation in each ovary.Ovarian follicle equipped with ovum generates Estrogen.Finally, an ovarian follicle is in the ascendance and keeps mature, and remaining ovarian follicle starts to disintegrate.The dominant follicle Estrogen level rise in former extremely big width of ovulating, until reaching critical level.This high estrogen is horizontal, triggers another Kind hormone is known as the surge of metakentrin (LH).Triggering ovulation, causes ovum to discharge from ovary.Indicate the knot of ovulatory cycle Beam.

The estrogen and the peak LH and ovary ovulation period successively occurred in menstrual cycle has substantial connection.Once estrogen Or the appearance at the peak LH, ovulation typically occur in 12-48 hours following.Therefore, estrogen or LH are monitored in the menstrual cycle Peak value may be used to determine best Time to pregnancy, or need to take the time of contraceptives.

Before preovulatory, the appearance at e surge and the peak LH not can confirm that ovulation actually occurs because estrogen increase sharply and It is not always to cause to ovulate that LH, which discharges peak,.Even with real ovulation, but due to after ovum lack of luteal phase or luteal phase it is short, And cause infertile.This phenomenon that having ovulation but cannot becoming pregnant, be the infertile most common reason in current women.

Commercialization inspection currently on the market is limited only to hormone blood examination or LH urine examination, since hormone blood examination needs large-scale instrument Device, it is both inconvenient, and diagnosis of the inspection result for infertility, and without unified standard.And LH urine examination (including Kunming Yunnan is big CN00112779.9 patent, the CN200610200333 patent of Gansu Lanzhou University and Wu Han Jing deep all places hall CN20110072240.9 patent) then it is mainly used in the preliminary estimation of the onset of ovulation.Therefore, up to now, there are no one both at home and abroad Kind easy-to-use infertile external non-invasive diagnosis product of commercialization, be used to Quantitative Monitoring women luteal function, determine whether there is or not It can become pregnant after ovulation, ovulation and relevant female fertility and hormone infertility.

Summary of the invention

The object of the present invention is to according to hormone progesterone production quantity and ovulation and the close pass become pregnant in female urine during ovulation System, come measure ovulation after progesterone urine metabolin-pregnanediol glucuronate (pregnanediol glucuronide PdG) life Cheng Liang, thus provide a kind of hospital, family and it is personal carry out it is complete quantitatively or semi-quantitatively, inexpensive convenient immunoassay method and inspection Test agent box.Using the onset of ovulation as reference point, by the concentration of PdG in being urinated in 6 days after quantitative determination ovulation, to monitor luteal function Variation, to verify, can whether there is or not become pregnant after true ovulation and ovulation.(1) as pregnanediol value < 2.0mg/24h, show nothing Ovulation or Luteinized unraptured Follicle (luteinized unruptured follicle, LUF).(2) if pregnanediol value exists When between 2.0-3.0mg/24h, indicates ovulation, but lack of luteal phase or short after ovulation, cannot become pregnant.(3) only work as pregnant two When alcohol number > 3.0mg/24h, which is only the ovulatory cycle with complete fertility.Therefore, pregnanediol value in 6 days after ovulation It is the judgment criteria that women has complete fertility and can normally become pregnant more than 3.0mg/24h.

If use ovulation after in 6 days pregnanediol value whether more than 3.0mg/24h as standard, the present invention can also be exempted from qualitative Epidemic disease analysis is such as colloidal gold semi-quantitative immunoreagent item, to determine whether women there is complete fertility can normally become pregnant (> 3.0mg/ For 24 hours) or suffer from infertility (≤3.0mg/24h).

The present invention by pregnanediol glucuronate is connected by attachment (linker) with coupling protein to be formed it is pregnant The protein hybridization couplet of glycol glucosiduronic acid, and the protein hybridization couplet of pregnanediol glucuronate is measured, to monitor corpus luteum The variation of function, to verify, can whether there is or not become pregnant after true ovulation and ovulation.

Attachment of the present invention is divided into hydrophobicity (such as simple non-resilient carbochain) and hydrophily (the miscellaneous original of such as oxygen-containing, sulphur, nitrogen The elastic carbochain of son), the length is 1-100 carbon atom, preferably 5-25 carbon atom, more preferable 6-20 carbon atom is optimal 6-18 carbon atom is selected, wherein one or more carbon atoms can be replaced by the hetero atom selected from oxygen, sulphur, nitrogen etc..Although connection The hydrophily or hydrophobic performance of the length of object and it, while the sensitive of immunoassay is affected in different ways, to some extent Degree and specificity.But relationship between the two there is no so far systematically to be studied, more without report with regard to how to efficiently use attachment Length and hydrophilic/hydrophobic energy are closed for the development and application of the business application such as immunoassay kits of immunoassay The design of reason ground.

The present invention can control pregnanediol glucuronate and be coupled by changing the hydrophilic and hydrophobic of attachment and the length of chain Connection quantity on albumen also just has adjusted density of the pregnanediol glucuronate in reagent strip detection line, i.e. density is higher, line Property range is wider, and density is lower, and sensitivity is better.Meanwhile the length and type of carbochain also influence pregnanediol glucuronate-coupling The combination quality of protein hybridization couplet and antibody, can effectively avoid the non-specific binding with antibody, and suitably long carbon Chain can reduce sterically hindered with antibody combination.

First embodiment of the invention, be related to it is a kind of for measuring the protein hybridization couplet of pregnanediol glucuronate, Include pregnanediol glucuronate and coupling protein, which is characterized in that the attachment also comprising both connections.

Second embodiment of the invention is related to the couplet of first embodiment, which is characterized in that the attachment is Comprising 1-100 carbon atom, preferably 5-25 carbon atom, more preferable 6-20 carbon atom, most preferably 6-18 carbon atom it is straight Chain or branched group.

Third embodiment of the invention is related to the couplet of the second embodiment, which is characterized in that the attachment One or more carbon atoms are exchanged for heteroatoms.

Fourth embodiment of the invention is related to the couplet of third embodiment, which is characterized in that the hetero atom choosing From oxygen, nitrogen and sulphur.

Fifth embodiment of the invention, is related to the couplet of any one of the first to the 4th embodiment, and feature exists In the coupling protein is selected from ovalbumin (OVA), bovine serum albumin(BSA) (BSA), hemocyanin (KLH).

Sixth embodiment of the invention is related to the couplet of any one of the first to the 4th embodiment, the coupling Body is selected from:

(1) pregnanediol glucuronate -- 6 atom carbochain -- ovalbumin couplet

(2) pregnanediol glucuronate -- 13 atom carbochain -- ovalbumin couplet

(3) pregnanediol glucuronate -- 20 atom carbochain -- ovalbumin couplet

(4) pregnanediol glucuronate -- 18 atom carbochain -- ovalbumin couplet.

Seventh embodiment of the invention, is related to the couplet of any one of the first to the 4th embodiment, and feature exists In the pregnanediol glucuronate and attachment and coupling protein ratio are 1:1 to 100:1.

Eighth embodiment of the invention is related to a kind of for detecting the kit of pregnanediol glucuronate, the reagent Box includes the protein hybridization couplet of any one of first to the 7th embodiment of the invention, which is characterized in that also comprising with The antibody of the protein hybridization couplet specific binding.

Ninth embodiment of the invention is related to the kit of the 8th embodiment, which is characterized in that the antibody is single Clonal antibody or polyclonal antibody.

Tenth embodiment of the invention is related to the kit of the 9th embodiment, which is characterized in that also containing with it is described The detectable marker of antibody connection.

Eleventh embodiment of the invention is related to the kit of the 9th embodiment, which is characterized in that also contains and institute State the secondary antibody of antibody specificity combination.

Twelfth embodiment of the invention is related to the kit of the 11st embodiment, which is characterized in that the antibody Ratio with secondary antibody is 1:2 to 2:1.

Thirteenth embodiment of the invention is related to the kit of the 11st or the 12nd embodiment, which is characterized in that Also containing the detectable marker being connect with the secondary antibody.

Fourteenth embodiment of the invention, be related to the tenth or the 13rd embodiment kit, which is characterized in that institute It states detectable marker and is selected from colloid, chromophore, chemiluminescent groups, fluorogen, isotope or enzyme.

Fifteenth embodiment of the invention is related to a kind of for monitoring the kit of luteal function, which is characterized in that packet Couplet or kit containing any one of the first to the 14th embodiment of the invention.

The beneficial effects of the present invention are for the women with complete fertility, the present invention is without suspected of natural conception Accurate information is provided at the beginning of ideal time and gestation.For suffering from infertility and women being treated, this hair It is bright that also to doctor and patient, the whether successful ideal tools of therapeutic effect can be judged by providing one simultaneously.In reproductive center, originally The measurement of progesterone provided by inventing is without suspected of the Best Times for determining artificial insemination, and test-tube baby's (IVF) early stage of development is for promoting The monitoring and determination of gonadal hormone induced ovulation ovulate and the time of ovum are taken to give huge to improve Pregnancy Success rate It helps.

It should be understood that within the scope of the present invention, above-mentioned various technical characteristics of the invention and below in (embodiment) specifically It can be combined with each other between each technical characteristic of description, to form a new or preferred technical solution.

Detailed description of the invention

Fig. 1 .PdG immunity test strip and quantitative fluorescence analysis schematic diagram

As illustrated in fig. 1, test strips of the invention, can be by nitrocellulose filter, sample pad, water absorption pad and PVC or PS Bottom plate etc. is constituted.Drawing on nitrocellulose filter has a two lines: one is the coated detection line of PdG- protein conjugates, another For the coated internal standard nature controlling line of independent two antiantibodys.Colloidal gold (20nm-100nm) or various fluorescent reagents both can be marked directly PdG monoclonal antibody can also first mark secondary antibody polyclonal antibody, then again in conjunction with PdG monoclonal antibody immunity.As preferred Scheme first marks secondary antibody polyclonal antibody, then, then adjusts and optimize the best ratio of secondary antibody marker Yu PdG monoclonal antibody Example, to obtain most effective immune combination.It is suitable for the invention general with colloidal gold or fluorogenic quantitative detection test strips Portable quantitative analysis instrument (Shanghai TSR3000 Jie Ning Biotechnology Co., Ltd), can be to colloid golden red color on reagent strip The depth or fluorescence intensity level carry out quick and precisely quantitative analysis.The weight and female urine of its fluorescence intensity level or colloid golden red Middle PdG concentration is inversely.

Testing principle of the invention: by by a certain percentage dilute after female urine drop to sample pad on (a step dry method), Or be added in a test tube, (two step wet process) is dripped in sample pad again after mixing well.Contain PdG in sample pad or in test tube Single anti-mouse antibody and the sheep anti mouse secondary antibody and goat-anti rabbit secondary antibody for using fluorescent marker respectively (as internal standard).In sample pad or test tube In, PdG is first in conjunction with fluorescence PdG monoclonal antibody in urine sample.Through capillary action, remaining fluorescence PdG monoclonal antibody flows again in sample pad Detection line on reagent strip carries out immune combination with the PdG- protein conjugates being coated in detection line in advance, to make to examine Survey line shows fluorescence.And the goat-anti rabbit secondary antibody fluorescence in sample pad, it will flow on nature controlling line in conjunction with coated rabbit antibody, make matter Control line also shows fluorescence.After fluorescent test paper strip is inserted into portable quantitative analysis instrument, to glimmering on test strips detection line, nature controlling line Luminous intensity carries out quantitative analysis simultaneously, and on application detection line and nature controlling line fluorescence intensity ratio, come determine urinate in PdG it is dense Degree.No matter whether hormone PdG is contained in urine sample, nature controlling line shows fluorescence always, if nature controlling line does not show fluorescence, shows test strips In vain.It will be understood by those skilled in the art that above-mentioned quantitative immunoassay is former when colloidal gold replaces fluorescence to carry out two antiantibody of label Reason is equally applicable to colloid gold immune test paper quantitative analysis.

The range of linearity analysis chart of Fig. 2 albumen addition colloid gold immune analysis

As shown in Fig. 2, sensitivity can be controlled in 5-100ng/ml (neurophysin coupling down to 1ng/ml, the range of linearity Body 1,2) or 1-100ng/ml between (neurophysin couplet 4) (see Fig. 2), colloidal gold quantitative testing test paper item can expire completely The Quantitative Monitoring of podiatrist institute and household person to PdG (i.e. luteal function).

The range of linearity analysis chart of Fig. 3 albumen addition fluoroimmunoassay

As shown in figure 3, detecting PdG with unsaturated labelled antibody, up to 0.1ng/ml, the range of linearity is controllable for sensitivity Between 0.5-20ng/ml (neurophysin couplet 1,2) or 0.1-50ng/ml (neurophysin couplet 4), fluorescent quantitation inspection Test paper slip can satisfy hospital and household person to the Quantitative Monitoring of PdG (i.e. luteal function) completely.

Foregoing invention content of the invention is not intended to describe each disclosed embodiment or every kind of embodiment party of the invention Case.It is described below and exemplary embodiment is more particularly exemplified.Throughout the specification, guidance, these realities are provided by example Example can it is various combination be subject to using.In either case, exemplified content is as just representative example, without that should manage Solution is exclusiveness example.

Specific embodiment

The present inventor after extensive and in-depth study, has been surprisingly found that for the first time, the egg of pregnanediol glucuronate of the invention White hybridized coupling body and its detection method and detection kit can be with by changing the hydrophilic and hydrophobic of attachment and the length of chain Connection quantity of the pregnanediol glucuronate on coupling protein is controlled, so that measurement sensitivity is up to 0.1ng/ml, the range of linearity It can be controlled between 0.1-50ng/ml, so as to complete the present invention.

Attachment

Herein described " attachment " is the connection for referring to link together pregnanediol glucuronate and coupling protein That records in base, including but not limited to US7521425 and US8288349 can be used as the compound of linker.

Coupling protein

Herein described " coupling protein " is to refer to the attachment through the invention to connect with pregnanediol glucuronate Protein together, including but not limited to ovalbumin, bovine serum albumin(BSA), hemocyanin.

Couplet

Herein described " couplet " refers to that the attachment connects through the invention for pregnanediol glucuronate and coupling protein The adduct being connected together, including but not limited to:

Pregnanediol glucuronate -- 6 atom carbochain -- ovalbumin couplet (1)

Pregnanediol glucuronate -- 13 atom carbochain -- ovalbumin couplet (2)

Pregnanediol glucuronate -- 20 atom carbochain -- ovalbumin couplet (3)

Pregnanediol glucuronate -- 18 atom polyethylene glycol carbochain -- ovalbumin couplet (4)

Antibody

Herein described " antibody ", be refer to specifically with pregnanediol in pregnanediol glucuronate and/or couplet The antibody that glucosiduronic acid combines, in this application, term " antibody " and " specific antibody " are used interchangeably.The invention also includes There is the polyclonal antibody and monoclonal antibody of specificity, especially monoclonal antibody to pregnanediol glucuronate.Here, " special It is anisotropic " refer to that antibody can be incorporated into pregnanediol glucuronate, but nonrecognition and it is incorporated into other non-related molecules.The present invention is not only Including complete monoclonal or polyclonal antibody, but also including with immunocompetent antibody fragment, such as Fab' or (Fab)₂ Segment；Heavy chain of antibody；Antibody light chain；Genetically engineered Single Chain Fv Molecule A (Ladner etc., United States Patent (USP) No.4,946, 778)；Or chimeric antibody, such as there is mouse antibody binding specificity but retain the antibody of the antibody moiety from people.Of the invention is anti- Body can be prepared by various technologies known to those skilled in the art.

Detectable marker

Herein described " detectable marker " refers to the substance that can produce detection signal, the detectable marker packet Include but be not limited to colloid, chromophore, chemiluminescent groups, fluorogen, isotope or enzyme.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.