Immunoassay kit and determination method and preparation method thereof
阅读说明：本技术 一种免疫检测试剂盒及其测定方法和制备方法 (Immunoassay kit and determination method and preparation method thereof ) 是由 熊亮 郑兰花 洪礼清 蒋奎胜 庾琼 刘仁源 刘勇娥 李建霖 于 2019-10-08 设计创作，主要内容包括：本发明提供一种免疫检测试剂盒,包括1)缀合物载体,包括配体、标记物和固相载体,所述配体用于与待测分析物发生特异性反应,所述标记物用于信号检测,所述配体与标记物相连接,所述固相载体用于承载配体和标记物；以及2)免疫层析试纸条；其中,所述缀合物载体与所述免疫层析试纸条分体式设置。本试剂盒具备既可在常温保存、运输,同时又能有良好精密度的特点,且操作方法简单快捷,适合即时检测使用。本试剂盒测定时先将样品溶液与缀合物载体混均匀后再加入免疫层析试纸条中进行检测,缀合物释放更为充分。本试剂盒制备方便、快捷、制造成本低廉,能与现有产线设备很好的兼容,可以不需要复杂的工艺及昂贵的设备。(The invention provides an immunoassay kit, which comprises 1) a conjugate carrier, a detection probe and a detection reagent, wherein the conjugate carrier comprises a ligand, a marker and a solid phase carrier, the ligand is used for carrying out a specific reaction with an analyte to be detected, the marker is used for signal detection, the ligand is connected with the marker, and the solid phase carrier is used for carrying the ligand and the marker; and 2) an immunochromatographic test strip; wherein, the conjugate carrier and the immunochromatographic test strip are arranged in a split mode. The kit has the characteristics of normal-temperature storage and transportation, good precision, simple and quick operation method and suitability for instant detection. When the kit is used for determination, the sample solution and the conjugate carrier are mixed uniformly and then added into the immunochromatography test strip for detection, so that the conjugate is released more fully. The kit is convenient and quick to prepare, low in manufacturing cost, and well compatible with the existing production line equipment, and complex processes and expensive equipment are not needed.)
1. An immunoassay kit, comprising:
1) the conjugate carrier comprises a ligand, a marker and a solid phase carrier, wherein the ligand is used for performing specific reaction with an analyte to be detected, the marker is used for signal detection, the ligand is connected with the marker, and the solid phase carrier is used for carrying the ligand and the marker; and the number of the first and second groups,
2) the immunochromatography test strip comprises a first sample pad, a nitrocellulose membrane, a water absorption pad and a bottom plate, wherein the first sample pad, the nitrocellulose membrane and the water absorption pad are sequentially arranged on the bottom plate along the liquid chromatography direction;
wherein, the conjugate carrier and the immunochromatographic test strip are arranged in a split mode.
2. The immunoassay kit of claim 1, wherein the label is any one of time-resolved fluorescent microspheres, ordinary fluorescent microspheres, magnetic microspheres, colloidal gold, colored latex microspheres, quantum dots, time-resolved fluorescent dyes, ordinary fluorescein, enzymes, biotin-avidin, and microsphere amplification systems.
3. The immunoassay kit according to claim 1, wherein the solid support is any one of a glass fiber, a polyester film and a nonwoven fabric.
4. The immunoassay kit of claim 1, wherein the immunochromatographic test strip further comprises a second sample pad disposed between the first sample pad and the nitrocellulose membrane.
5. The immunoassay kit of claim 1, wherein the conjugate carrier is stored and transported at ambient temperature in a sealed chamber.
6. The immunoassay kit of claim 5, wherein the chamber is any one of a centrifuge tube, a plastic tube, or a microplate.
7. An immunochromatographic assay method using the immunoassay kit according to any one of claims 1 to 6, comprising the steps of:
dissolving the conjugate: adding the sample solution into a conjugate carrier, dissolving the conjugate and fully reacting with the sample solution to obtain a mixed solution;
dropwise adding the mixed solution: taking the mixed solution fully reacted in the step, and dripping the mixed solution on a first sample pad in the immunochromatography test strip;
and (3) detection: and collecting signals of the T line and the C line by using an immunoassay instrument, calculating a T/C signal value, and obtaining the concentration of the analyte from the calibration curve.
8. The immunochromatographic assay method according to claim 7, further comprising, before the step of dissolving the conjugate:
obtaining a calibration curve: and taking standard sample curves of the analyte with different concentrations to obtain corresponding T line and C line signals, establishing a standard curve of the analyte concentration corresponding to the T line and C line signals to obtain a calibration curve, and storing the calibration curve in the ID chip.
9. A method of making an immunoassay kit according to any of claims 1 to 6, comprising the steps of:
s100, preparing a conjugate carrier;
s200, preparing immunochromatographic test paper;
s300, packaging an immunodetection kit;
in step S100, preparing a conjugate carrier includes:
s110, combining the ligand and the marker to obtain a conjugate;
s120, preparing a diluent, and dispersing the obtained conjugate in the diluent;
s130, fixing the diluent in a solid phase carrier in a spraying or smearing mode, and drying to obtain a conjugate carrier;
s140, cutting the conjugate carrier into fixed sizes and subpackaging the conjugate carrier in sealed chambers.
10. The method of preparing an immunoassay kit according to claim 9, wherein the mass concentration of the conjugate in the diluent is 0.05 to 0.5; the diluent comprises buffer pairs, proteins, saccharides, high polymers, a blocking agent, a preservative and a surfactant.
Technical Field
The invention relates to the field of immunodetection, in particular to an immunodetection kit, and a determination method and a preparation method thereof.
Background
Immunochromatography is a method capable of qualitatively and quantitatively analyzing trace analytes in a short time using an antigen-antibody reaction, and has been used for diagnosing or detecting various diseases and a variety of fields including medicine, agriculture, animal husbandry, food, military industry, and environmental fields.
Immunochromatographic assays are generally performed using assay strips containing reactive substances that change in response to the analyte to be detected, or using assay devices that contain assay strips mounted in plastic housings. Conventional assay test strips include: the sample pad, the combination pad, the nitrocellulose membrane and the absorbent pad which are connected in sequence are sequentially overlapped and assembled on the bottom plate, wherein the sample pad is used for receiving a liquid sample, namely an analyte; the conjugate pad contains a conjugate, i.e., a detection reagent, to which the analyte can bind; a detection part is fixed on the nitrocellulose membrane, and an antibody or an antigen is fixed on the detection part, namely the detection part can perform specific reaction with the analyte combined with the conjugate; the absorbent pad is used to ultimately receive a liquid sample. In the immunochromatographic assay using the assay strip, when a liquid sample is dropped on a sample pad, it moves through a conjugate pad and a nitrocellulose membrane by capillary phenomenon, and is finally received in a water absorbent pad. The conjugate in the conjugate pad also moves as a mobile phase with the liquid sample, and if the analyte to be detected is present in the liquid sample, the conjugate will bind to the antigen or antibody on the nitrocellulose membrane by the analyte (generally referred to as a "sandwich reaction"), or the conjugate and the analyte compete for binding to the antigen or antibody (generally referred to as a "competition reaction"), and thus, the presence of the analyte in the sample can be visually perceived or perceived by the sensor.
However, the conventional assay strip has problems in that a liquid sample moving by capillary phenomenon is not uniformly bound to a dry conjugate immobilized on a conjugate pad and the conjugate is not completely released in the conjugate pad, and thus, there may be variations between assay strips, which may cause the accuracy and reproducibility of quantitative analysis of the sample to be lowered.
An immunochromatographic test strip, which can be constituted of a conjugate pad and an insoluble membrane support, is provided in patent CN103314297A, because a conjugate portion is formed in a linear shape and there is a certain positional relationship between the conjugate portion and the insoluble membrane support, to achieve excellent releasability of the conjugate from the conjugate pad, to complete the reaction in a shorter time, and also to achieve excellent sensitivity. However, the liquid sample moved by capillary phenomenon is still bound to the conjugate in the present patent, and thus effective release of the conjugate cannot be guaranteed.
Provided in patent CN104428675A are a lyophilized conjugate structure for point-of-care testing (POCT) immunochromatography, an immunoassay kit comprising the same, and a method for analysis using the kit, which can perform quantitative analysis with higher reproducibility and linearity depending on concentration, compared to a conventional assay method using an immunochromatographic test strip comprising a conjugate pad prepared by adsorbing a conjugate, because a sample is subjected to immunochromatography after uniformly reacting with the lyophilized conjugate structure prepared separately from the outside according to the present invention on the outside. However, the freeze-dried conjugate structure body needs to be obtained through a freeze-drying method, and the freeze-drying method has the advantages of long storage period, good uniformity, capability of storing the biological activity of the marker to the maximum extent, high cost and long production period, and the freeze dryer needs to be operated by professional personnel, and has high requirements on the components of the marker diluent.
In addition, the label bound with the antibody/antigen can be stored in a liquid reagent form to improve the accuracy and reproducibility of sample analysis, and the label is diluted with a diluent and then stored in a centrifuge tube. However, when the liquid reagent containing the antibody and the antigen is stored for a long time, the affinity of the antigen-antibody reaction is reduced, or the antigen and the antibody are denatured, the liquid reagent has a complicated process formula, the difficulty in maintaining good stability is high, the stability can be maintained only by cold storage and transportation at 2-8 ℃, and the production, transportation, storage and other costs are increased.
Based on the above situation, there is a need to design an immunoassay device that can solve the above problems.
Disclosure of Invention
The invention aims to provide an immunoassay kit, which comprises a conjugate carrier and an immunochromatographic test strip, has the characteristics of normal-temperature storage and transportation, good precision, simple and quick operation method, and suitability for instant detection, and solves the problem of inaccurate result caused by excessive retention and insufficient release of a conjugate on a binding pad in the conventional test strip.
The second purpose of the invention is to provide an immunochromatographic assay method, wherein the sample solution and the conjugate carrier are mixed uniformly and then added into an immunochromatographic test strip for detection, so that the conjugate is released more fully.
The third purpose of the invention is to provide a preparation method of the immunoassay kit, wherein the preparation of the conjugate carrier is convenient and rapid, the manufacturing cost is low, the immunochromatographic test strip keeps the original structure, and the immunoassay kit can be well compatible with the existing production line equipment without complex process and expensive equipment.
In order to achieve the above object, the present invention provides in a first aspect an immunoassay kit comprising:
1) the conjugate carrier comprises a ligand, a marker and a solid phase carrier, wherein the ligand is used for performing a specific reaction with an analyte to be detected, the marker is used for detecting a signal, the ligand is connected with the marker, and the solid phase carrier is used for carrying the ligand and the marker;
2) the immunochromatography test strip comprises a first sample pad, a nitrocellulose membrane, a water absorption pad and a bottom plate, wherein the first sample pad, the nitrocellulose membrane and the water absorption pad are sequentially arranged on the bottom plate along the liquid chromatography direction;
wherein, the conjugate carrier and the immunochromatographic test strip are arranged in a split mode.
Specifically, the ligand is linked to the label by chemical and/or physical force.
Specifically, the conjugate carrier is provided separately from the immunochromatographic strip, which means that the conjugate carrier is not contained on the immunochromatographic strip, for example, the conjugate carrier is packaged separately from the immunochromatographic strip. When the immunoassay kit is used for detection, a sample solution can be mixed with a conjugate carrier in a cavity, and then the mixed sample solution is added into an immunochromatography test strip to obtain a detection result, so that the release capacity of a conjugate can be improved, the reaction sufficiency of an analyte to be detected and a ligand can be improved, and the accuracy of the detection result can be further improved.
Preferably, the marker is any one of a time-resolved fluorescent microsphere, a common fluorescent microsphere, a magnetic microsphere, colloidal gold, a colored latex microsphere, a quantum dot, a time-resolved fluorescent dye, common fluorescein, an enzyme, biotin-avidin and a microsphere amplification system.
Preferably, the solid phase carrier is any one of glass fiber, polyester film and nonwoven fabric.
Preferably, the immunochromatographic test strip further comprises a second sample pad, and the second sample pad is arranged between the first sample pad and the nitrocellulose membrane.
Preferably, the conjugate carrier is stored and transported in a sealed chamber at normal temperature.
Preferably, the chamber is any one of a centrifuge tube, a plastic tube or a microplate.
Preferably, the diluent comprises buffer pairs, proteins, saccharides, polymers, blocking agents, preservatives, and surfactants.
The second aspect of the present invention provides an immunochromatographic assay method using the above immunoassay kit, comprising the steps of:
dissolving the conjugate: adding the sample solution into a conjugate carrier, dissolving the conjugate and fully reacting with the sample solution to obtain a mixed solution;
dropwise adding the mixed solution: taking the mixed solution fully reacted in the step, and dripping the mixed solution on a first sample pad in the immunochromatography test strip;
and (3) detection: and collecting signals of the T line and the C line by using an immunoassay instrument, calculating a T/C signal value, and obtaining the concentration of the analyte from the calibration curve.
Preferably, before the step of dissolving the conjugate, the method further comprises:
obtaining a calibration curve: and taking standard sample curves of the analyte with different concentrations to obtain corresponding T line and C line signals, establishing a standard curve of the analyte concentration corresponding to the T line and C line signals to obtain a calibration curve, and storing the calibration curve in the ID chip.
The third aspect of the invention provides a preparation method of an immunoassay kit, which uses the immunoassay kit and comprises the following steps:
s100, preparing a conjugate carrier;
s200, preparing immunochromatographic test paper;
s300, packaging immunoassay kit
In step S100, preparing a conjugate carrier includes:
s110, combining the ligand and the marker to obtain a conjugate;
s120, preparing a diluent, and dispersing the obtained conjugate in the diluent;
s130, fixing the diluent in a solid phase carrier in a spraying or smearing mode, and drying to obtain a conjugate carrier;
s140, cutting the conjugate carrier into fixed sizes and subpackaging the conjugate carrier in sealed chambers.
Preferably, the mass concentration of the conjugate in the diluent is 0.05-0.5 per mill; the diluent comprises buffer pairs, proteins, saccharides, high polymers, a blocking agent, a preservative and a surfactant.
The invention has the beneficial effects that:
1. the conjugate is treated by using a proper diluent, and is dispersed in a solid phase carrier, and the conjugate carrier is obtained after drying, so that the preparation method of the conjugate carrier is simple, the conjugate carrier can be stored and transported for a long time at normal temperature, and the cost is reduced;
2. the conjugate carrier and the immunochromatographic test strip are arranged in a split mode, so that the conjugate carrier can be independently packaged in a closed cavity, can be simply stored without pollution, and is easy to carry;
3. the conjugate can be quickly and fully dissolved in the sample solution to fully react with the analyte to be detected, the conjugate can not generate nonspecific aggregation after being redissolved, the releasing capacity of the conjugate and the reaction sufficiency of the analyte to be detected and the ligand can be effectively improved by redissolving the conjugate in the sample solution, and therefore the accuracy of the detection result is further improved;
4. compared with the conventional immunochromatographic test paper for solidifying the conjugate on the binding pad, the conjugate provided by the invention has stable titer, and shows better precision; compared with the immunochromatographic test paper for storing the marker in a liquid state or a freeze-dried state, the conjugate carrier is dried, is convenient and quick to prepare, has low manufacturing cost, and can be stored at normal temperature for a long time;
5. the immunoassay kit can keep the original chromatography test strip structure, can be well compatible with the existing production line equipment, and can not need complex processes and expensive equipment.
Drawings
FIG. 1 is a schematic diagram of an immunochromatographic test strip in the prior art;
FIG. 2 is a schematic view of an immunoassay kit of the present invention;
FIG. 3 is a schematic view of another configuration of the immunoassay kit of the present invention;
FIG. 4 is a flow chart of the assay method of the immunoassay kit of the present invention;
FIG. 5 is a flow chart of a method of making the immunoassay kit of the present invention;
FIG. 6 is a flowchart of the preparation of a conjugate carrier by the immunoassay kit of the present invention.
Wherein, in fig. 1: 1. a sample pad; 2. a nitrocellulose membrane; 21. detecting lines; 22. a quality control line; 3. a water absorbent pad; 4. a base plate; 5. a bonding pad;
in fig. 2 and 3: 1. a first sample pad; 2. a nitrocellulose membrane; 21. detecting lines; 22. a quality control line; 3. a water absorbent pad; 4. a base plate; 5. a second sample pad; 7. a chamber; 8 a conjugate carrier.
Detailed Description
(conventional immunochromatography test strip)
As shown in fig. 1, the conventional immunochromatographic test strip includes: the
(immunoassay kit)
The immunoassay kit provided by the present invention, as shown in fig. 2, comprises:
1) The
The label may produce a signal that is visually perceptible or perceptible using a sensor, may produce a signal by an inherent property of the label (e.g., luminescence), or may produce a signal by an external stimulus (e.g., fluorescence).
In some embodiments of the invention, the label is any one of a time-resolved fluorescent microsphere, a normal fluorescent microsphere, a magnetic microsphere, colloidal gold, a colored latex microsphere, a quantum dot, a time-resolved fluorochrome, a normal fluorescein, an enzyme, a biotin-avidin, and a microsphere amplification system.
The ligand may specifically bind to the analyte, and any substance may be used as a ligand in the present invention as long as it exhibits the property of specifically binding to a specific receptor. The ligand is bound to a label to form a conjugate, and the label and the ligand may be physically or chemically linked to each other.
In some embodiments of the invention, the ligand is an antibody that specifically binds to an antigen or an antigen that specifically binds to an antibody.
The solid phase carrier is used for bearing the conjugate, the solid phase carrier does not react with the conjugate, namely, the performance of the conjugate is not adversely affected, and long-term storage at normal temperature and the characteristics of the conjugate can be realized by preparing the conjugate into a solution with predetermined components and concentrations and then dispersing the solution on the surface of the solid phase carrier. At the same time, the solid support does not react with the sample solution or the analyte.
In some embodiments of the present invention, the solid support is any one of glass fiber, polyester film, and non-woven fabric.
In some embodiments of the invention, the conjugate carrier is stored separately and hermetically in
The
In some embodiments of the invention, the
In some embodiments of the invention, the
In some embodiments of the invention, the conjugate carrier is formed by: combining the ligand and the marker, diluting the marker combined with the ligand to a certain concentration by a diluent, uniformly distributing the marker on a solid phase carrier in a spraying or soaking mode, drying, cutting into a fixed size, and subpackaging in a sealed chamber, namely storing at normal temperature.
In some embodiments of the present invention, the dilution to a certain concentration means that the mass concentration of the conjugate in the diluent is 0.05% o to 0.5% o. In some embodiments of the invention, the mass concentration of the conjugate in the diluent is between 0.02% and 0.1%.
In some embodiments of the invention, the diluent comprises a buffer pair, a protein, a saccharide, a polymer, a blocking agent, a preservative, a surfactant.
The buffer pair in the diluent is Tris-HCl or PB, and the concentration is 0.01-0.05M; the protein is BSA or casein, and the concentration is 0.5 to 2 percent; the sugar is sucrose and/or trehalose, and the concentration is 5% -25%; the high polymer is any one or the combination of PVP, PVA or PEG, and the concentration is 0.5-2%; the blocking agent is Roche MAK series or Scantibodies HBR series, and the concentration is 1-5 mg/mL; the preservative is Proclin300 or sodium azide with the concentration of 0.05-0.1%; the surfactant is Tween-20, Triton-100 or S9, and the concentration is 0.1-1%.
The pH of the dilution is 7.0-9.0, preferably 9.0.
2) The immunochromatographic test strip comprises a
Unlike the conventional immunochromatographic test strip, the present invention does not have a conjugate pad, but retains a sample pad, a nitrocellulose membrane and a water absorbent pad, and the structures thereof have been disclosed in the prior art and are not described herein.
Wherein, the cellulose nitrate membrane is coated with a detection line (T line) 21 and a quality control line (C line) 22, the
As shown in fig. 3, in some embodiments of the present invention, the immunochromatographic test strip further comprises a
In order to enable the sample solution to be uniformly transferred to the nitrocellulose membrane from the sample pad and facilitate the reduction of the influence of interference substances in the sample, two layers of sample pads can be arranged, a second sample pad is arranged between the first sample pad and the nitrocellulose membrane, and the material of the second sample pad can be the same as that of the first sample pad and can also be different from that of the first sample pad. Through this setting, the migration speed and the degree of consistency of steerable sample solution promote product property ability.
In some embodiments of the invention, the immunoassay kit further comprises a housing, the housing comprises an upper housing and a lower housing, the immunochromatographic test strip is mounted between the upper housing and the lower housing, the upper housing comprises a sample adding hole and an observation port, the sample adding hole corresponds to the first sample pad so as to facilitate the addition of a sample solution, and the observation port corresponds to the T line and the C line of the nitrocellulose membrane so as to facilitate the observation and the acquisition of the detection result. When the immunoassay kit is used for detection, a sample solution dissolved with a conjugate is added from a sample adding hole to carry out chromatography reaction, and signal acquisition is carried out at an observation port after a period of time.
The conjugate pad of the conventional immunochromatographic test strip is used for immobilizing a conjugate, wherein the conjugate is also stored in a solid state, when a sample solution migrates from the sample pad to the conjugate pad by capillary force, the immobilized conjugate is dissolved in the sample solution, while the amount of the sample solution dropped into the sample pad during detection is small, and meanwhile, the sample solution flows with the capillary force, so that the conjugate immobilized in all the conjugate pads cannot be completely released, namely, the conjugate in the conjugate pad remains after the detection is finished, and the accuracy of the final detection result is affected; meanwhile, after the detection of different immunochromatographic test strips is finished, the detection results of different immunochromatographic test strips are deviated due to different residual amounts of the conjugates in the binding pads.
In the invention, the conjugate is fixed in the conjugate carrier, and the conjugate carrier has two main advantages, namely 1) the conjugate is diluted and then uniformly distributed on the solid phase carrier in a spraying or soaking way, and the like, and can be stored at normal temperature after being dried and can also keep good stability after being stored for a long time; 2) the sample solution is firstly mixed with the conjugate carrier in the chamber, so that the releasing capacity of the conjugate can be improved, the reaction sufficiency of the analyte to be detected and the ligand can be improved, and the accuracy of the detection result can be further improved.
(immunochromatographic assay method)
The immunochromatographic assay method provided by the present invention, as shown in fig. 4, uses the above immunoassay kit, and comprises the following steps:
dissolving the conjugate: adding the sample solution into a conjugate carrier, dissolving the conjugate and fully reacting with the sample solution to obtain a mixed solution;
dropwise adding the mixed solution: taking the mixed solution fully reacted in the step, and dripping the mixed solution on a first sample pad in the immunochromatography test strip;
and (3) detection: and collecting signals of the T line and the C line by using an immunoassay instrument, calculating a T/C signal value, and obtaining the concentration of the analyte from the calibration curve.
In some embodiments of the invention, prior to the step of solubilizing the conjugate, further comprising:
obtaining a calibration curve: and taking standard sample curves of the analyte with different concentrations to obtain corresponding T line and C line signals, and establishing a standard curve corresponding to the concentration of the analyte and the T line and C line signals.
The invention can obtain the qualitative detection result and/or the quantitative detection result according to the signal intensity of the T line and the C line.
Specifically, when the analyte to be detected does not exist in the sample solution, the T line does not generate a signal, when the analyte to be detected exists in the sample solution, the T line generates a signal, the C line is a quality control line, and the C line generates a signal no matter whether the analyte to be detected exists in the sample, and the C line signal is a standard for judging whether the sample is enough or not and whether the chromatography process is normal or not. Therefore, the detection result can be qualitatively obtained.
Meanwhile, because the signal generated by the T line is in positive correlation with the concentration of the analyte to be detected in a certain range, according to the correlation curve of the signal generated by the T line and the concentration of the analyte to be detected, the invention can also carry out intensity analysis on the corresponding signals generated by the T line and the C line through auxiliary detection equipment, and then convert the signals into the concentration of the analyte to be detected through a certain corresponding relation, so that the detection result can be obtained quantitatively.
Of course, if the immunoassay instrument for auxiliary detection already has a corresponding calibration curve, or has already obtained a calibration curve once, the detection process can be completed without repeatedly obtaining the calibration curve in the subsequent detection process, and a quantitative detection result can be obtained.
An immunochromatographic assay method utilizing a combination of an immunoreaction principle based on an antigen-antibody reaction and an immunochromatographic principle in which a sample and a reagent move along a medium through a mobile phase. In the general sense, an immune response refers to an antigen-antibody reaction, but in the broad sense of the present invention it is meant to include not only antigen-antibody reactions, but also reactions between a receptor and a ligand to which it specifically binds. Further, without being limited thereto, it also includes a reaction between substances that specifically recognize each other, such as an enzyme-substrate reaction.
The analyte, also referred to as an analyte to be measured, includes viruses and physiologically active substances that can be generally measured by an antigen-antibody reaction.
In some embodiments of the invention, the virus comprises influenza viruses such as influenza a virus, influenza b virus, hepatitis c virus, human immunodeficiency virus, and the like.
In some embodiments of the present invention, the physiologically active substance includes human hemoglobin, hepatitis B antibody, hepatitis C antibody, human immunodeficiency virus antibody, tumor marker (AFP/CEA/PSA/CA199), infectious agent (CRP/PCT/SAA/IL6), and the like.
Sample, also called sample solution, refers to a sample that may contain an analyte, including all biological samples isolated from a mammal, preferably a human.
In some embodiments of the invention, the sample comprises whole blood, blood cells, serum, plasma, bone marrow spinal fluid, sweat, urine, tears, saliva, skin, mucous membrane, and hair. Preferably, the sample may be whole blood, serum, plasma.
In some embodiments of the invention, the sample may be buffered prior to being sufficiently reacted with the treated sample solution added to the conjugate carrier to sufficiently solubilize the conjugate to ensure complete dissolution of the conjugate and complete reaction with the analyte.
The immunoassay method of the present invention can be used for analyzing blood glucose levels and diagnosing diseases using serum as a sample. Examples of such diseases include malaria antigen (Ag), AIDS, hepatitis c, hepatitis b, syphilis, gastric ulcer-causing microorganisms, cancer markers (AFP, PSA, CEA), tuberculosis, SAS, dengue fever, and leprosy. Preferably, the analyte may be a cancer marker (AFP, PSA, CEA), but is not limited thereto.
(method of preparing immunoassay kit)
The method for preparing the immunoassay kit provided by the invention comprises the following steps as shown in figure 5:
s100, preparing a conjugate carrier;
s200, preparing immunochromatographic test paper;
s300, packaging the immunoassay kit.
As shown in fig. 6, the preparation of the conjugate carrier in step S100 includes:
s110, combining the ligand and the marker to obtain a conjugate;
s120, preparing a diluent, dispersing the obtained conjugate in the diluent, and diluting the conjugate to a certain concentration;
s130, fixing the diluent in a solid phase carrier in a spraying or smearing mode, and drying to obtain a conjugate carrier;
s140, cutting the conjugate carrier into fixed sizes and subpackaging the conjugate carrier in sealed chambers.
Wherein, in step S200, the preparation of the immunochromatographic test strip comprises:
s210, preparing a sample pad buffer solution, soaking the sample pad in the sample pad buffer solution in a distributed manner, taking out the sample pad, and drying the sample pad;
s220, coating a detection line T line and a quality detection line C line on the nitrocellulose membrane, and drying;
s230, sequentially arranging the sample pad, the nitrocellulose membrane and the water absorption pad on a bottom plate;
s240, clamping the immunochromatographic test strip between the upper shell and the lower shell to obtain the immunochromatographic test strip.
In some embodiments of the present invention, the dilution of the conjugate to a certain concentration in step S120 means that the mass concentration of the conjugate in the diluent is 0.05% o to 0.5% o. Preferably, the mass concentration of the conjugate in the diluent is 0.02% to 0.1%.
Wherein, in step S300, the packaged immunodetection kit comprises:
packaging the cavity containing the conjugate carrier obtained in the step S100, the immunochromatographic test strip obtained in the step S200 and a drying agent in an aluminum foil bag, sealing by heat sealing, and storing at normal temperature; or
And (4) respectively packaging the cavity containing the conjugate carrier obtained in the step (S100) and the immunochromatographic test strip obtained in the step (S200), respectively packaging the cavities and the immunochromatographic test strip together with a drying agent in an aluminum foil bag, sealing the aluminum foil bag in a heat sealing manner, and storing the aluminum foil bag at normal temperature.