Fluorescence immune test strip for joint quantitative detection of multiple indexes of sex hormone

Abstract

The invention provides a fluorescence immunoassay test strip for joint quantitative detection of multiple indexes of sex hormones, and belongs to the technical field of medical detection. The test strip comprises a piece of absorbent paper 1, a nitrocellulose membrane 1, a binding pad 1, a sample pad, a binding pad 2, a nitrocellulose membrane 2 and a piece of absorbent paper 2 which are sequentially connected to a PVC (Polyvinyl Chloride) bottom plate, wherein two detection lines are sequentially arranged on the nitrocellulose membrane 1 in parallel and are respectively coated with a second follicle-stimulating hormone monoclonal antibody and a second anti-mullerian hormone monoclonal antibody; a monoclonal antibody mixed solution marked by fluorescent microspheres is sprayed on the combination pad 1; the monoclonal antibody mixed solution comprises a first follicle-stimulating hormone monoclonal antibody and a first anti-mullerian hormone monoclonal antibody; a monoclonal antibody mixed solution marked by fluorescent microspheres is sprayed on the combination pad 2; the monoclonal antibody mixed solution comprises a first luteinizing hormone monoclonal antibody and a quality control line monoclonal antibody; a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane 2 in parallel; the detection line is coated with a second luteinizing hormone monoclonal antibody; the fluorescence immunoassay test strip is simple to operate, low in error, reliable in detection result and high in accuracy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a fluorescent immunoassay test strip for combined quantitative detection of multiple sex hormone indexes. BACKGROUND

[0002] Ovarian reserve function refers to the ability of follicle growth, development and formation of fertilizable oocytes in the ovarian cortex, i.e. the quantity and quality of oocytes in the ovary. With the growth of age or other factors, the quantity of oocytes decreases and the quality of oocytes declines, i.e. the decline of ovarian reserve function. There are many factors affecting the ovarian reserve function, such as age, genetic factors, autoimmune factors, surgery of ovary and surrounding tissue, social psychological factors, etc. The decline of ovarian reserve function is a gradual process, and early detection and diagnosis and timely treatment intervention can effectively improve the ovarian function of women and improve the pregnancy ability and pregnancy outcome. At present, the reference indexes or methods of ovarian reserve function of women used in the clinic include age, hormone level, ultrasonic examination, stimulation test, cytokine detection, etc. However, there is still no index that can meet the detection requirements. It is very important to establish a detection method with simple operation, less harm to human body, economy and good prediction value to comprehensively detect multiple ovarian reserve function biological indexes.

[0003] Follicle-stimulating hormone (FSH) is secreted by the adenohypophyseal follicle-stimulating hormone cells, and is collectively called luteinizing hormone (LH) as a gonadotropin. The basal FSH refers to the serum FSH level on the 2-5th day of menstruation (i.e. the early follicular phase), which can reflect the ovarian reserve function. It is generally considered that the serum basal FSH < 10 U / L indicates normal ovarian reserve function, and if FSH > 10-15 U / L, it indicates the decline of ovarian reserve function. The FSH and LH secreted by the pituitary increase with the decrease of ovarian reserve function in the physiological state, and in the early stage of the decline of ovarian reserve function, FSH rises more significantly than LH, so their ratio changes first, which is manifested as the increase of the ratio. Most scholars believe that when the basal FSH / LH > 2, the patient often has poor response to superovulation, and FSH / LH > 3.6 is regarded as a marker point for evaluating the decline of ovarian function.

[0004] Anti-Mullerian hormone (AMH) is also known as Mullerian inhibitory substance, belonging to the transforming growth factor-beta (TGF-beta) family. AMH is mainly secreted by granulosa cells of early growth follicle in ovary, and its concentration is affected by the recruitment speed of primordial follicle and the follicle reserve: therefore, AMH can reflect the number of primordial follicle and pre-antral follicle, and is not affected by the gonadotropin secreted by hypothalamus. Studies have shown that AMH does not change with the female menstrual cycle, and its evaluation value for ovarian reserve function is better than that of basic endocrine hormones. Studies have found that the basic AMH level does not change much before the age of 35 in women, and starts to decrease when the age is greater than 30, and decreases linearly after the age of 35: when the serum AMH level is less than or equal to 1.3 ng / ml, the number of oocytes is significantly reduced. Low AMH value is related to low ovarian response, low embryo quality and poor pregnancy outcome. Compared with FSH and LH on the third day of the cycle, AMH value is more closely related to the number of early antral follicles, and AMH is currently considered to be a reliable indicator for predicting ovarian reserve and reproductive potential.

[0005] At present, the methods for detecting the above biological indicators mainly include radioimmunoassay (RIA), high-sensitivity immunoradiometric assay (IRMA), enzyme immunoassay (EIA), microparticle enzyme immunoassay (MEIA), colloidal gold method and the like. RIA method has radioactive pollution, and IRMA method is time-consuming and laborious, and has low degree of automation. The above methods cannot simply and quickly detect multiple indicators related to ovarian reserve function. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a fluorescence immunoassay test strip for joint quantitative detection of multiple sex hormone indicators.

[0007] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0008] The fluorescence immunoassay test strip for joint quantitative detection of multiple sex hormone indicators comprises, in sequence, a water absorption paper 1, a nitrocellulose membrane 1, a combination pad 1, a sample pad, a combination pad 2, a nitrocellulose membrane 2 and a water absorption paper 2 connected in sequence on a PVC base plate:

[0009] Two detection lines are arranged in sequence and in parallel on the nitrocellulose membrane 1: the two detection lines are respectively coated with a second follicle-stimulating hormone monoclonal antibody and a second anti-Mullerian hormone monoclonal antibody;

[0010] The second follicle-stimulating hormone monoclonal antibody and the second anti-Mullerian hormone monoclonal antibody coated on the two detection lines recognize different antigen epitopes;

[0011] The combination pad 1 is sprayed with a fluorescent microsphere-labeled monoclonal antibody mixture:

[0012] The fluorescent microsphere-labeled monoclonal antibody mixture comprises a fluorescent microsphere-labeled first follicle stimulating hormone monoclonal antibody and a fluorescent microsphere-labeled first anti-Mullerian hormone monoclonal antibody.

[0013] The binding pad 2 is sprayed with a fluorescent microsphere-labeled antibody:

[0014] The fluorescent microsphere-labeled antibody mixture comprises a fluorescent microsphere-labeled first luteinizing hormone monoclonal antibody and a fluorescent microsphere-labeled chicken IgY polyclonal antibody.

[0015] The nitrocellulose membrane has one detection line and one quality control line arranged in parallel in sequence: the one detection line is coated with a second luteinizing hormone monoclonal antibody

[0016] Preferably, the mass ratio of the first follicle stimulating hormone monoclonal antibody, the first luteinizing hormone monoclonal antibody, the first anti-Mullerian hormone monoclonal antibody and the chicken IgY polyclonal antibody to the fluorescent microspheres is independently (0.2-0.5):1.

[0017] Preferably, the spraying concentration of the fluorescent microsphere-labeled antibody mixture on the binding pad is 2-6 μL / cm.

[0018] Preferably, the sample pad is a whole blood sample pad.

[0019] Preferably, the width of the detection line and the quality control line is independently 1-2 mm.

[0020] Preferably, the spacing between adjacent detection lines is independently 4-8 mm; the spacing between the detection line and the quality control line is 4-8 mm.

[0021] Preferably, the quality control line is sprayed with a rabbit anti-chicken IgY polyclonal antibody.

[0022] Preferably, the test strip further comprises a shell; the shell comprises a bottom clamping groove and a clamping shell.

[0023] Preferably, the shell is provided with a sample addition hole and two result detection windows.

[0024] The beneficial effects of the present application: the fluorescence immunoassay test strip provided by the present application for the joint quantitative detection of multiple sex hormone indicators, two detection lines are arranged in parallel on the nitrocellulose membrane 1 in turn: the second follicle stimulating hormone monoclonal antibody and the second anti-Mullerian hormone monoclonal antibody are coated respectively; the binding pad 1 is sprayed with a mixed solution of fluorescent microsphere-labeled monoclonal antibodies: the mixed solution of monoclonal antibodies includes the first follicle stimulating hormone monoclonal antibody and the first anti-Mullerian hormone monoclonal antibody; the binding pad 2 is sprayed with a mixed solution of fluorescent microsphere-labeled antibodies: the mixed solution of antibodies includes the first luteinizing hormone monoclonal antibody and chicken IgY polyclonal antibody; one detection line and one quality control line are arranged in parallel on the nitrocellulose membrane 2 in turn: the second luteinizing hormone monoclonal antibody is coated on the detection line, and the concentrations of FSH, AMH and LH in the sample can be detected at the same time, and the results can be read by an immunofluorescence analyzer, which has the advantages of convenience, simple operation, accurate results and the like, and is suitable for clinical diagnosis and POCT detection. The fluorescence immunoassay test strip provided by the present application integrates FSH, AMH and LH detection in the same detection test paper, not only reduces the error caused by multiple detection of each indicator, simplifies the complex operation steps of multiple indicator detection, but also reduces the detection cost. And according to the embodiment, the correlation between the detection results of the immunization test strip and the detection results of the clinical chemical luminescence method is very high, between 0.9523-0.9901: therefore the detection result is reliable and the accuracy is high. In summary, the fluorescence immunoassay test strip provided by the present application has significant clinical significance in the early diagnosis and treatment intervention of ovarian reserve function decline, and the improvement of female fertility and pregnancy outcome. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The internal side view of the fluorescence immunoassay test strip for joint quantitative detection of multiple indicators of ovarian reserve function;

[0026] Figure 2 The top view of the fluorescence immunoassay test strip for joint quantitative detection of multiple indicators of ovarian reserve function;

[0027] Figure 3 FSH clinical consistency comparison chart;

[0028] Figure 4 AMH clinical consistency comparison chart;

[0029] Figure 5 LH clinical consistency comparison chart. DETAILED DESCRIPTION

[0030] The application provides a fluorescent immunoassay test strip for combined quantitative detection of multiple sex hormone indexes, which comprises, in sequence, water absorption paper 1, nitrocellulose membrane 1, combination pad 1, sample pad, combination pad 2, nitrocellulose membrane 2 and water absorption paper 2 connected to a PVC base plate.

[0031] Two detection lines are arranged in sequence on the nitrocellulose membrane 1, and the two detection lines are coated with second follicle stimulating hormone monoclonal antibody and second anti-Mullerian hormone monoclonal antibody respectively.

[0032] The second follicle stimulating hormone monoclonal antibody and the second anti-Mullerian hormone monoclonal antibody coated on the two detection lines recognize different antigen epitopes.

[0033] The combination pad 1 is sprayed with a mixed solution of fluorescent microsphere-labeled monoclonal antibodies.

[0034] The mixed solution of fluorescent microsphere-labeled monoclonal antibodies comprises fluorescent microsphere-labeled first follicle stimulating hormone monoclonal antibody and fluorescent microsphere-labeled first anti-Mullerian hormone monoclonal antibody.

[0035] The combination pad 2 is sprayed with a mixed solution of fluorescent microsphere-labeled antibodies.

[0036] The mixed solution of fluorescent microsphere-labeled antibodies comprises fluorescent microsphere-labeled first luteinizing hormone monoclonal antibody and fluorescent microsphere-labeled chicken IgY polyclonal antibody.

[0037] One detection line and one quality control line are arranged in sequence on the nitrocellulose membrane 2, and the one detection line is coated with second luteinizing hormone monoclonal antibody.

[0038] In the application, the fluorescent immunoassay test strip comprises a PVC base plate, and the source and specifications of the PVC base plate are not specially required in the application, and a conventional PVC base plate for test strips can be used.

[0039] In the application, the PVC base plate is sequentially connected with water absorption paper, nitrocellulose membrane, combination pad, sample pad, combination pad, nitrocellulose membrane and water absorption paper.

[0040] In the application, the source and specifications of the water absorption paper 1 are not specially required, and a conventional water absorption paper can be used.

[0041] In the present application, the first follicle stimulating hormone monoclonal antibody and the first anti-Mullerian hormone monoclonal antibody can be obtained from commercially available products or prepared by the inventors. In the specific implementation of the present application, the two monoclonal antibodies are preferably prepared by the inventors. Specifically, the human FSH protein and the human AMH protein purchased from the British National Biological Product Testing Institute are used as immunizing antigens to immunize mice, and then the hybridoma technology is used to prepare and screen specific cell strains producing specific monoclonal antibodies. The specific cell strains are injected into the abdominal cavity of mice to produce the two monoclonal antibodies. In the screening process of the present application, the following corresponding antigens are preferably used as non-specific antigens to screen specific cell strains: follicle stimulating hormone, luteinizing hormone, anti-Mullerian hormone, serum inhibin B, thyroid stimulating hormone, human chorionic gonadotropin, estradiol, human placental lactogen, and prolactin. The mouse ascites after injection of the specific cell strains is collected, and the mouse ascites is purified by the caprylic acid-ammonium sulfate precipitation method to obtain specific monoclonal antibodies. In the present application, the titer of the two monoclonal antibodies is preferably 4x10 or more, and the subtype is preferably IgG1, IgG2a, or IgG2b. After protein purification, the content of the purified protein is preferably 5.2-21.3 mg / mL.

[0042] In the present application, the nitrocellulose membrane 1 is overlapped on the water-absorbing paper 1, and two detection lines are arranged on the nitrocellulose membrane in sequence and parallel. The two detection lines are coated with a second follicle stimulating hormone monoclonal antibody and a second anti-Mullerian hormone monoclonal antibody, respectively. The coating order of the second follicle stimulating hormone monoclonal antibody and the second anti-Mullerian hormone monoclonal antibody is not particularly limited in the present application. In the specific implementation of the present application, the coating order is preferably the second follicle stimulating hormone monoclonal antibody and the second anti-Mullerian hormone monoclonal antibody.

[0043] In the present application, the preparation method of the second follicle stimulating hormone monoclonal antibody and the second anti-Mullerian hormone monoclonal antibody is the same as the preparation method of the first follicle stimulating hormone monoclonal antibody and the first anti-Mullerian hormone monoclonal antibody. In the present application, the second follicle stimulating hormone monoclonal antibody and the second anti-Mullerian hormone monoclonal antibody coated on the detection line recognize different antigen epitopes corresponding to the first follicle stimulating hormone monoclonal antibody and the first anti-Mullerian hormone monoclonal antibody, respectively. In the present application, the recognition of different antigen epitopes by the corresponding monoclonal antibodies is preferably determined according to the additive index. The additive index of the corresponding monoclonal antibodies is preferably ≥95%. The additive index AI = [(2A1+2) / (A1+A2)-1]x100%, AI > 50% indicates different antigen determinants, and AI < 50% indicates the same antigen determinant.

[0044] In the present application, the spraying of the detection line is preferably carried out by using a membrane scratching and gold spraying instrument; the width of the detection line is independently preferably 1-2 mm; the spacing between adjacent detection lines is independently preferably 4-8 mm. In the present application, the coating concentration of the FSH and AMH monoclonal antibodies is preferably (0.4-0.8) mg / mL and (0.6-1.2) mg / mL, respectively, and more preferably 0.6 mg / mL and 1.0 mg / mL, respectively. In the present application, the membrane scratching amount of the gold spraying instrument is preferably (0.8-1.2) μL / cm, and more preferably 1 μL / cm.

[0045] In the present application, after the binding pad 1 is overlapped on the nitrocellulose membrane 1, a fluorescent microsphere-labeled monoclonal antibody mixture is sprayed on the binding pad: the monoclonal antibody mixture includes a first follicle-stimulating hormone monoclonal antibody and a first anti-Mullerian hormone monoclonal antibody: in the present application, the mass ratio of the first follicle-stimulating hormone monoclonal antibody and the first anti-Mullerian hormone monoclonal antibody to the fluorescent microspheres is independently preferably (0.2-0.5): 1, and more preferably (0.2-0.3): 1. 1. In the present application, the volume ratio of the fluorescent microsphere-labeled first follicle-stimulating hormone monoclonal antibody and the fluorescent microsphere-labeled first anti-Mullerian hormone monoclonal antibody in the monoclonal antibody mixture is preferably 1:1. In the present application, the spraying concentration of the fluorescent microsphere-labeled monoclonal antibody mixture on the binding pad is preferably 2-6 μL / cm, and more preferably 4 μL / cm.

[0046] In the present application, the sample pad is preferably a whole blood filtration sample pad. The material of the sample pad is preferably a glass cellulose membrane: the preparation method of the sample pad preferably includes the following steps: soaking the glass cellulose membrane in a sealing liquid and then drying: the soaking time is preferably 3-7 min, and more preferably 5 min: the sealing liquid preferably includes the following components at the following concentrations: 0.05wt% Tris, 1wt% Tween-20, 0.5wt% PEG6000, 0.4wt% bovine serum albumin, and 0.02wt% mouse anti-human red blood cells.

[0047] In the application, the binding pad 2 is overlapped with the sample pad, and the binding pad is sprayed with a mixture of fluorescent microsphere-labeled antibodies, which includes a first luteinizing hormone monoclonal antibody and chicken IgY polyclonal antibody. In the application, the mass ratio of the first luteinizing hormone monoclonal antibody and chicken IgY polyclonal antibody to the fluorescent microspheres is preferably (0.2-0.5):1, and more preferably (0.2-0.3):1. In the application, the volume ratio of the fluorescent microsphere-labeled first luteinizing hormone monoclonal antibody and the fluorescent microsphere-labeled chicken IgY polyclonal antibody in the mixture is preferably 1:1. In the application, the spraying concentration of the mixture of fluorescent microsphere-labeled antibodies on the binding pad is preferably 2-6 μL / cm, and more preferably 4 μL / cm.

[0048] In the application, the first luteinizing hormone monoclonal antibody can be obtained from commercially available products or prepared by oneself. In the specific implementation of the application, the first luteinizing hormone monoclonal antibody is preferably prepared by oneself. Specifically, the human LH protein purchased from the National Institute for Biological Standards and Control is used as an immunizing antigen, and the specific cell strain producing the specific monoclonal antibody is prepared and screened by using the hybridoma technology after immunizing mice. The specific cell strain is injected into the abdominal cavity of the mouse to produce the first luteinizing hormone monoclonal antibody. In the screening process, the following antigens are preferably used as non-specific antigens: follicle-stimulating hormone, luteinizing hormone, anti-Mullerian hormone, serum inhibin B, thyroid-stimulating hormone, human chorionic gonadotropin, estradiol, human placental lactogen, and prolactin. The specific cell strain is obtained by screening. The mouse ascites after injection of the specific cell strain is collected, and the specific monoclonal antibody is obtained by purification through the caprylic acid-ammonium sulfate precipitation method. In the application, the titer of the first luteinizing hormone monoclonal antibody is preferably 4×10 or more, the subtype is preferably IgG1, IgG2a, or IgG2b, and the protein content after purification is preferably 3.9-9.3 mg / mL.

[0049] In the application, the nitrocellulose membrane 2 is overlapped with the binding pad 2, and one detection line and one quality control line are arranged on the nitrocellulose membrane 2 in sequence and in parallel. The second luteinizing hormone monoclonal antibody is coated on the detection line. The coating sequence is the second luteinizing hormone monoclonal antibody, rabbit anti-chicken IgY antibody, and the like.

[0050] In the present application, the preparation method of the second luteinizing hormone monoclonal antibody is the same as the preparation method of the first luteinizing hormone monoclonal antibody. In the present application, the second luteinizing hormone monoclonal antibody coated on the detection line recognizes a different antigen epitope than the first luteinizing hormone monoclonal antibody. In the present application, the different antigen epitopes recognized by the corresponding monoclonal antibodies are preferably determined according to the additive index; the additive index of the corresponding monoclonal antibody is preferably ≥ 95%. The additive index AI = [(2A1+2) / (A1+A2)-1]x100%, AI > 50% indicates different antigenic determinants, and AI < 50% indicates the same antigenic determinant.

[0051] In the present application, the spraying of the detection line is preferably performed using a membrane scratching gold spraying instrument; the width of the detection line is independently preferably 1-2 mm; and the spacing between the detection line and the quality control line is 4-8 mm. In the present application, the rabbit anti-chicken IgY polyclonal antibody is preferably sprayed on the quality control line. In the present application, the coating concentration of the LH monoclonal antibody is preferably (0.4-0.6) mg / mL, and more preferably 0.6 mg / mL. In the present application, the coating concentration of the rabbit anti-chicken IgY polyclonal antibody on the quality control line is preferably (0.8-1.2) mg / mL, and more preferably 0.8 mg / mL. In the present application, the membrane scratching amount of the gold spraying instrument is preferably (0.8-1.2) μL / cm, and more preferably 1 μL / cm.

[0052] In the present application, the nitrocellulose membrane 2 is overlapped with the water absorption paper 2, and the present application does not have special requirements for the source and specifications of the water absorption paper, and a conventional water absorption paper in the art can be used.

[0053] In the present application, the width of the fluorescent immunoassay test strip is preferably 3-5 mm, and more preferably 3 mm. The immunoassay test strip preferably further comprises a shell; the shell preferably comprises a bottom clamping groove and a clamping shell; and the shell preferably is provided with a sample addition hole and two result detection windows. The present application does not have special requirements for the material of the shell, and a conventional material in the art can be used.

[0054] In the present application, the water absorption paper 1, the nitrocellulose membrane 1, the combination pad 1, the sample pad, the combination pad 2, the nitrocellulose membrane 2 and the water absorption paper 2 are sequentially adhered to the PVC bottom plate from top to bottom in the direction perpendicular to the PVC bottom plate; the above materials are assembled and cut to obtain the fluorescent immunoassay test strip of the present application.

[0055] The specific structure of the fluorescent immunoassay test strip of the present application is shown in Figure 1 The top view is shown in Figure 2As shown: 1 is PVC bottom plate, 2 is water absorption paper 1, 3 is NC film 1, 4 is binding pad 1, 5 is sample pad, 6 is binding pad 2, 7 is NC film 2, 8 is water absorption paper 2, 9-1 is FSH detection line, 9-2 is AMH detection line, 9-3 is LH detection line, 10 is quality control line, 11 is card shell, 12 is result detection window 1, 13 is sample addition hole, and 14 is result detection window 2.

[0056] The detection principle and method of the fluorescent immunoassay test strip are as follows:

[0057] The detection principle of the fluorescence immunoassay test strip for joint quantitative detection of multiple indexes of ovarian reserve function is a double antibody sandwich method, the fluorescence immunoassay test strip contains multiple detection lines, in the specific detection process, the sample mixture simultaneously chromatographs to two sides, first, the sample chromatographs upward, the AMH antigen in the sample combines with the AMH antibody labeled with fluorescent microspheres in the combination pad 1 to form a complex under the chromatography of the nitrocellulose membrane 1, the complex moves to the first detection line (9-2) of the nitrocellulose membrane 1 under the chromatography, the detection line is coated with an antibody recognizing another epitope of the AMH antigen, forming a double antibody sandwich complex of fluorescent microspheres-AMH antibody-antigen-another AMH antibody structure, the complex gathers at the detection line (9-2); at this time, the sample that has not combined with the AMH antibody at the detection line 9-2 continues to chromatograph and moves to the second detection line of the nitrocellulose membrane 1, the FSH antigen in the sample (which has combined with the fluorescent microsphere-labeled FSH monoclonal antibody) also specifically combines with another FSH monoclonal antibody coated at the second detection line in a double antibody sandwich manner and gathers at the detection line (9-1). Meanwhile, the sample chromatographs downward from the sample well, the LH antigen in the sample combines with the LH antibody labeled with fluorescent microspheres in the combination pad 2 to form a complex under the chromatography of the nitrocellulose membrane 2, the complex moves to the first detection line (9-3) of the nitrocellulose membrane 2 under the chromatography, the detection line is coated with an antibody recognizing another epitope of the LH antigen, forming a double antibody sandwich complex of fluorescent microspheres-LH antibody-antigen-another LH antibody structure, the complex gathers at the detection line (9-3); at this time, the fluorescent microspheres combined with the chicken IgY polyclonal antibody continue to chromatograph and move to the quality control line, and specifically combine with the rabbit anti-chicken IgY polyclonal antibody coated at the quality control line and gather at the quality control line. Each complex gathers at the detection line, is excited by a light source to release emission light of a corresponding wavelength, the higher the concentration of the antigen in the sample, the higher the intensity of the emission light of the detection line. The fluorescence detection system converts the light signal into a digital signal, detects the enrichment fluorescence intensity of the detection line and the quality control line, plots a standard curve with the concentration as the abscissa and the signal value ratio of the detection line to the quality control line (T / C) as the ordinate, so that the concentrations of various antigens of FSH, AMH and LH in the sample can be accurately calculated.

[0058] The technical solutions provided by the present application are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0059] Example 1

[0060] Structure of the fluorescence immunoassay test strip for joint quantitative detection of multiple indexes of sex hormones

[0061] A fluorescence immunoassay test strip for combined quantitative detection of multiple sex hormone indicators, the test strip comprising, in sequence, a water absorption paper 1, a nitrocellulose membrane 1, a bonding pad 1, a sample pad, a bonding pad 2, a nitrocellulose membrane 2 and a water absorption paper 2 connected in sequence on a PVC base plate. Figure 1 As shown in the structure: wherein the PVC base plate of the test strip has, in sequence from top to bottom, a water absorption paper 1 (2), a nitrocellulose membrane 1 (3), a bonding pad 1 (4), a sample pad (5), a bonding pad 2 (6), a nitrocellulose membrane 2 (7) and a water absorption paper 2 (8) adhered in sequence: wherein the nitrocellulose membrane 1 (3) has, in sequence, FSH detection lines (9-1) and AMH detection lines (9-2) each 1 mm wide drawn in parallel, the nitrocellulose membrane 2 (7) has, in sequence, LH detection lines (9-3) and a quality control line (10) each 1 mm wide drawn in parallel, the spacing between each detection line and between the quality control line and the detection lines is 4 mm: after assembly, cutting is performed according to a width of 3-5 mm to obtain a fluorescence immunoassay test strip for combined quantitative detection of multiple indicators of ovarian reserve function: the test strip is installed in a shell, and the shell has a sample hole and two result detection windows (12 mm x 3 mm) provided thereon.

[0062] Method for preparing a fluorescence immunoassay test strip for combined quantitative detection of multiple indicators of ovarian reserve function

[0063] (1) Preparation and purification of monoclonal antibodies: human FSH protein, human LH protein and human AMH protein purchased from the National Biological Product Testing Institute of the United Kingdom are used as immunizing antigens to immunize 6-week-old healthy BALB / c female mice, hybridoma technology is used to prepare and screen monoclonal antibody cell strains, and the following corresponding antigens are selected in the screening process: follicle-stimulating hormone, luteinizing hormone, anti-Mullerian hormone, serum inhibin B, thyroid-stimulating hormone, human chorionic gonadotropin, estradiol, human placental lactogen and prolactin are used as non-specific antigens for screening: the specific antibody cell strains obtained by screening are injected into mice to prepare ascites, and the ascites is purified by the caprylic acid-ammonium sulfate precipitation method to obtain monoclonal antibodies.

[0064] (2) Screening of three paired monoclonal antibodies: The obtained monoclonal cell lines were required to have a titer of 4×10 or higher by ELISA. Monoclonal antibodies with subtypes IgG1, IgG2a, and IgG2b were selected. After protein purification, the purified protein content was 3.9-21.3 mg / ml. Paired antibodies were screened by ELISA additive assay. The additive index (AI) was calculated according to the following formula: AI = [(2A1+2) / (A1+A2)-1]*100%. AI > 50% indicates that they target different antigenic determinants, and AI < 50% indicates that they target the same antigenic determinant. In this invention, the additive index AI of each paired antibody is required to be above 95% to ensure that the two paired monoclonal antibodies recognize different antigenic epitopes. The paired antibodies selected in this embodiment are: α-FSH MH00103 and β-FSH MH00104 antibodies; α-LH MH00303 and β-LH MH00304; AMH 6B3 and AMH 2C10.

[0065] (3) Antibody-labeled fluorescent microspheres

[0066] The 300nm fluorescent microspheres provided by Yocon Biotechnology were diluted with MES (0.1mol / L), activated with 1 / 50 EDC (40mg / mL) and 1 / 50 NHS (20mg / mL), centrifuged, and the supernatant was discarded. The microspheres were then resuspended in 0.1mol / L MES by sonication. Antibody was added at a ratio of (0.2–0.5):1, and the mixture was stirred for 2 hours. Then, 5% bovine serum albumin was added for blocking, and the mixture was stirred for 1 hour. After centrifugation, the supernatant was discarded, and the microspheres were washed twice. The fluorescent microspheres were then sonicated and resuspended in 0.01M PBS (pH 7.4) containing 5% sucrose, 1% bovine serum albumin, and 0.04% sodium azide. The mixture was stored at 4°C for later use.

[0067] (4) Preparation of bonding pad 1

[0068] The conjugate pad is made of glass fiber membrane. First, monoclonal antibodies labeled with FSH and AMH fluorescent microspheres were prepared at a ratio of 250g monoclonal antibody / 1mg microspheres. Then, the two types of monoclonal antibodies labeled with fluorescent microspheres were mixed evenly at a volume ratio of 1:1 and set aside. The gold spraying apparatus was turned on, the "gold spraying" program was selected, and the spraying volume was set to 4μL / cm. The mixture was evenly and continuously sprayed onto the glass fiber filter membrane. After thorough drying in a 37℃ incubator, the conjugate pad was obtained.

[0069] (5) Preparation of bonding pad 2

[0070] The binding pad is made of glass fiber cellulose membrane. First, prepare the LH and chicken IgY fluorescent microsphere-labeled antibodies according to the ratio of 250 g of monoclonal antibody to 1 mg of microspheres, then mix the two kinds of fluorescent microsphere-labeled antibodies in a volume ratio of 1:1, and use them as needed. Turn on the membrane cutting and gold spraying instrument, select the "gold spraying" program, set the spraying amount to 4 μL / cm, and uniformly and continuously spray it on the glass fiber filter membrane. After drying in a 37°C incubator, the binding pad is prepared.

[0071] (6) Preparation of sample pad

[0072] The sample pad is a whole blood filtration sample pad made of glass fiber cellulose membrane. It is prepared by soaking in a blocking solution for 5 minutes and drying in a 37°C incubator. The blocking solution contains 0.05% Tris, 1% Tween-20, 0.5% PEG6000, 0.4% bovine serum albumin, and 0.02% mouse anti-human red blood cells.

[0073] (7) Coating of detection line and quality control line on NC membrane

[0074] FSH and AMH monoclonal antibodies are coated on NC membrane 1 in parallel to form two detection lines: FSH detection line (9-1) and AMH detection line (9-2). LH monoclonal antibody and rabbit anti-chicken IgY polyclonal antibody are coated on NC membrane 2 to form LH detection line (9-3) and quality control line (10). The line width is 1 mm, and the spacing between adjacent detection lines and between detection lines and quality control line is 4 mm. The coating concentrations of FSH, AMH, LH, and chicken IgY monoclonal antibodies are 0.6 mg / mL, 1.0 mg / mL, and 0.6 mg / mL, respectively. The coating concentration of rabbit anti-chicken IgY polyclonal antibody is 0.8 mg / mL. Use the membrane cutting and gold spraying instrument to set the cutting amount to 1 μL / cm, and require the cutting line to be a uniform, continuous, and uninterrupted straight line. After drying in a 37°C incubator, the membrane is prepared.

[0075] (8) Preparation of test strip

[0076] The water-absorbing paper 1, nitrocellulose membrane 1, binding pad 1, sample pad, binding pad 2, nitrocellulose membrane 2, and water-absorbing paper 2 are sequentially adhered to the PVC base plate from top to bottom, cut according to a width of 3-5 mm, and installed into the corresponding card shell to prepare the test strip. The test strip and desiccant are sealed in an aluminum foil bag and dried.

[0077] Example 2

[0078] (1) Preparation of standard curve

[0079] Standard antigen linear dilution: the FSH and LH standard antigen were linearly diluted with negative bovine serum: 120 mIU / mL, 100 mIU / mL, 75 mIU / mL, 50 mIU / mL, 25 mIU / mL, 10 mIU / mL, 5 mIU / mL, 1 mIU / mL, 0 mIU / mL; the AMH standard antigen was linearly diluted with negative bovine serum: 15 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.25 ng / mL, 0.1 ng / mL, 0 ng / mL; the standard sample was added to the test paper sample well, the same batch of test paper and reagent was used, each concentration was repeated for more than 3 times, the fluorescence intensity data of the detection line and the quality control line were obtained by using the fluorescence immunoassay analyzer, the data were analyzed, the concentration of the standard was used as the horizontal coordinate, the fluorescence intensity ratio (T / C) of the detection line and the quality control line was used as the vertical coordinate, a standard curve was fitted, and the software program of the analyzer was written for quantitative detection of sample concentration.

[0080] (2) Sample detection

[0081] Prepare the sample: serum, plasma, whole blood sample or standard solution, if stored at low temperature, it needs to be restored to room temperature in advance;

[0082] Prepare the test paper: open the aluminum foil bag and take out the test paper, if stored at low temperature, it needs to be restored to room temperature in advance:

[0083] Detection: add the sample to the sample well of the test paper, and click "start" to detect.

[0084] Interpret the results: after 15 min, the fluorescence immunoassay analyzer automatically displays the numerical value and unit of the concentration of each detection index. According to the above sample dilution method, various samples were prepared for detection, and the results showed that the detection sensitivity of FSH was 1 mIU / mL, the maximum detection range was 120 mIU / mL; the detection sensitivity of LH was 1 mIU / mL, the maximum detection range was 100 mIU / mL; the detection sensitivity of AMH was 0.1 ng / mL, the maximum detection range was 15 ng / mL. The linear correlation coefficients R of the three detection indexes were all greater than 0.99.

[0085] (3) Verification of correlation with clinical chemical luminescence detection

[0086] 60 blood samples were randomly selected from the reproductive medicine or obstetrics and gynecology departments of 4 provincial hospitals in Guangdong Province, and the fluorescence immunoassay test strip for jointly quantitatively detecting multiple indexes of ovarian reserve function and the chemiluminescence method used in the hospital were compared. The results of the chemiluminescence method used in the hospital were taken as the abscissa, and the results of the fluorescence immunoassay test strip were taken as the ordinate, and a correlation curve was drawn. The results prove that: in the detection range of FSH, the correlation R2 of the two detection methods is 0.9808 (y=1.0142x+0.0855); in the detection range of AMH, the correlation R of the two detection methods is 0.987 (y=1.0337x-0.0972); in the detection range of LH, the correlation R2 of the two detection methods is 0.9771 (y=0.9465x+1.0887).

[0087] As can be seen from the above examples, the fluorescence immunoassay test strip provided by the present application integrates FSH, AMH and LH detection in the same detection test strip, is simple to operate and has low error; the correlation with the detection results of the clinical chemiluminescence method is very high, the detection results are reliable, and the accuracy is high.

[0088] The above only describes the preferred embodiments of the present application, and it should be pointed out that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A fluorescent immunoassay test strip for quantitative detection of multiple sex hormone indicators, the test strip comprising, in order, an absorbent paper 1, a nitrocellulose membrane 1, a binding pad 1, a sample pad, a binding pad 2, a nitrocellulose membrane 2, and an absorbent paper 2, which are sequentially connected to a PVC base plate: two detection lines are arranged in parallel on the nitrocellulose membrane 1 in order: the two detection lines are respectively coated with a second follicle-stimulating hormone monoclonal antibody and a second anti-Mullerian hormone monoclonal antibody; the second follicle-stimulating hormone monoclonal antibody and the second anti-Mullerian hormone monoclonal antibody coated on the two detection lines recognize different antigen epitopes; the binding pad 1 is sprayed with a fluorescent microsphere-labeled monoclonal antibody mixture: the fluorescent microsphere-labeled monoclonal antibody mixture includes a fluorescent microsphere-labeled first follicle-stimulating hormone monoclonal antibody and a fluorescent microsphere-labeled first anti-Mullerian hormone monoclonal antibody; the binding pad 2 is sprayed with a fluorescent microsphere-labeled antibody mixture: the fluorescent microsphere-labeled antibody mixture includes a fluorescent microsphere-labeled first luteinizing hormone monoclonal antibody and a fluorescent microsphere-labeled chicken IgY polyclonal antibody; one detection line and one quality control line are arranged in parallel on the nitrocellulose membrane in order: the one detection line is coated with a second luteinizing hormone monoclonal antibody.

2. The fluorescent immunostrip according to claim 1, characterized in that, The mass ratio of the first follicle-stimulating hormone monoclonal antibody, the first luteinizing hormone monoclonal antibody, the first anti-Mullerian hormone monoclonal antibody, and the chicken IgY polyclonal antibody to the fluorescent microspheres is independently (0.2-0.5):

1.

3. The fluorescent immunostrip of claim 2, wherein, The spraying concentration of the fluorescent microsphere-labeled antibody mixture on the binding pad is 2-6 μL / cm.

4. The fluorescent immunostrip according to claim 1 or 2, characterized in that, The sample pad is a whole blood sample pad.

5. The fluorescent immunostrip according to claim 1 or 2, characterized in that, The width of the detection line and the quality control line is independently 1-2 mm.

6. The fluorescent immunostrip of claim 5, wherein, The spacing between the two detection lines is independently 4-8 mm; the spacing between the detection line and the quality control line is 4-8 mm.

7. The fluorescent immunostrip of claim 1, wherein, The quality control line is sprayed with a rabbit anti-chicken IgY polyclonal antibody.

8. The fluorescent immunostrip of claim 1, wherein, The test strip further comprises a shell; the shell comprises a bottom clamping groove and a clamping shell.

9. The fluorescent immunostrip of claim 8, wherein, The shell is provided with a sample addition hole and two result detection windows.

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