CN110940817A

CN110940817A - Enzyme linked immunosorbent assay kit for detecting anti-mullerian hormone content and detection method thereof

Info

Publication number: CN110940817A
Application number: CN201911313451.XA
Authority: CN
Inventors: 邓陈玲; 华权高
Original assignee: CUSABIO BIOTECH Co Ltd
Current assignee: CUSABIO BIOTECH Co Ltd
Priority date: 2019-12-19
Filing date: 2019-12-19
Publication date: 2020-03-31
Anticipated expiration: 2039-12-19
Also published as: CN110940817B

Abstract

The invention provides a double-antibody sandwich enzyme-linked immunoassay kit for detecting the content of anti-mullerian hormone and a detection method thereof, wherein the kit comprises an enzyme label plate coated by a capture antibody, a sample diluent, a standard substance, a biotin-labeled detection antibody, horse radish peroxidase-labeled avidin, a concentrated washing solution, a substrate solution and a stop solution; wherein the capture antibody is a murine anti-human AMH antibody; the sample diluent was 1 XPBS + 1% BSA + 0.05% Tween-20+ 0.1% Proclin 300; the standard substance is a human AMH freeze-dried standard substance; the detection antibody is an anti-human AMH antibody; the concentrated wash was 25 XPBST containing 25 XPPBS + 1.25% Tween20+ 2.5% Proclin300, at pH 7.6; the substrate solution is a TMB chromogenic substrate solution; the stop solution is 2M sulfuric acid. The kit and the detection method thereof reduce the cost of manpower and equipment, reduce the usage amount of raw materials and samples, and reduce the cost, thereby improving the use efficiency.

Description

Enzyme linked immunosorbent assay kit for detecting anti-mullerian hormone content and detection method thereof

Technical Field

The invention belongs to the field of biological detection, and particularly relates to an enzyme linked immunosorbent assay kit for detecting the anti-mullerian hormone content and a detection method thereof.

Background

The Anti-mullerian hormone is also called mullerian-inhibiting factor (Anti-mueller-hormone AMH), belongs to a member of the super-family of transforming growth factor β (TGF- β), and is a polypeptide hormone connected by disulfide bonds.A human AMH gene is positioned in the short arm of chromosome 19, contains 5 exons, codes a protein precursor of 560 amino acids, the carboxyl end of the protein precursor is an active end, is formed by connecting disulfide bonds, and is a dimeric glycoprotein with the molecular weight of 140 kDa.AMH is a homologous dimeric glycoprotein formed by connecting two monomers with the molecular weight of 70kDa by disulfide bonds, and the activation of AMH needs to hydrolyze and release a carboxyl terminal dimer so as to have the biological activity of promoting the degradation of mullerian tube and can inhibit the growth and differentiation of certain tumors.

AMH inhibits granulosa cell proliferation and follicle maturation, decreases the sensitivity of the follicle to Follicle Stimulating Hormone (FSH), inhibits follicle growth and selection of dominant follicles, thereby slowing down the consumption of the follicular population, so that excess or deficiency of AMH will result in disordered serum in follicular dysplasia and reproductive function.

Polycystic ovarian syndrome is a common endocrine and metabolic disorder disease of women in the reproductive age, the domestic incidence rate is 5% -10%, the polycystic ovarian syndrome is the most common cause of anovulatory infertility, and the serum AMH level can accurately reflect the number of antral follicles, can be used as a tool for diagnosing PCOS and evaluating the curative effect of PCOS, and can be used as a substitute for B-ultrasonic diagnosis of PCOS.

At present, the quantitative analysis method of serum AMH comprises an enzyme-linked immunosorbent assay (ELISA), a chemiluminescence method, an electrochemiluminescence method, a colloidal gold method, a fluorescence immunochromatography method, a latex enhanced turbidimetry method and the like. The chemiluminescence method and the electrochemical luminescence method have higher sensitivity but require expensive special equipment, and are not suitable for small-batch detection or common laboratories; the colloidal gold method has simple operation, short reaction time and convenient use, but is not suitable for accurate quantitative analysis; when the colloidal gold method and the fluorescence immunochromatography method are combined for use, the requirements on the test strip are more, and the treatment process is troublesome; the latex enhanced immunoturbidimetry has simple operation, rapid reaction and accurate quantification, can be used for clinic, but has poor low-value repeatability, needs a special biochemical instrument and is expensive.

The enzyme-linked immunosorbent assay adopts a 96 micro-porous plate as a solid phase carrier to coat an antibody, and can realize quantitative detection by utilizing antigen-antibody immunoreaction; the method is widely applied due to the advantages of good specificity, high sensitivity, accurate quantification, low requirement on experimental equipment and the like, but has the defects of long reaction time, complicated steps, poor repeatability and the like.

A double-antibody sandwich incubation three-step method is mostly adopted in a quantitative ELISA kit on the market, namely, a coating capture antibody is fixed in a 96-hole micropore plate to prepare a solid phase carrier, a standard substance or a sample to be detected is respectively added into micropores and incubated for 90min at 37 ℃, a target antigen is combined with the capture antibody connected on the solid phase carrier, the solid phase carrier is dried by beating after being washed for 3 times, a biotinylated detection antibody is added to be incubated for 60min at 37 ℃, the solid phase carrier is dried by beating after being washed for 3 times, then HRP-labeled avidin is added to be incubated for 30min at 37 ℃, TMB substrate is added to be thoroughly washed for 5 times and is developed for 15 min. TMB is converted to blue by the catalysis of peroxidase and to the final yellow by the action of an acid. The shade of the color is positively correlated with the antigen in the sample. And (4) measuring the absorbance (OD value) by using a microplate reader at the wavelength of 450nm, and calculating the content of the sample to be detected.

Disclosure of Invention

In view of the above problems, the invention provides a double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content and a detection method thereof, and the invention has the advantages that a double-antibody sandwich room-temperature oscillation two-step method is adopted, wherein the first step is to add a standard substance/sample + biotin-labeled detection antibody (60-120min), and the second step is to add horseradish peroxidase (30-45 min). The prior art is a three-step method: the first step is to add standard/sample (90min), the second step is to add biotin antibody (60min), and the third step is to add horseradish peroxidase (30 min). The reaction steps are two steps, the sample loading amount of the standard substance/sample to be detected and the detection antibody is 50uL, the reaction time is shortened to 2.5h, the labor and equipment cost is reduced, the use amount of raw materials and samples is reduced, the cost is reduced, and the use efficiency is improved.

The technical scheme for realizing the purpose is as follows:

the invention provides a double-anti sandwich enzyme-linked immunosorbent assay kit for detecting the content of anti-mullerian hormone, which comprises an enzyme label plate coated by a capture antibody, sample diluent, a standard substance, a biotin-labeled detection antibody, horse radish peroxidase-labeled avidin, a concentrated washing solution, a substrate solution and a stop solution;

wherein the capture antibody is a murine anti-human AMH antibody;

the sample diluent was 1 XPBS + 1% BSA + 0.05% Tween-20+ 0.1% Proclin 300;

the standard substance is a human AMH freeze-dried standard substance;

the detection antibody is an anti-human AMH antibody;

the concentrated wash was 25 XPBST containing 25 XPPBS + 1.25% Tween20+ 2.5% Proclin300, at pH 7.6;

the substrate solution is a TMB chromogenic substrate solution;

the stop solution is 2M sulfuric acid.

In one embodiment, in the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content, the preparation method of the ELISA plate coated by the capture antibody comprises the following steps:

(1) diluting a mouse anti-human AMH antibody to 1-4 ug/mL by using a buffer solution, and then coating an enzyme label plate; wherein the buffer solution is selected from a carbonate buffer solution with the pH of 9.6 and/or a phosphate buffer solution with the pH of 7.2-7.4;

wherein the preparation steps of the carbonate buffer solution are as follows: 2.352g of NaHCO₃And 2.332g Na₂CO₃Mixing, adding water to 1L; the preparation steps of the phosphate buffer solution are as follows: mixing 8g NaCl, 0.2g KCl, 1.44g Na₂HPO₄And 0.44g KH₂PO₄Mixing, adding water to 1L;

(2) placing the enzyme label plate treated in the step (1) at 4 ℃ overnight or placing the enzyme label plate in a thermostat at 37 ℃ for 2h and then at 4 ℃ overnight;

(3) adding sealing liquid into each hole for sealing after drying, then placing in a constant temperature box at 37 ℃ for 2h, drying again, and then adding 10% sucrose solution into each hole for fixing for 0.5 h; wherein the confining liquid is selected from 1-5% of bovine serum albumin, 2-5% of skimmed milk powder and/or 5-10% of calf serum.

(1) diluting a mouse anti-human AMH antibody to 2ug/mL by using a carbonate buffer solution with the pH value of 9.6, and coating an enzyme label plate, wherein each hole is 100 ug/mL;

(2) placing the enzyme label plate treated in the step (1) at 4 ℃ overnight;

(3) patting to dry, adding 2% bovine serum albumin blocking solution into each hole for blocking, then placing in a 37 ℃ constant temperature box for 2h, patting to dry again, and then adding 10% sucrose solution into each hole for fixing for 0.5 h.

In one embodiment, in the double-antibody sandwich enzyme-linked immunoassay kit for detecting the anti-mullerian hormone content, the preparation steps of 25 × PBS in the concentrated washing solution are as follows: 200g of NaCL, 5g of KCL and 36g of Na₂HPO₄And 11g KH₂PO₄Mixing, adding water to 1L, and adjusting the pH to 6.0-6.5.

In one embodiment, in the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content, the concentration of the biotin-labeled detection antibody is 0.1-0.4 ug/mL.

In one embodiment, in the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the content of the anti-mullerian hormone, the concentration of the biotin-labeled detection antibody is 0.2 ug/mL.

In one embodiment, in the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content, the concentration of the horse radish peroxidase-labeled avidin is 0.125-0.5 ug/mL.

In one embodiment, in the double-antibody sandwich enzyme-linked immunosorbent kit for detecting the anti-mullerian hormone content, the concentration of the horse radish peroxidase-labeled avidin is 0.25 ug/mL.

The invention also provides a detection method of the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content, which is characterized by comprising the following steps of:

(1) preparing a sample treatment solution to be detected, wherein the sample to be detected is selected from a serum sample, a plasma sample, a cell culture supernatant sample, a urine sample or a tissue lysate sample;

(2) preparing a standard solution, a biotin-labeled detection antibody diluent, a horseradish peroxidase-labeled avidin diluent and a concentrated washing solution working solution;

(3) balancing the sample treatment solution to be detected, the standard solution, the biotin-labeled detection antibody diluent, the horseradish peroxidase-labeled avidin diluent, the concentrated washing solution working solution, the TMB chromogenic substrate solution and the 2M sulfuric acid at 18-25 ℃ for 15-30 min;

(4) taking the ELISA plate coated by the capture antibody, respectively arranging a standard substance hole and a sample hole to be detected, adding 50uL of the standard substance solution to each standard substance hole, and adding 50uL of the sample treatment solution to be detected to each sample hole to be detected; then adding 50uL of biotin-labeled detection antibody diluent into each hole, uniformly mixing, and oscillating for 60-120min at 18-25 ℃;

(5) discarding liquid in each hole, spin-drying, adding 200uL of the concentrated washing liquid working solution into each hole, keeping for 1-2 min, repeating for 3 times, and spin-drying;

(6) adding 100uL of the horseradish peroxidase-labeled avidin diluent into each hole, and oscillating for 30-45min at 18-25 ℃;

(7) discarding liquid in each hole, spin-drying, adding 200uL of the concentrated washing liquid working solution into each hole, keeping for 1-2 min, repeating for 5 times, and spin-drying;

(8) adding 50-100 uL of the TMB chromogenic substrate solution into each hole, and developing in a dark place at 18-25 ℃ for 15-30 min;

(9) adding 50-100 uL of the 2M sulfuric acid into each hole, and stopping the reaction;

(10) and (4) measuring the optical density value of the elisa plate processed in the step (9) at the wavelength of 450nm by using an elisa reader.

In one embodiment, in the detection method of the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content, the preparation method of the sample treatment solution to be detected in the step (1) comprises the following steps:

standing the serum sample at room temperature for 2 hours or standing overnight at 4 ℃, centrifuging at 2-8 ℃ for 15min at 1000Xg, and taking supernatant for detection;

or, centrifuging the collected plasma sample at 2-8 ℃ at 1000Xg for 15min, and taking the supernatant for detection;

or collecting the cell culture supernatant sample or urine sample, centrifuging at 2-8 ℃ for 15min at 1000Xg, and taking the supernatant for detection;

or, 100mg of the tissue lysate sample is taken, washed by 1 XPBS, then added into 1mL of 1 XPBS to prepare homogenate, then placed at-20 ℃ overnight, repeatedly frozen and thawed for 2 times, then centrifuged for 5min at 2-8 ℃ at 5000Xg, and the supernatant is taken for detection.

In one embodiment, in the detection method of the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content, the supernatant for detection obtained in the process of preparing the sample treatment solution to be detected is stored at-20 ℃ or-80 ℃ for detection.

In one embodiment, in the detection method of the double-antibody sandwich enzyme-linked immunoassay kit for detecting the anti-mullerian hormone content, the preparation method of the standard solution in the step (2) comprises the following steps: centrifuging 4ng of an AMH freeze-dried standard substance at 6000-10000rpm for 30-60 seconds, and dissolving with 1mL of the sample diluent to obtain a standard substance solution S7, wherein the concentration of the standard substance solution S7 is 4000 pg/mL; adding 250uL of sample diluent into 7 1.5mL centrifuge tubes respectively, and then sucking 250uL of standard solution S7 into a centrifuge tube S6 to obtain standard solution S6, wherein the concentration of the standard solution S6 is 2000 pg/mL; then sucking 250uL of the standard solution S6 into a centrifuge tube S5 to obtain a standard solution S5, wherein the concentration of the standard solution S5 is 1000 pg/mL; then sucking 250uL of the standard solution S5 into a centrifuge tube S4 to obtain a standard solution S4, wherein the concentration of the standard solution S4 is 500 pg/mL; then sucking 250uL of the standard solution S4 into a centrifuge tube S3 to obtain a standard solution S3, wherein the concentration of the standard solution S3 is 250 pg/mL; then sucking 250uL of the standard solution S3 into a centrifuge tube S2 to obtain a standard solution S2, wherein the concentration of the standard solution S2 is 125 pg/mL; then sucking 250uL of the standard solution S2 into a centrifuge tube S1 to obtain a standard solution S1, wherein the concentration of the standard solution S1 is 62.5 pg/mL; the standard solution S0 was 250uL of the sample dilution, and the concentration of the standard solution S1 was 0pg/mL, as shown in table 1:

table 1: the concentration of the standard solution S0-S7

Numbering	S7	S6	S5	S4	S3	S2	S1	S0
									pg/mL	4000	2000	1000	500	250	125	62.5	0

In one embodiment, in the detection method of the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content, the preparation step of the biotin-labeled detection antibody diluent in the step (2) is as follows: diluting the biotin-labeled detection antibody with the sample diluent to obtain a biotin-labeled detection antibody diluent; wherein the volume ratio of the biotin-labeled detection antibody to the biotin-labeled detection antibody diluent is 1: 1000.

In one embodiment, in the detection method of the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content, the preparation step of the horse radish peroxidase-labeled avidin diluent in the step (2) is as follows: diluting the horse radish peroxidase-labeled avidin with the sample diluent to obtain horse radish peroxidase-labeled avidin diluent; wherein the volume ratio of the horseradish peroxidase-labeled avidin to the horseradish peroxidase-labeled avidin diluent is 1: 4000.

In one embodiment, in the detection method of the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content, the preparation step of the working solution of the concentrated washing solution in the step (2) is as follows: diluting the concentrated washing solution with deionized water to obtain a concentrated washing solution working solution; wherein the volume ratio of the concentrated washing liquid to the concentrated washing liquid working solution is 1: 25.

The invention also provides a double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content and application of a detection method of the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content in detection of the anti-mullerian hormone content.

The double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content and the detection method thereof have the beneficial effects that: in the prior art, the kit mostly adopts a double-antibody sandwich incubation three-step method, the reaction time is 3.5h, the reaction steps are three steps, the sample loading amount of a standard substance/sample to be detected and a detection antibody is 100uL, and the experimental equipment is a 37 ℃ constant temperature incubator; the method has the advantages that a double-antibody sandwich room-temperature oscillation two-step method is adopted, wherein the reaction steps are two steps, the sample loading amount of a standard substance/sample to be detected and a detection antibody is 50uL, the reaction time is shortened to 2.5h, the labor and equipment cost is reduced, the raw material and sample usage amount is reduced, the cost is reduced, and the use efficiency is improved as shown in Table 2:

table 2: compared with the prior art, the double-resistance sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content

	The invention	Prior Art
			Reaction time	2.5h	3.5h
Reaction step	Two-step process	Three-step method
			Reaction temperature	At room temperature	37℃
Using instruments	Microplate oscillator	37 ℃ thermostat
			Sample loading of standard/sample to be tested	50uL	100uL
Detecting the amount of antibody loaded	50uL	100uL

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a standard curve diagram of the double antibody sandwich ELISA method for detecting anti-Mullerian hormone content according to the present invention in example 12;

FIG. 2 is a standard curve diagram of the room temperature shaking two-step method according to the present invention in example 13;

FIG. 3 is a standard three-step incubation graph of the prior art in example 13.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagent materials used in the following examples are all commercially available products unless otherwise specified.

Example 1: the invention relates to a double-antibody sandwich enzyme-linked immunoassay kit for detecting the anti-mullerian hormone content

The invention relates to a double-anti sandwich enzyme-linked immunosorbent assay kit for detecting the content of anti-mullerian hormone, which comprises an enzyme label plate coated by a capture antibody, a sample diluent, a standard substance, a biotin-labeled detection antibody, horseradish peroxidase-labeled avidin, a concentrated washing solution, a substrate solution and a stop solution;

wherein the capture antibody is a murine anti-human AMH antibody; the sample diluent was 1 XPBS + 1% BSA + 0.05% Tween-20+ 0.1% Proclin 300; the standard substance is a human AMH freeze-dried standard substance; the detection antibody is an anti-human AMH antibody; the concentrated wash was 25 XPBST containing 25 XPPBS + 1.25% Tween20+ 2.5% Proclin300, at pH 7.6; the substrate solution is a TMB chromogenic substrate solution; the stop solution is 2M sulfuric acid; the preparation steps of the 25 XPBS are as follows: 200g of NaCL, 5g of KCL and 36g of Na₂HPO₄And 11g KH₂PO₄Mixing, adding water to 1L, and adjusting pH to 6.0; wherein the concentration of the biotin-labeled detection antibody is 0.1 ug/mL; the concentration of the horseradish peroxidase-labeled avidin is 0.125 ug/mL.

Example 2: the invention relates to a double-resistance sandwich enzyme-linked immunosorbent assay for detecting the anti-mullerian hormone contentEpidemic disease reagent kit

wherein the capture antibody is a murine anti-human AMH antibody; the sample diluent was 1 XPBS + 1% BSA + 0.05% Tween-20+ 0.1% Proclin 300; the standard substance is a human AMH freeze-dried standard substance; the detection antibody is an anti-human AMH antibody; the concentrated wash was 25 XPBST containing 25 XPPBS + 1.25% Tween20+ 2.5% Proclin300, at pH 7.6; the substrate solution is a TMB chromogenic substrate solution; the stop solution is 2M sulfuric acid; the preparation steps of the 25 XPBS are as follows: 200g of NaCL, 5g of KCL and 36g of Na₂HPO₄And 11g KH₂PO₄Mixing, adding water to 1L, and adjusting pH to 6.5; wherein the concentration of the biotin-labeled detection antibody is 0.4 ug/mL; the concentration of the horseradish peroxidase-labeled avidin is 0.5 ug/mL.

Example 3: the invention relates to a double-antibody sandwich enzyme-linked immunoassay kit for detecting the anti-mullerian hormone content

wherein the capture antibody is a murine anti-human AMH antibody; the sample diluent was 1 XPBS + 1% BSA + 0.05% Tween-20+ 0.1% Proclin 300; the standard substance is a human AMH freeze-dried standard substance; the detection antibody is an anti-human AMH antibody; the concentrated wash was 25 XPBST containing 25 XPPBS + 1.25% Tween20+ 2.5% Proclin300, at pH 7.6; the substrate solution is a TMB chromogenic substrate solution; the stop solution is 2M sulfuric acid; the preparation steps of the 25 XPBS are as follows: 200g of NaCL, 5g of KCL and 36g of Na₂HPO₄And 11g KH₂PO₄Mixing, adding water to 1L, and adjusting pH to 6.5; wherein the concentration of the biotin-labeled detection antibody is 0.2 ug/mL; the concentration of the horseradish peroxidase-labeled avidin is 0.25 ug/mL.

Example 4: the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content Preparation method of ELISA plate

(1) Diluting a mouse anti-human AMH antibody to 1ug/mL by using a carbonate buffer solution with the pH value of 9.6, and then coating an enzyme label plate; wherein the preparation steps of the carbonate buffer solution are as follows: 2.352g of NaHCO₃And 2.332g Na₂CO₃Mixing, adding water to 1L; (2) placing the enzyme label plate treated in the step (1) at 4 ℃ overnight; (3) patting to dry, adding 1% bovine serum albumin blocking solution into each hole for blocking, then placing in a 37 ℃ constant temperature box for 2h, patting to dry, and then adding 10% sucrose solution into each hole for fixing for 0.5 h.

Example 5: the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content Preparation method of ELISA plate

(1) Diluting a mouse anti-human AMH antibody to 4ug/mL by using a phosphate buffer solution with the pH of 7.2-7.4, and then coating an enzyme label plate; wherein the preparation steps of the phosphate buffer solution are as follows: mixing 8g NaCl, 0.2g KCl, 1.44g Na₂HPO₄And 0.44gKH₂PO₄Mixing, adding water to 1L; (2) placing the enzyme label plate treated in the step (1) in a constant temperature box at 37 ℃ for 2h, and then standing overnight at 4 ℃; (3) patting to dry, adding 5% bovine serum albumin blocking solution into each hole for blocking, then placing in a 37 ℃ constant temperature box for 2h, patting to dry, and then adding 10% sucrose solution into each hole for fixing for 0.5 h.

Example 6: the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content Preparation method of ELISA plate

(1) Diluting a mouse anti-human AMH antibody to 2ug/mL by using a carbonate buffer solution with the pH value of 9.6, and then coating an enzyme label plate; wherein the preparation steps of the carbonate buffer solution are as follows: 2.352g of NaHCO₃And 2.332g Na₂CO₃Mixing, adding water to 1L; (2) will be provided withPlacing the enzyme label plate treated in the step (1) at 4 ℃ overnight; (3) patting to dry, adding 2% bovine serum albumin blocking solution into each hole for blocking, then placing in a 37 ℃ constant temperature box for 2h, patting to dry again, and then adding 10% sucrose solution into each hole for fixing for 0.5 h.

Example 7: the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content Preparation method of ELISA plate

(1) Diluting a mouse anti-human AMH antibody to 4ug/mL by using a carbonate buffer solution with the pH value of 9.6, and then coating an enzyme label plate; wherein the preparation steps of the carbonate buffer solution are as follows: 2.352g of NaHCO₃And 2.332g Na₂CO₃Mixing, adding water to 1L; (2) placing the enzyme label plate treated in the step (1) at 4 ℃ overnight; (3) and (3) patting to dry, adding 2-5% of skimmed milk powder sealing liquid into each hole for sealing, then placing in a 37 ℃ thermostat for 2h, patting to dry, and then adding 10% of sucrose solution into each hole for fixing for 0.5 h.

Example 8: the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content Preparation method of ELISA plate

(1) Diluting a mouse anti-human AMH antibody to 4ug/mL by using a carbonate buffer solution with the pH value of 9.6, and then coating an enzyme label plate; wherein the preparation steps of the carbonate buffer solution are as follows: 2.352g of NaHCO₃And 2.332g Na₂CO₃Mixing, adding water to 1L; (2) placing the enzyme label plate treated in the step (1) at 4 ℃ overnight; (3) and (3) drying, adding 5-10% of calf serum confining liquid into each hole for sealing, then placing in a 37 ℃ constant temperature box for 2h, drying again, and then adding 10% of sucrose solution into each hole for fixing for 0.5 h.

Example 9: the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content is used Line detection

(1) Preparing a sample treatment solution to be detected:

standing the serum sample at room temperature for 2 hours or standing overnight at 4 ℃, centrifuging at 2 ℃ for 15min at 1000Xg, and taking supernatant as sample treatment liquid to be detected for detection; or subpackaging the supernatant, and then storing at-20 ℃ for detection; repeated freeze thawing should be avoided, and the thawed sample treatment solution to be detected should be centrifuged again and then used for detection;

or, after collecting the plasma sample, centrifuging the plasma sample at 2 ℃ at 1000xg for 15min, and taking the supernatant as a sample treatment fluid to be detected for detection; or subpackaging the supernatant, and then storing at-20 ℃ for detection; repeated freeze thawing should be avoided, and the thawed sample treatment solution to be detected should be centrifuged again and then used for detection;

or collecting the cell culture supernatant sample or urine sample, centrifuging at 2 ℃ for 15min at 1000Xg, and taking the supernatant as a sample treatment solution to be detected for detection; or subpackaging the supernatant, and then storing at-20 ℃ for detection; repeated freezing and thawing should be avoided; wherein the supernatant of the urine sample to be detected is centrifuged again before detection so as to remove the possible sediment of the supernatant of the sample during the storage period;

or, 100mg of the tissue lysate sample is taken, washed by 1 XPBS, cut into small pieces and placed into a tissue grinder (homogenate tube), then the small pieces are added into 1mL of 1 XPBS to prepare homogenate, then the homogenate is placed at minus 20 ℃ for overnight, then the repeated freeze thawing is carried out for 2 times to destroy cell membranes, then the centrifugation is carried out for 5min at 2 ℃ and 5000Xg, and the supernatant is taken for detection; or subpackaging the supernatant, then storing at-20 ℃ for detection, and centrifuging the thawed sample treatment fluid to be detected again;

it should be noted that hemolysis of the sample can affect the final test result, and therefore, the hemolyzed sample is not suitable for the test.

the preparation method of the standard solution comprises the following steps: centrifuging 4ng of a human AMH freeze-dried standard substance at 6000rpm for 30 seconds, and then dissolving with 1mL of the sample diluent to obtain a standard substance solution S7, wherein the concentration of the standard substance solution S7 is 4000 pg/mL; adding 250uL of sample diluent into 7 1.5mL centrifuge tubes respectively, and then sucking 250uL of standard solution S7 into a centrifuge tube S6 to obtain standard solution S6, wherein the concentration of the standard solution S6 is 2000 pg/mL; then sucking 250uL of the standard solution S6 into a centrifuge tube S5 to obtain a standard solution S5, wherein the concentration of the standard solution S5 is 1000 pg/mL; then sucking 250uL of the standard solution S5 into a centrifuge tube S4 to obtain a standard solution S4, wherein the concentration of the standard solution S4 is 500 pg/mL; then sucking 250uL of the standard solution S4 into a centrifuge tube S3 to obtain a standard solution S3, wherein the concentration of the standard solution S3 is 250 pg/mL; then sucking 250uL of the standard solution S3 into a centrifuge tube S2 to obtain a standard solution S2, wherein the concentration of the standard solution S2 is 125 pg/mL; then sucking 250uL of the standard solution S2 into a centrifuge tube S1 to obtain a standard solution S1, wherein the concentration of the standard solution S1 is 62.5 pg/mL; the standard solution S0 is 250uL of the sample diluent, and the concentration of the standard solution S1 is 0 pg/mL;

the preparation method of the biotin-labeled detection antibody diluent comprises the following steps: diluting the biotin-labeled detection antibody with the sample diluent to obtain a biotin-labeled detection antibody diluent; wherein the volume ratio of the biotin-labeled detection antibody to the biotin-labeled detection antibody diluent is 1: 1000;

the preparation method of the horseradish peroxidase-labeled avidin diluent comprises the following steps: diluting the horse radish peroxidase-labeled avidin with the sample diluent to obtain horse radish peroxidase-labeled avidin diluent; wherein the volume ratio of the horse radish peroxidase-labeled avidin to the horse radish peroxidase-labeled avidin diluent is 1: 4000;

the preparation steps of the concentrated washing liquid working solution are as follows: diluting the concentrated washing solution with deionized water to obtain a concentrated washing solution working solution; wherein the volume ratio of the concentrated washing liquid to the concentrated washing liquid working solution is 1: 25;

(3) balancing the sample treatment solution to be detected, the standard solution, the biotin-labeled detection antibody diluent, the horseradish peroxidase-labeled avidin diluent, the concentrated washing solution working solution, the TMB chromogenic substrate solution and the 2M sulfuric acid at 18 ℃ for 15 min;

(4) taking the ELISA plate coated by the capture antibody, respectively arranging a standard substance hole and a sample hole to be detected, adding 50uL of the standard substance solution to each standard substance hole, and adding 50uL of the sample treatment solution to be detected to each sample hole to be detected; then adding 50uL of the biotin-labeled detection antibody diluent into each hole, slightly shaking and uniformly mixing, covering with a new plate, and oscillating for 90min at 18-25 ℃ by using a microplate oscillator;

(5) discarding the liquid in each hole, spin-drying, adding 200uL of the concentrated washing liquid working solution into each hole, keeping for 1min, repeating for 3 times, and spin-drying;

(6) adding 100uL of the horse radish peroxidase-labeled avidin dilution into each well, covering with a new plate patch, and oscillating for 30min at 18 ℃;

(7) discarding the liquid in each hole, spin-drying, adding 200uL of the concentrated washing liquid working solution into each hole, keeping for 1min, repeating for 5 times, and spin-drying;

(8) adding 90uL of the TMB chromogenic substrate solution into each hole, and developing for 15min in the dark at 18 ℃;

(9) adding 50uL of the 2M sulfuric acid to each well, and terminating the reaction;

(10) measuring the optical density value (OD) of the ELISA plate treated in the step (9) at the wavelength of 450nm by using an ELISA reader₄₅₀Value).

Example 10: the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content is used Line detection

(1) Preparing a sample treatment solution to be detected:

standing the serum sample at room temperature for 2 hours or standing overnight at 4 ℃, centrifuging at 8 ℃ for 15min at 1000Xg, and taking supernatant as sample treatment liquid to be detected for detection; or subpackaging the supernatant, and then storing at-80 ℃ for detection; repeated freeze thawing should be avoided, and the thawed sample treatment solution to be detected should be centrifuged again and then used for detection;

or, centrifuging the plasma sample at 1000xg at 8 ℃ for 15min after collecting the plasma sample, and taking the supernatant as a sample treatment fluid to be detected for detection; or subpackaging the supernatant, and then storing at-80 ℃ for detection; repeated freeze thawing should be avoided, and the thawed sample treatment solution to be detected should be centrifuged again and then used for detection;

or collecting the cell culture supernatant sample or urine sample, centrifuging at 8 ℃ for 15min at 1000Xg, and taking the supernatant as a sample treatment solution to be detected for detection; or subpackaging the supernatant, and then storing at-80 ℃ for detection; repeated freezing and thawing should be avoided; wherein the supernatant of the urine sample to be detected is centrifuged again before detection so as to remove the possible sediment of the supernatant of the sample during the storage period;

or, 100mg of the tissue lysate sample is taken, washed by 1 XPBS, cut into small pieces and placed into a tissue grinder (homogenate tube), then the small pieces are added into 1mL of 1 XPBS to prepare homogenate, then the homogenate is placed at minus 20 ℃ for overnight, then the repeated freeze thawing is carried out for 2 times to destroy cell membranes, then the centrifugation is carried out for 5min at 8 ℃ and 5000Xg, and the supernatant is taken for detection; or subpackaging the supernatant, then storing at-80 ℃ for detection, and centrifuging the thawed sample treatment fluid to be detected again;

the preparation method of the standard solution comprises the following steps: centrifuging 4ng of a human AMH freeze-dried standard substance at 10000rpm for 60 seconds, and dissolving the standard substance with 1mL of the sample diluent to obtain a standard substance solution S7, wherein the concentration of the standard substance solution S7 is 4000 pg/mL; adding 250uL of sample diluent into 7 1.5mL centrifuge tubes respectively, and then sucking 250uL of standard solution S7 into a centrifuge tube S6 to obtain standard solution S6, wherein the concentration of the standard solution S6 is 2000 pg/mL; then sucking 250uL of the standard solution S6 into a centrifuge tube S5 to obtain a standard solution S5, wherein the concentration of the standard solution S5 is 1000 pg/mL; then sucking 250uL of the standard solution S5 into a centrifuge tube S4 to obtain a standard solution S4, wherein the concentration of the standard solution S4 is 500 pg/mL; then sucking 250uL of the standard solution S4 into a centrifuge tube S3 to obtain a standard solution S3, wherein the concentration of the standard solution S3 is 250 pg/mL; then sucking 250uL of the standard solution S3 into a centrifuge tube S2 to obtain a standard solution S2, wherein the concentration of the standard solution S2 is 125 pg/mL; then sucking 250uL of the standard solution S2 into a centrifuge tube S1 to obtain a standard solution S1, wherein the concentration of the standard solution S1 is 62.5 pg/mL; the standard solution S0 is 250uL of the sample diluent, and the concentration of the standard solution S1 is 0 pg/mL;

(3) balancing the sample treatment solution to be detected, the standard solution, the biotin-labeled detection antibody diluent, the horseradish peroxidase-labeled avidin diluent, the concentrated washing solution working solution, the TMB chromogenic substrate solution and the 2M sulfuric acid at 25 ℃ for 30 min;

(4) taking the ELISA plate coated by the capture antibody, respectively arranging a standard substance hole and a sample hole to be detected, adding 50uL of the standard substance solution to each standard substance hole, and adding 50uL of the sample treatment solution to be detected to each sample hole to be detected; then adding 50uL of the biotin-labeled detection antibody diluent into each hole, slightly shaking and uniformly mixing, covering with a new plate, and oscillating for 120min at 25 ℃ by using a microplate oscillator;

(5) discarding the liquid in each hole, spin-drying, adding 200uL of the concentrated washing liquid working solution into each hole, keeping for 2min, repeating for 3 times, and spin-drying;

(6) adding 100uL of the horse radish peroxidase-labeled avidin diluent into each well, covering with a new plate patch, and oscillating for 45min at 25 ℃;

(7) discarding the liquid in each hole, spin-drying, adding 200uL of the concentrated washing liquid working solution into each hole, keeping for 2min, repeating for 5 times, and spin-drying;

(8) adding 100uL of the TMB chromogenic substrate solution into each hole, and carrying out dark color development for 30min at 25 ℃;

(9) adding 100uL of the 2M sulfuric acid to each well, and terminating the reaction;

Example 11: determining optimal detection conditions

(1) Optimal conditions for screening coated capture antibodies

Respectively coating the capture antibody with a carbonate buffer solution with the pH of 9.6 and a phosphate buffer solution with the pH of 7.2-7.4 to prepare an ELISA plate, directly placing the ELISA plate at 4 ℃ overnight or placing the ELISA plate in a thermostat at 37 ℃ for 2h and then at 4 ℃ overnight, and arranging 3 multiple holes according to the OD of the S7/S0 hole₄₅₀The highest value was the best buffer, and the results are shown in Table 3:

table 3:

and (4) conclusion: optimal coating conditions included the use of carbonate buffer at pH 9.6 overnight at 4 ℃.

(2) Screening for optimal blocking conditions

Respectively adding 2% of bovine serum albumin, 5% of skim milk powder or 10% of calf serum confining liquid into each hole of the ELISA plate, wherein 3 holes are formed, then placing the ELISA plate in a 37 ℃ thermostat for 2 hours, and after drying, adding 10% of sucrose solution into each hole for fixing for 0.5 hour; OD according to S7/S0 well₄₅₀The highest value is the best sealing condition, and the results are shown in Table 4:

table 4: contrast of occlusion effect

Closed condition(s)	2% bovine serum albumin	5% defatted milk powder	10% Calf serum
				S7	2.653	2.464	2.653
S0	0.125	0.141	0.125
				S7/S0	21.224	17.475	21.224

And (4) conclusion: as can be seen from Table 4, both calf serum and bovine serum albumin can achieve good blocking effect, but the influence of the batch on the calf serum is larger than that of the bovine serum albumin, which is not as stable as the bovine serum albumin, so 2% of the bovine serum albumin is preferably selected to prepare the blocking solution of the present invention.

(3) Screening for optimal concentration of coating antibody and optimal concentration of Biotin-labeled detection antibody

Under the condition that the standard substance and the use condition of horse radish peroxidase labeled avidin (Streptavidin-HRP) are fixed, different coating antibody concentrations and biotin labeled detection antibody concentrations are respectively set by adopting a chessboard titration method, and the OD is determined according to S7/S0 holes₄₅₀The highest value was the optimal coating antibody concentration and biotin-labeled detection antibody concentration, and the results are shown in table 5:

table 5: comparison of the Effect of coating antibody concentration and Biotin-labeled detection antibody concentration

And (4) conclusion: as can be seen from Table 5, the optimal conditions of the present invention are to select the concentration of the coated antibody to be 2ug/mL, and to select the concentration of the biotin-labeled detection antibody to be 0.2 ug/mL.

(4) Screening for optimal concentration of Standard solution S7 and optimal concentration of horse radish peroxidase-labeled avidin

Under the condition that the concentration of the coated antibody and the concentration of a biotin-labeled detection antibody are fixed, a chessboard titration method is adopted to respectively set a standard solution S7 with different concentrations and horse radish peroxidase-labeled avidin (Streptavidin-HRP), and the standard solution S7 with the lowest concentration and OD (OD) is selected₄₅₀The value is more than or equal to 2.5 and the OD of S0 hole₄₅₀The optimum value is 0.15 or less, and the results are shown in Table 6:

table 6: comparison of the effects of the concentration of S7 in the Standard solution and the concentration of horse radish peroxidase-labeled avidin

And (4) conclusion: as can be seen from Table 6, the concentration range of the standard solution S7 of the present invention can be 1-10 ng/mL, and is optimally 4ng/mL, that is, 4ng of the human AMH lyophilized standard is diluted with 1mL of the sample diluent.

Example 12: experimental parameter verification

According to the experimental conditions, the method standard of the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content is established, wherein the verification experimental parameters are as follows:

(1) establishment of a Standard Curve

Inventive Standard Curve OD₄₅₀The value results are shown in table 7:

table 7:

standard curve (pg/mL)	OD₄₅₀ 1	OD₄₅₀ 2	Average OD₄₅₀
				4000	2.617	2.641	2.629
2000	1.893	1.891	1.892
				1000	1.291	1.256	1.274
500	0.897	0.901	0.899
				250	0.599	0.612	0.606
125	0.408	0.407	0.408
				62.5	0.265	0.258	0.262
0	0.141	0.143	0.142

Rational function: y ═ a + bx)/(1+ cx + dx ^2)

Coefficient data: a is-84.464; 487.949; c is-0.416; d is 0.057;

and (4) conclusion: the standard curve correlation linearity was obtained by fitting a standard curve as shown in figure 1: r > 0.9998, which meets the industry requirement R > 0.9900, hereinafter denoted R > 0.9900.

(2) The minimum detection limit (sensitivity) was 15.56pg/mL, which is the concentration corresponding to the mean plus two standard deviations of the measurements of 20 blank samples (i.e. the sample dilutions).

(3) The test of the sample to be tested comprises the following steps:

randomly testing 56 parts of the stock solution of the normal human serum sample, and treating the sample to be tested according to the serum sample treatment OD₄₅₀The values are shown in Table 8:

table 8: the serum sample OD₄₅₀Value of

OD is obtained according to the fitting formula y ═ a + bx)/(1+ cx + dx ^2)₄₅₀The concentrations ng/mL for the values are shown in Table 9:

table 9: the serum sample concentration (ng/ml)

3.551	5.564	1.959	0.008	5.497	0.541	5.189
							0.746	2.018	3.789	0.458	0.554	1.954	0.677
0.162	1.390	5.439	0.929	1.064	2.024	4.814
							1.148	1.832	0.888	1.151	0.857	2.805	0.460
5.170	5.401	1.197	1.052	0.376	4.111	1.354
							0.010	6.007	0.639	5.793	0.876	0.246	0.421
4.876	5.525	0.616	0.096	1.604	0.469	3.532
							0.239	1.011	2.424	0.132	4.677	0.038	0.314

The concentration range of the serum samples was calculated as shown in table 10:

table 10:

number of serum samples	Serum sample Range (ng/mL)
		n＝8	＜0.25
n＝15	0.25-0.90
		n＝33	0.91-6.10

OD of 20 serum samples₄₅₀The value was not less than 2.0, and the calculated concentration was greatly deviated, and the value (concentration ng/mL) measured after diluting 5-fold with serum sample diluent was as shown in Table 11:

table 11: serum sample OD₄₅₀Value and concentration

OD450 value	1.211	1.763	1.692	1.899	1.795	1.984	1.865	1.305	1.831	1.052
											Concentration ng/mL	4.367	8.741	8.068	10.132	9.056	11.072	9.772	4.983	9.420	3.428

OD₄₅₀Value of	0.851	1.902	1.425	1.854	0.981	1.324	1.764	1.596	1.201	1.542
											Concentration ng/mL	2.407	10.165	5.841	9.657	3.048	5.113	8.751	7.213	4.305	6.760

And (4) conclusion: as can be seen from the above, 56 samples of normal human serum were randomly tested, and the range of samples is shown in Table 12, which is consistent with the data (Mayo clinical official website data) being queried. Therefore, the kit provided by the invention has qualified accuracy and is suitable for detecting a sample to be detected.

Table 12:

number of serum samples	Serum sample Range (ng/mL)
		n＝8	＜0.25
n＝15	0.25-0.90
		n＝23	0.91-7.25
n＝10	7.25-11.10

From the Mayo clinical official website data, AMH reference values are shown in table 13:

table 13:

reference packet	Reference range (ng/mL)
		Male:<24 months	14-466
Male: 24 months to 12 years	7.4-243
		Male:>12 years old	0.7-19
Female:<24 months	<4.7
		Female: 24 months to 12 years	<8.8
Female: 13-45 years old	0.9-9.5
		Female:>45 years old	1.0
Women with menopausal or premature ovarian failure	<0.25

(4) Specificity detection

Selecting serum of 12 different species as sample for detection, and detecting according to OD₄₅₀The value corresponds to the concentration to calculate the crossing rate, the specific condition of the kit is judged according to the size of the crossing rate, and the data is shown in a table 14:

table 14:

and (4) conclusion: the detection of specificity among species shows that the kit of the invention has no cross reaction with other species and has strong specificity.

(5) Detection of precision

The precision testing method in the same batch comprises the following steps: repeating the measurement of the same batch of products by using low, medium and high value samples to be measured for 20 times (n), calculating the average value and Standard Deviation (SD) of the 20 measurement results, and calculating the Coefficient of Variation (CV) according to the formula CV as standard deviation/average value multiplied by 100%; the precision testing method among different batches comprises the following steps: the measurement was repeated 20 times (n) for each of the 3 batches of the product using the low, medium, and high value samples to be measured, and the average value and Standard Deviation (SD) of the 20 measurement results were calculated, and the coefficient of variation CV was calculated according to the formula CV ═ standard deviation/average value × 100%, as shown in table 15:

table 15:

and (4) conclusion: as can be seen from the above, the kit of the invention has good precision, and meets the industrial requirements that the CV in the same batch is less than or equal to 8 percent and the CV in different batches is less than or equal to 10 percent.

(6) Stability detection

The test method comprises the following steps: the reagents (an ELISA plate coated by a capture antibody, a biotin-labeled detection antibody, a standard substance and horse radish peroxidase-labeled avidin) in the kit are placed at 37 ℃ for 4 days and 7 days to carry out thermal destruction experiments, then the reagents are compared with the reagents placed at 4 ℃ to set a standard curve and a sample to be tested, the reduction percentage is calculated to judge the stability, and the validity period is calculated, as shown in Table 16:

table 16:

wherein: 7-day drop rate at 37 ℃ (1-37 ℃ for 7-day OD₄₅₀OD at/4 ℃₄₅₀)×100％

4-day decrease rate at 37 ℃ (1-37 ℃ (4-day OD)₄₅₀OD at/4 ℃₄₅₀)×100％

And (4) conclusion: from the above, the stability of the kit provided by the invention is good, the average reduction rate of the standard sample and the sample to be tested is 11% after the kit is thermally damaged for 7 days at 37 ℃, and the kit meets the index requirements in the proposed technical requirements, so that the stability of the kit provided by the invention is inferred to be 1 year.

The parameters of the kit of the invention can be obtained through the detailed data as follows: the standard curve is linear: r is more than 0.9900; minimum detection limit (sensitivity): 15.56 pg/mL; specificity: no other species and no cross precision: CV in the same batch is less than or equal to 8 percent, and CV in different batches is less than or equal to 10 percent; stability: the validity period is one year;

from the above, the double-resistance sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content, which is prepared by the invention, has the advantages of strong specificity, good precision and good stability, can accurately detect the AMH concentration, and can assist in evaluating the fertility of the ovarian function and the premature senility symptoms according to the quantitative analysis of the detection result.

Example 13: comparative experiment of the present invention with the prior art

The three-step method of double antibody sandwich incubation of the prior art and the two-step method of double antibody sandwich incubation of the present invention were compared for the kit parameters (standard curve linearity, range of samples to be tested, minimum detection limit, specificity, precision, stability) with detailed data as shown in tables 17, 18, 19 and 20:

(1) linear comparison of standard curves

Table 17: linear comparison of standard curves

The standard curve of the room temperature oscillation two-step method is shown in the attached figure 2, and the formula is as follows:

rational function: y ═ a + bx)/(1+ cx + dx ^2)

Coefficient data:

a＝-77.600

b＝472.407

c＝-0.430

d＝0.061

the standard curve of the prior art three-step incubation method is shown in FIG. 3, and the formula is as follows:

rational function: y ═ a + bx)/(1+ cx + dx ^2)

Coefficient data:

a＝-104.479

b＝508.708

c＝-0.403

d＝0.057

and (4) conclusion: from the above, the correlation linearity of the standard curve of the invention (room temperature oscillation two-step method) is as follows: r is more than 0.9999; standard curve correlation linearity of prior art (three-step incubation): r is more than 0.9998;

therefore, the linear R of the invention (a room temperature oscillation two-step method) and the prior art (a three-step incubation method) is more than 0.9900, meets the industrial requirement standard, and is better than the prior art.

(2) Range comparison of samples to be tested

Table 18: range comparison of samples to be tested

Wherein:

the concentration is calculated by a formula obtained by a corresponding fitting curve of the invention (room temperature oscillation two-step method) and a fitting curve of the prior art (incubation three-step method);

the deviation rate is (1-room temperature oscillation two-step method to be measured sample concentration/three-step incubation method to be measured sample concentration) x 100%

And (4) conclusion: it can be seen from the above that, the deviation rate of the method and the prior art is less than or equal to 15%, the deviation rate is smaller, and the accuracy test is qualified.

(3) The lowest detection limits (sensitivities) were compared as follows:

the lowest limit of detection is the concentration corresponding to the mean plus two standard deviations of the measurements of 20 blank samples (i.e., the sample dilutions). The higher the sensitivity, the lower the minimum detection limit.

The lowest detection limit value of the room temperature oscillation two-step method is 15.438 pg/mL; the lowest detection limit value of the prior art three-step incubation method is 17.598 pg/mL; therefore, the room temperature shaking two-step method has higher sensitivity than the prior incubation three-step method.

(4) Comparison of specificity tests

Selecting 12 different species of sera as samples to be detected for detection, and detecting according to OD₄₅₀Calculating the crossing rate corresponding to the concentration, and judging the specificity condition of the kit according to the size of the crossing rate.

And (4) conclusion: through species specificity detection, the room temperature oscillation two-step method and the prior art incubation three-step method have no cross reaction with other species and have strong specificity.

(5) Comparison of precision

The precision testing method in the same batch comprises the following steps: the measurement of the same batch of products was repeated 20 times (n) using low, medium, and high value samples to be measured, and the average value and Standard Deviation (SD) of the 20 measurement results were calculated, and the Coefficient of Variation (CV) was calculated according to the formula CV standard deviation/average value × 100%.

The precision testing method among different batches comprises the following steps: the measurement was repeated 20 times (n) for each of the products between 3 batches using low, medium, and high value samples to be measured, and the average and Standard Deviation (SD) of the 20 measurement results were calculated, and the Coefficient of Variation (CV) was calculated according to the formula CV standard deviation/average × 100%.

The precision within the same batch is shown in table 19:

table 19:

the precision between the different batches is shown in table 20:

table 20:

and (4) conclusion: the room temperature shaking two-step method and the prior art incubation three-step method both meet the industrial requirements, namely: CV in the same batch is less than or equal to 8 percent, and CV in different batches is less than or equal to 10 percent. The room temperature shaking two-step method of the invention has higher precision than the three-step incubation method in the prior art).

(6) Comparison of stability tests

The test method comprises the following steps: all reagents (an ELISA plate coated by a capture antibody, a biotin-labeled detection antibody, a standard substance and horse radish peroxidase-labeled avidin) in the kit are placed at 37 ℃ for 4 days and 7 days to carry out thermal destruction experiments, then the reagents are compared with all the reagents placed at 4 ℃ to set a standard curve and a sample to be detected, the reduction percentage is calculated, the stability is judged according to the reduction percentage, and the validity period is calculated.

And (4) conclusion: after stability tests are carried out on the room temperature oscillation two-step method and the prior art incubation three-step method, the average reduction rate of the standard substance and the sample to be tested is 11 percent after thermal destruction is carried out for 7 days at 37 ℃, the standard substance and the sample to be tested both meet the index requirements in the drawn up technical requirements, and the stability of the reagent boxes of the two methods is deduced to be 1 year.

In summary, the following steps: the kit is obtained through various parameter verification comparison experiments, the room temperature oscillation two-step method and the prior art incubation three-step method both meet the standard in the industry, and various parameters of the kit are better than those of the prior art, so that the kit is shorter in reaction time, simple to operate, less in raw material and sample to be detected, lower in equipment and experimental requirements, more suitable for being used in common laboratories, wider in audience and larger in market benefit.

Therefore, compared with the prior art, the kit and the detection method thereof have the beneficial effects that: the invention adopts a double-antibody sandwich room temperature oscillation two-step method, shortens the reaction time to 2.5h, has two reaction steps, the sample loading amount of a standard substance/a sample to be detected and a detection antibody is 50uL, and the experimental equipment is a microplate oscillator, thereby reducing the cost of manpower and equipment, reducing the usage amount of raw materials and the sample to be detected, lowering the cost and improving the use efficiency of the sample to be detected which is difficult to collect or more precious. The kit and the detection method thereof have the advantages of simple reaction steps, simple operation and convenient use, and the detection result can accurately calculate the concentration of AMH and carry out quantitative analysis on the AMH, as shown in Table 21:

table 21:

in conclusion, the above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes or modifications according to the present invention without departing from the spirit of the present invention, which falls within the scope of the appended claims.

Claims

1. A double-anti sandwich enzyme-linked immunosorbent assay kit for detecting the content of anti-mullerian hormone comprises an ELISA plate coated by a capture antibody, a sample diluent, a standard substance, a biotin-labeled detection antibody, horseradish peroxidase-labeled avidin, a concentrated washing solution, a substrate solution and a stop solution;

wherein the capture antibody is a murine anti-human AMH antibody;

the sample diluent was 1 XPBS + 1% BSA + 0.05% Tween-20+ 0.1% Proclin 300;

the standard substance is a human AMH freeze-dried standard substance;

the detection antibody is an anti-human AMH antibody;

the substrate solution is a TMB chromogenic substrate solution;

the stop solution is 2M sulfuric acid.

2. The double-anti sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content according to claim 1, wherein the preparation method of the ELISA plate coated by the capture antibody comprises the following steps:

(1) diluting a mouse anti-human AMH antibody to 1-4 ug/mL by using a buffer solution, and then coating an enzyme label plate, wherein each hole is 100 ug/mL; wherein the buffer solution is selected from a carbonate buffer solution with the pH of 9.6 and/or a phosphate buffer solution with the pH of 7.2-7.4;

wherein the preparation steps of the carbonate buffer solution are as follows: 2.352g of NaHCO₃And 2.332g Na₂CO₃Mixing, adding water to 1L;

the preparation steps of the phosphate buffer solution are as follows: mixing 8g NaCl, 0.2g KCl, 1.44g Na₂HPO₄And 0.44g KH₂PO₄Mixing, adding water to 1L;

(3) adding sealing liquid into each hole for sealing after drying, then placing in a constant temperature box at 37 ℃ for 2h, drying again, and then adding 10% sucrose solution into each hole for fixing for 0.5 h; wherein the confining liquid is selected from 1-5% of bovine serum albumin, 2-5% of skimmed milk powder and/or 5-10% of calf serum;

preferably, the preparation method of the elisa plate coated by the capture antibody comprises the following steps:

(2) placing the enzyme label plate treated in the step (1) at 4 ℃ overnight;

(3) and (3) drying, adding 2% bovine serum albumin blocking solution into each hole for blocking, then placing in a 37 ℃ constant temperature box for 2h, drying again, and then adding 10% sucrose solution into each hole for fixing for 0.5 h.

3. The double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content according to claim 1, wherein the 25 x PBS in the concentrated washing solution is prepared by the following steps: 200g of NaCL, 5g of KCL and 36g of Na₂HPO₄And 11gKH₂PO₄Mixing, adding water to 1L, and adjusting the pH to 6.0-6.5;

preferably, the concentration of the biotin-labeled detection antibody is 0.1-0.4 ug/mL;

preferably, the concentration of the biotin-labeled detection antibody is 0.2 ug/mL;

preferably, the concentration of the horseradish peroxidase-labeled avidin is 0.125-0.5 ug/mL;

preferably, the concentration of the horseradish peroxidase-labeled avidin is 0.25 ug/mL.

4. A detection method using the double-antibody sandwich enzyme-linked immunoassay kit for detecting the anti-mullerian hormone content according to any one of claims 1 to 3, characterized in that the detection method comprises the following steps:

5. The detection method of the double-antibody sandwich enzyme-linked immunoassay kit for detecting the anti-mullerian hormone content according to claim 4, wherein the preparation method of the sample treatment solution to be detected in the step (1) is as follows:

or taking 100mg of the tissue lysate sample, washing with 1 XPBS, adding the tissue lysate sample into 1mL of 1 XPBS to prepare homogenate, repeatedly freezing and thawing for 2 times after standing overnight at-20 ℃, centrifuging for 5min at 5000Xg at 2-8 ℃, and taking supernatant for detection;

preferably, the supernatant for detection is stored at-20 ℃ or-80 ℃ to be detected.

6. The detection method of the double-antibody sandwich enzyme-linked immunoassay kit for detecting the anti-mullerian hormone content according to claim 4, wherein the preparation method of the standard solution in the step (2) is as follows: centrifuging 4ng of an AMH freeze-dried standard substance at 6000-10000rpm for 30-60 seconds, and dissolving with 1mL of the sample diluent to obtain a standard substance solution S7, wherein the concentration of the standard substance solution S7 is 4000 pg/mL; adding 250uL of sample diluent into 7 1.5mL centrifuge tubes respectively, and then sucking 250uL of standard solution S7 into a centrifuge tube S6 to obtain standard solution S6, wherein the concentration of the standard solution S6 is 2000 pg/mL; then sucking 250uL of the standard solution S6 into a centrifuge tube S5 to obtain a standard solution S5, wherein the concentration of the standard solution S5 is 1000 pg/mL; then sucking 250uL of the standard solution S5 into a centrifuge tube S4 to obtain a standard solution S4, wherein the concentration of the standard solution S4 is 500 pg/mL; then sucking 250uL of the standard solution S4 into a centrifuge tube S3 to obtain a standard solution S3, wherein the concentration of the standard solution S3 is 250 pg/mL; then sucking 250uL of the standard solution S3 into a centrifuge tube S2 to obtain a standard solution S2, wherein the concentration of the standard solution S2 is 125 pg/mL; then sucking 250uL of the standard solution S2 into a centrifuge tube S1 to obtain a standard solution S1, wherein the concentration of the standard solution S1 is 62.5 pg/mL; the standard solution S0 was 250uL of the sample dilution, and the concentration of the standard solution S1 was 0 pg/mL.

7. The detection method of the double-antibody sandwich enzyme-linked immunoassay kit for detecting the anti-mullerian hormone content according to claim 4, wherein the preparation of the biotin-labeled detection antibody diluent in the step (2) comprises the following steps: diluting the biotin-labeled detection antibody with the sample diluent to obtain a biotin-labeled detection antibody diluent; wherein the volume ratio of the biotin-labeled detection antibody to the biotin-labeled detection antibody diluent is 1: 1000.

8. The detection method of the double-antibody sandwich enzyme-linked immunoassay kit for detecting the anti-mullerian hormone content according to claim 4, wherein the preparation of the horse radish peroxidase-labeled avidin diluent in the step (2) comprises the following steps: diluting the horse radish peroxidase-labeled avidin with the sample diluent to obtain horse radish peroxidase-labeled avidin diluent; wherein the volume ratio of the horseradish peroxidase-labeled avidin to the horseradish peroxidase-labeled avidin diluent is 1: 4000.

9. The detection method of the double-antibody sandwich enzyme-linked immunoassay kit for detecting the anti-mullerian hormone content according to claim 4, wherein the preparation steps of the concentrated washing solution working solution in the step (2) are as follows: diluting the concentrated washing solution with deionized water to obtain a concentrated washing solution working solution; wherein the volume ratio of the concentrated washing liquid to the concentrated washing liquid working solution is 1: 25.

10. The double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content according to any one of claims 1 to 3 and the application of the detection method of the double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting the anti-mullerian hormone content according to any one of claims 1 to 3 in detecting the anti-mullerian hormone content.