Anti-Mullerian hormone antibody-enzyme marker and its preparation method and application

Abstract

The invention discloses a method for preparing an anti-Mullerian hormone antibody-enzyme marker, comprising the following steps: maleamidation of alkaline phosphatase, sulfhydrylation of an AMH antibody, crosslinking, blocking, and purification. The preparation method optimizes and improves the conventional step of preparing an enzyme-labeled antibody using a heterobifunctional crosslinker. In the method, ultrafiltration is used to purify the maleamidated alkaline phosphatase, the sulfhydrylated AMH antibody, and the labeled AMH antibody-alkaline phosphatase marker, replacing the conventional purification method of dialysis. This not only eliminates the complicated dialysis process, shortens the preparation time of the enzyme-labeled antibody, but also reduces raw material loss. Also disclosed are the anti-Mullerian hormone antibody-enzyme marker prepared by the preparation method and the use of the anti-Mullerian hormone antibody-enzyme marker in preparing an AMH enzyme-labeled antibody detection reagent. The prepared AMH enzyme-labeled antibody detection reagent has high sensitivity and good stability in detecting AMH.

Description

Technical Field

[0001] The present invention relates to the technical field of medical diagnosis, and in particular to an anti-Mullerian hormone antibody-enzyme marker, a preparation method and application thereof. Background Art

[0002] Anti-Müllerian hormone (AMH) is a dimeric glycoprotein belonging to the transforming growth factor β (TGFβ) family. The earliest commercialized AMH kit was an enzyme-linked immunosorbent assay (ELISA) developed by Anshlabs, which used Anshlab's patented AMH antibody to develop an N-terminal-C-terminal mouse monoclonal antibody ELISA kit. Regarding the registered enzyme-linked immunosorbent assay (ELISA), quantum dot fluorescence, and fluorescence chromatography, the ELISA requires manual or semi-manual operation, which is cumbersome, time-consuming, and labor-intensive, and is prone to cross-contamination due to human factors. The colloidal gold lateral immunochromatography assay is simple and quick to operate, but due to limitations of the method itself, it has low sensitivity and is prone to large inter- and intra-batch variations.

[0003] In the prior art, there are problems such as decreased antibody sensitivity, unsatisfactory luminescence intensity, and unstable labeled complexes when AMH antibodies are labeled with chemiluminescent signal intensity indicators. Antibodies, like all proteins, are composed of amino acid residues. The amino groups of proteins (including lysine side chains and amino termini) are usually used to covalently link labels. Although a variety of labeling methods have been reported in the literature, there are actually only four most commonly used labeling methods, and the only difference is the way in which the label is converted into a reactive derivative. Regardless of the method, almost without exception, the label is covalently linked to the lysine residue of the antibody molecule. The four commonly used labeling methods include sodium periodate, succinyl ester (NHS), carbodiimide, and homotypic or heterotypic bifunctional reagents. When the label is a protein molecule (such as alkaline phosphatase ALP), the antibody labeling process is slightly complicated because the antibody and the labeling molecule have multiple amines.

[0004] In enzymatic chemiluminescent immunoassays, antigen or antibody enzyme labels play a crucial role in the sensitivity and specificity of the immunoassay and are key reagents in these assays. Alkaline phosphatase (ALP) is a widely used enzyme in ELISAs. Binding it to antibodies through certain technical means is a crucial step in the development of ELISAs. Generally, there are several methods for cross-linking ALP to antibodies: glutaraldehyde, sodium periodate, and heterobifunctional groups.

[0005] Alkaline phosphatase, as a marker enzyme, is widely used in quantitative diagnosis of clinical immunology. The marker solution of alkaline phosphatase is one of the components of in vitro diagnostic kits, and the stability of alkaline phosphatase activity is an important factor affecting the quality and performance of the kits. Like other biological enzymes, alkaline phosphatase has a short half-life and is easily inactivated. Changes in acid, base, salt ions, and temperature conditions will change or even completely inactivate the enzyme. The long-term stable storage technology of alkaline phosphatase in solution is a key technology for in vitro diagnostic kit products. Technologies for improving enzyme stability include chemical modification technology and additive technology. However, the existing technology has the following defects:

[0006] The preparation of enzyme-labeled antibodies using heterobifunctional cross-linkers often requires dialysis to purify intermediates and products, a complex and time-consuming process with significant raw material loss.

[0007] 2) Existing methods for preparing enzyme-labeled antibody diluents cannot meet the stability requirements of enzyme-labeled antibodies prepared by different methods. There are few universal alkaline phosphatase enzyme marker stabilizers on the market, and there is a lack of targeted methods for preparing stable diluents.

[0008] Therefore, there is an urgent need to provide a novel method for preparing anti-Müllerian hormone antibody-enzyme marker and its application to solve the above problems. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide an anti-Mullerian hormone antibody enzyme marker and its preparation method and application, which optimizes and improves the conventional steps of preparing enzyme-labeled antibodies using heterobifunctional cross-linking agents, shortens the preparation time of enzyme-labeled antibodies, and the prepared AMH enzyme-labeled antibody detection reagent has high sensitivity and good stability in detecting AMH.

[0010] To solve the above technical problems, the first technical solution adopted by the present invention is to provide a method for preparing an anti-Müllerian hormone antibody-enzyme marker, comprising the following steps:

[0011] (1) Maleamidation of alkaline phosphatase

[0012] Prepare GMBS working solution by mixing ALP solution with GMBS working solution, react at room temperature in the dark for 30-60 minutes, and then purify by centrifugation in an ultrafiltration centrifuge tube 2-3 times;

[0013] (2) Thiolation of AMH antibodies

[0014] Prepare DTT working solution, mix the AMH antibody solution with DTT working solution, react at room temperature in the dark for 30-60 minutes, and then purify by centrifugation 3-5 times using an ultrafiltration centrifuge tube;

[0015] (3) Cross-linking and blocking

[0016] Mix maleamidated alkaline phosphatase and thiol-labeled AMH antibody and react at room temperature in the dark for 1-2 hours. After the reaction, add N-ethylmaleimide and L-cysteine ​​in a certain concentration, leave at room temperature in the dark for 30-60 minutes to block.

[0017] (4) Purification

[0018] The cross-linked product prepared in step (3) was purified 3 to 5 times by ultrafiltration centrifugation tube. The purified product was diluted to a suitable storage volume with 10 mM PBS at a pH of 7.0 to 7.5, and then an equal volume of glycerol was added and stored frozen at -20°C.

[0019] In a preferred embodiment of the present invention, the specific steps of step (1) include:

[0020] (11) Prepare 0.9 mg / ml GMBS working solution and protect from light

[0021] Add 0.0060g GMBS to 600ul DMSO, mix well, take 100ul and add to 1ml PBS (pH 7.45) and mix well;

[0022] (12) Add the prepared 0.9 mg / ml GMBS working solution to 5 mg / ml ALP and react at room temperature in the dark for 30–60 min. The molar ratio of GMBS to ALP is 10:1–50:1.

[0023] (13) The reaction solution was concentrated and purified 2 to 3 times using a 10 kD ultrafiltration centrifuge tube at 10,000 r / min for 10 min each time; the inner tube of the ultrafiltration tube was inverted at 2,000 r / min and centrifuged for 1 min to collect the purified solution to obtain maleamidated alkaline phosphatase.

[0024] In a preferred embodiment of the present invention, the specific steps of step (2) include:

[0025] (21) Prepare 10mM DTT working solution and protect from light

[0026] Dissolve 0.0154g DTT in 1ml 10mM PBS (pH 7.1), mix well, and add 100ul to 900ul PBS 7.1 to obtain 10mM DTT.

[0027] (22) Add 10 mM DTT working solution to 2 mg / ml AMH antibody and react at room temperature in the dark for 30–60 min. The molar ratio of DTT:AMH antibody is 100:1–600:1.

[0028] (23) The reaction solution was concentrated and purified 3 to 5 times using a 50 kD ultrafiltration centrifuge tube at 8000 r / min for 5 min each time; the inner tube of the ultrafiltration tube was inverted at 2000 r / min and centrifuged for 1 min to collect the purified solution to obtain the thiol-amylated AMH antibody.

[0029] In a preferred embodiment of the present invention, in step (3), the concentration of N-ethylmaleimide added is 1 mg N-ethylmaleimide per mg protein; the concentration of L-cysteine ​​added is 1 mg L-cysteine ​​per mg protein.

[0030] In a preferred embodiment of the present invention, the specific steps of step (4) include:

[0031] The cross-linked product prepared in step (3) was concentrated and purified 3 to 5 times using a 50 kD ultrafiltration centrifuge tube at 8000 r / min for 5 min / time; the inner tube of the ultrafiltration tube was inverted at 2000 r / min, centrifuged for 1 min, and the purified liquid was collected; the purified product was diluted to a suitable storage volume with 10 mM PBS with a pH of 7.0 to 7.5, and then an equal volume of glycerol was added and stored frozen at -20°C.

[0032] Furthermore, in the preparation process of anti-Müllerian hormone antibody-enzyme label, the cross-linking agent GMBS can be replaced by heterobifunctional cross-linking agents (amino-thiol) such as Sulfo-GMBS, SMCC, Sulfo-SMCC, SMPB, Sulfo-SMPB, SPDP, LC-SPDP, EMCS, AMAS, SIAB, and Sulfo-SIAB;

[0033] The reducing agent may be one or more of TCEP, DTT, mercaptoethanol, and sodium thiosulfate.

[0034] In order to solve the above technical problems, the second technical solution adopted by the present invention is to provide an anti-Mullerian hormone antibody-enzyme marker prepared by the method described in any one of the above items.

[0035] In order to solve the above technical problems, the third technical solution adopted by the present invention is: to provide an application of the anti-Mullerian hormone antibody-enzyme marker as described above to prepare an AMH enzyme-labeled antibody detection reagent.

[0036] In a preferred embodiment of the present invention, the method for preparing an AMH enzyme-labeled antibody detection reagent comprises the following steps:

[0037] (1) Prepare enzyme-labeled antibody diluent, which includes the following components by mass fraction:

[0038]

[0039] Dissolving the above components in pure water and adjusting the pH to 6.0-7.2 to obtain a dilution solution of the alkaline phosphatase marker;

[0040] (2) diluting the anti-Mullerian hormone antibody-enzyme marker storage solution with the enzyme-labeled antibody diluent prepared in step (1) at a ratio of 1:600 ​​to 1:4000 to the required concentration to obtain an AMH enzyme-labeled antibody detection reagent.

[0041] Furthermore, the buffer salt is one or more of MES, HEPES, and acetic acid / sodium acetate.

[0042] Furthermore, the surfactant is Brij35 or / and S9.

[0043] Furthermore, the polyol is one or more of sorbitol, inositol, and xylitol.

[0044] Furthermore, the non-reducing sugar is trehalose and / or sucrose.

[0045] Furthermore, the preservative is Proclin 300.

[0046] The beneficial effects of the present invention are:

[0047] (1) The present invention provides a method for preparing an alkaline phosphatase-labeled AMH antibody, which optimizes and improves the conventional step of preparing an enzyme-labeled antibody using a heterobifunctional cross-linker. In the method, ultrafiltration is used to purify maleamidated alkaline phosphatase, thiol-labeled AMH antibody, and labeled AMH antibody-alkaline phosphatase marker, replacing the conventional purification method of dialysis. This not only eliminates the complicated process of dialysis, shortens the preparation time of the enzyme-labeled antibody, but also reduces the loss of raw materials.

[0048] (2) The enzyme-labeled antibody diluent of the present invention introduces 2,6-pyridinedicarboxylic acid on the basis of the traditional diluent formula components and optimizes the formula. The diluent is suitable for enzyme-labeled antibodies prepared by the GMBS method and has specificity, filling the gap in the preparation method of the stable diluent required for AMH antibody-alkaline phosphatase enzyme-labeled antibody prepared by the GMBS method;

[0049] (3) The present invention also provides a method for preparing an enzyme-labeled antibody reagent for detecting anti-Mullerian hormone using a magnetic particle chemiluminescence method. The reagent is used to detect AMH with high sensitivity and good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is a linear curve graph of the AMH enzyme-labeled antibody detection reagent calibrator. DETAILED DESCRIPTION

[0051] The preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more precise definition of the protection scope of the present invention.

[0052] Example 1:

[0053] A method for preparing an anti-Mullerian hormone antibody-enzyme marker comprises the following steps:

[0054] (1) Maleamidation of alkaline phosphatase:

[0055] (11) Prepare 0.9 mg / ml GMBS working solution and protect from light

[0056] Add 0.0060g GMBS to 600ul DMSO, mix well, take 100ul and add to 1ml PBS (pH 7.45) and mix well;

[0057] (12) Add the prepared 0.9 mg / ml GMBS working solution to 5 mg / ml ALP and react at room temperature in the dark for 30–60 min. The labeling ratio (GMBS:ALP molar ratio) is 10:1–50:1.

[0058] (13) The reaction solution was concentrated and purified 2 to 3 times using a 10 kD ultrafiltration centrifuge tube at 10,000 r / min for 10 min each time; the inner tube of the ultrafiltration tube was inverted at 2,000 r / min and centrifuged for 1 min to collect the purified solution to obtain maleamidated alkaline phosphatase.

[0059] (2) Thiolation of AMH antibodies:

[0060] Prepare DTT working solution, mix the AMH antibody solution with DTT working solution, react at room temperature in the dark for 30-60 minutes, and then purify by centrifugation 3-5 times using an ultrafiltration centrifuge tube;

[0061] (21) Prepare 10mM DTT working solution and protect from light

[0062] Dissolve 0.0154g DTT in 1ml 10mM PBS (pH 7.1), mix well, and add 100ul to 900ul PBS 7.1 to obtain 10mM DTT.

[0063] (22) Add 10 mM DTT working solution to 2 mg / ml AMH antibody and react at room temperature in the dark for 30–60 min. The labeling ratio (molar ratio of DTT:AMH antibody) is 100:1–600:1.

[0064] (23) The reaction solution was concentrated and purified 3 to 5 times using a 50 kD ultrafiltration centrifuge tube at 8000 r / min for 5 min each time; the inner tube of the ultrafiltration tube was inverted at 2000 r / min and centrifuged for 1 min to collect the purified solution to obtain the thiol-amylated AMH antibody.

[0065] (3) Cross-linking and blocking:

[0066] Mix maleamidated alkaline phosphatase and thiol-modified AMH antibody and react at room temperature in the dark for 1-2 hours. After the reaction, add 1 mg of N-ethylmaleimide per milligram of protein and incubate at room temperature in the dark for 30-60 minutes to block unreacted thiol groups. Then, add 1 mg of L-cysteine ​​per milligram of protein and incubate at room temperature in the dark for 30-60 minutes to block unreacted maleimide groups.

[0067] (4) Purification:

[0068] The cross-linked product prepared in step (3) was concentrated and purified 3 to 5 times using a 50 kD ultrafiltration centrifuge tube at 8000 r / min for 5 min / time; the inner tube of the ultrafiltration tube was inverted at 2000 r / min, centrifuged for 1 min, and the purified liquid was collected; the purified product was diluted to a suitable storage volume with 10 mM PBS with a pH of 7.0 to 7.5, and then an equal volume of glycerol was added and stored frozen at -20°C.

[0069] Example 2:

[0070] A method for preparing an AMH enzyme-labeled antibody detection reagent using the anti-Mullerian hormone antibody-enzyme marker described in Example 1 comprises the following steps:

[0071] (1) Prepare enzyme-labeled antibody diluent, which includes the following components by mass fraction:

[0072]

[0073]

[0074] Dissolve the above components in pure water and adjust the pH to 7.2 to obtain a dilution solution of the alkaline phosphatase marker;

[0075] (2) diluting the anti-Mullerian hormone antibody-enzyme marker storage solution with the enzyme-labeled antibody diluent prepared in step (1) at a ratio of 1:600 ​​to 1:4000 to the required concentration to obtain an AMH enzyme-labeled antibody detection reagent.

[0076] Example 3:

[0077] A method for preparing an AMH enzyme-labeled antibody detection reagent using the anti-Mullerian hormone antibody-enzyme marker described in Example 1 comprises the following steps:

[0078] (1) Prepare enzyme-labeled antibody diluent, which includes the following components by mass fraction:

[0079]

[0080] Dissolve the above components in pure water and adjust the pH to 7.2 to obtain a dilution solution of the alkaline phosphatase marker;

[0081] (2) diluting the anti-Mullerian hormone antibody-enzyme marker storage solution with the enzyme-labeled antibody diluent prepared in step (1) at a ratio of 1:600 ​​to 1:4000 to the required concentration to obtain an AMH enzyme-labeled antibody detection reagent.

[0082] The experiment found that, compared with Example 2, 2,6-pyridinedicarboxylic acid had a significant effect on the activity preservation of the AMH antibody-alkaline phosphatase marker.

[0083] Example 4:

[0084] A method for preparing an AMH enzyme-labeled antibody detection reagent using the anti-Mullerian hormone antibody-enzyme marker described in Example 1 comprises the following steps:

[0085] (1) Prepare enzyme-labeled antibody diluent, which includes the following components by mass fraction:

[0086]

[0087] Dissolve the above components in pure water and adjust the pH to 6.5 to obtain a dilution solution of the alkaline phosphatase marker;

[0088] (2) diluting the anti-Mullerian hormone antibody-enzyme marker storage solution with the enzyme-labeled antibody diluent prepared in step (1) at a ratio of 1:600 ​​to 1:4000 to the required concentration to obtain an AMH enzyme-labeled antibody detection reagent.

[0089] The experiment found that compared with Example 3, the AMH antibody-alkaline phosphatase marker was more stable when stored in a slightly acidic diluent.

[0090] Example 5:

[0091] A method for preparing an AMH enzyme-labeled antibody detection reagent using the anti-Mullerian hormone antibody-enzyme marker described in Example 1 comprises the following steps:

[0092] (1) Prepare enzyme-labeled antibody diluent, which includes the following components by mass fraction:

[0093]

[0094]

[0095] Dissolve the above components in pure water and adjust the pH to 6.5 to obtain a dilution solution of the alkaline phosphatase marker;

[0096] (2) diluting the anti-Mullerian hormone antibody-enzyme marker storage solution with the enzyme-labeled antibody diluent prepared in step (1) at a ratio of 1:600 ​​to 1:4000 to the required concentration to obtain an AMH enzyme-labeled antibody detection reagent.

[0097] The test found that compared with Example 4, when the BSA concentration was changed from 1% to 3%, the results showed that the stabilization effect was not significantly improved.

[0098] Example 6:

[0099] A method for preparing an AMH enzyme-labeled antibody detection reagent using the anti-Mullerian hormone antibody-enzyme marker described in Example 1 comprises the following steps:

[0100] (1) Prepare enzyme-labeled antibody diluent, which includes the following components by mass fraction:

[0101]

[0102] Dissolve the above components in pure water and adjust the pH to 6.5 to obtain a dilution solution of the alkaline phosphatase marker;

[0103] (2) diluting the anti-Mullerian hormone antibody-enzyme marker storage solution with the enzyme-labeled antibody diluent prepared in step (1) at a ratio of 1:600 ​​to 1:4000 to the required concentration to obtain an AMH enzyme-labeled antibody detection reagent.

[0104] The experiment found that compared with Example 5, the sorbitol concentration in the solution was changed from 1% to 5%. The results showed that the stabilization effect of this example was significantly improved, and sorbitol had a great influence on the activity preservation of the AMH antibody-alkaline phosphatase marker.

[0105] Example 7:

[0106] A method for preparing an AMH enzyme-labeled antibody detection reagent using the anti-Mullerian hormone antibody-enzyme marker described in Example 1 comprises the following steps:

[0107] (1) Prepare enzyme-labeled antibody diluent, which includes the following components by mass fraction:

[0108]

[0109] Dissolve the above components in pure water and adjust the pH to 6.5 to obtain a dilution solution of the alkaline phosphatase marker;

[0110] (2) diluting the anti-Mullerian hormone antibody-enzyme marker storage solution with the enzyme-labeled antibody diluent prepared in step (1) at a ratio of 1:600 ​​to 1:4000 to the required concentration to obtain an AMH enzyme-labeled antibody detection reagent.

[0111] The experiment found that compared with Example 6, the trehalose concentration in the solution was changed from 1% to 10%. The results showed that the stabilization effect of this example was significantly improved. The trehalose concentration had a great influence on the activity preservation of the AMH antibody-alkaline phosphatase marker.

[0112] Embodiment of detecting AMH using the prepared AMH enzyme-labeled antibody detection reagent:

[0113] Required reagents: calibrator / sample, AMH antibody-coated magnetic bead working solution, AMH enzyme-labeled antibody working solution, sample diluent, cleaning solution, substrate. Use the AMH detection enzyme-labeled antibody reagent described in the present invention and APS-5 as the luminescent substrate.

[0114] Detection process: The sample is tested on a fully automatic chemiluminescence immunoassay (sandwich chemiluminescence immunoassay). The detection principle is as follows:

[0115] Step 1: Add the sample, superparamagnetic particles (magnetic beads) coated with anti-AMH monoclonal antibodies, and an anti-AMH monoclonal antibody-alkaline phosphatase marker to a reaction tube. After incubation, AMH in the sample binds to the anti-AMH monoclonal antibody coated on the magnetic beads. Simultaneously, the anti-AMH monoclonal antibody-alkaline phosphatase marker binds to another site of AMH in the sample, forming an antigen-antibody sandwich immune complex. After the reaction is complete, a wash step is performed to remove unbound substances.

[0116] Step 2: Add the chemiluminescent substrate solution (APS-5) to the reaction tube. The luminescent substrate is decomposed by alkaline phosphatase to produce chemiluminescence. The number of photons generated by the reaction is measured by the radiomultiplier tube. The number of photons generated is proportional to the concentration of AMH in the sample. The concentration of AMH in the sample is calculated using the calibration curve of the detector.

[0117] Performance evaluation

[0118] (1) Linearity:

[0119] The prepared AMH calibrator was measured to obtain the calibrator curve as shown in Figure 1 As shown, the linearity is good.

[0120] Calibrator data:

[0121]

[0122] (2) Sensitivity:

[0123] Take 0 ng / mL of standard solution and repeat the test 20 times with the same batch of reagents to obtain the average luminescence value M and standard deviation SD. Perform two-point regression fitting on the luminescence values ​​corresponding to the concentrations between 0 ng / ml AMH and 0.5 ng / ml to obtain a linear equation. Substitute the luminescence value corresponding to M+2SD into the above equation to obtain the corresponding concentration value, which is the minimum detection limit of the detection reagent, which is calculated to be 0.0373 ng / mL.

[0124]

[0125] (3) Precision:

[0126] Two AMH samples with concentrations of 32 ng / mL and 0.5 ng / mL within the linear range were tested using three batches of reagents, and the intra-batch and inter-batch differences were calculated. The results showed that the inter-batch and intra-batch differences of the kit were both less than 5%.

[0127]

[0128]

[0129] (4) Thermal stability test

[0130] The enzyme-labeled working solution prepared in Example 7 was placed in a 37°C constant temperature and humidity incubator (40-60% relative humidity) for an accelerated stability test. Using a 32 ng / ml AMH calibrator as a sample, the luminescence value was measured before placement and for 7 consecutive days (in combination with other components required for AMH detection). The remaining proportion of enzyme-labeled antibody activity in the solution was calculated (the ratio of the luminescence value after the accelerated test to the luminescence value before storage at 37°C).

[0131] Detection process: The sample is tested on a fully automatic chemiluminescence immunoassay (sandwich chemiluminescence immunoassay) Acceleration days: Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

[0132] Active remaining ratio 102.1% 98.5% 101.2% 97.5% 98.3% 97.1% 97.2%

[0133] From the results in the table above, it can be seen that after the AMH enzyme-labeled antibody reagent is accelerated, the activity loss of the enzyme-labeled antibody is 2.8% after 7 days.

[0134] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0135] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. An application of an anti-Mullerian hormone antibody-alkaline phosphatase marker to prepare an AMH enzyme-labeled antibody detection reagent, characterized in that: The method for preparing an AMH enzyme-labeled antibody detection reagent comprises the following steps: (1) Prepare enzyme-labeled antibody diluent, including the following components: Buffer salt 10mM~50mM BSA 1%~3% Magnesium ion 5mM Zinc ion 0.1mM NaCl 0.9% 2,6-pyridinedicarboxylic acid 0.1% Brij35 0.05% Sorbitol 1%~5% Trehalose 10% Preservatives 0.03%~0.05% Among them, the percentage is the mass fraction; Dissolving the above components in pure water and adjusting the pH to 6.0-6.5 to obtain a dilution of the anti-Mullerian hormone antibody-alkaline phosphatase marker; (2) The anti-Mullerian hormone antibody-alkaline phosphatase marker stock solution is diluted to the desired concentration at a ratio of 1:600 ​​to 1:4000 using the anti-Mullerian hormone antibody-alkaline phosphatase marker diluent prepared in step (1) above to obtain an AMH enzyme-labeled antibody detection reagent.

2. The use according to claim 1, characterized in that The method for preparing the anti-Mullerian hormone antibody-alkaline phosphatase marker comprises the following steps: (1) Maleamidation of alkaline phosphatase Prepare GMBS working solution by mixing ALP solution with GMBS working solution. After reacting at room temperature in the dark for 30-60 minutes, purify the mixture by centrifugation in an ultrafiltration centrifuge tube 2-3 times. (2) Thiolation of AMH antibodies Prepare DTT working solution, mix the AMH antibody solution with DTT working solution, react at room temperature in the dark for 30-60 minutes, and then purify by centrifugation 3-5 times using an ultrafiltration centrifuge tube; (3) Cross-linking and blocking Mix maleamidated alkaline phosphatase and thiol-labeled AMH antibody and react at room temperature in the dark for 1-2 hours. After the reaction, add N-ethylmaleimide and L-cysteine ​​in a certain concentration, and let it react at room temperature in the dark for 30-60 minutes to block the reaction. (4) Purification The cross-linked product prepared in step (3) was purified 3 to 5 times by ultrafiltration centrifuge tube centrifugation. The purified product was diluted to a suitable storage volume with 10 mM PBS at a pH of 7.0 to 7.5, and then an equal volume of glycerol was added and stored frozen at -20°C.

3. The use according to claim 2, characterized in that The specific steps of step (1) include: (11) Prepare 0.9 mg / ml GMBS working solution and protect from light Add 0.0060g GMBS to 600ul DMSO, mix well, take 100ul and add to 1ml PBS (pH 7.45) and mix well; (12) Add the prepared 0.9 mg / ml GMBS working solution to 5 mg / ml ALP and react at room temperature in the dark for 30–60 min. The molar ratio of GMBS to ALP is 10:1–50:

1. (13) The reaction solution was concentrated and purified 2 to 3 times using a 10 kD ultrafiltration centrifuge tube at 10,000 r / min for 10 min each time; the inner tube of the ultrafiltration tube was inverted at 2,000 r / min and centrifuged for 1 min to collect the purified solution to obtain maleamidated alkaline phosphatase.

4. The use according to claim 2, characterized in that The specific steps of step (2) include: (21) Prepare 10mM DTT working solution and protect from light Dissolve 0.0154g DTT in 1ml 10mM PBS (pH 7.1), mix well, and add 100ul to 900ul PBS 7.1 to obtain 10mM DTT. (22) Add 10 mM DTT working solution to 2 mg / ml AMH antibody and react at room temperature in the dark for 30-60 min. The molar ratio of DTT:AMH antibody is 100:1-600:

1. (23) The reaction solution was concentrated and purified 3 to 5 times using a 50 kD ultrafiltration centrifuge tube at 8000 r / min for 5 min each time; the inner tube of the ultrafiltration tube was inverted at 2000 r / min and centrifuged for 1 min to collect the purified solution to obtain the thiol-modified AMH antibody.

5. The use according to claim 2, characterized in that In step (3), the concentration of N-ethylmaleimide added is 1 mg per mg of protein; the concentration of L-cysteine ​​added is 1 mg per mg of protein.

6. The use according to claim 2, characterized in that The specific steps of step (4) include: The cross-linked product prepared in step (3) was concentrated and purified 3 to 5 times using a 50 kD ultrafiltration centrifuge tube at 8000 r / min for 5 min / time; the inner tube of the ultrafiltration tube was inverted at 2000 r / min and centrifuged for 1 min to collect the purified solution; the purified product was diluted to a suitable storage volume with 10 mM PBS at a pH of 7.0 to 7.5, and then an equal volume of glycerol was added and stored frozen at -20°C.

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