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Preparation method of anti-AMH specific antibody and application of antibody in AMH detection kit

A specific and antibody technology, applied in the application of antibodies in AMH detection kits and the preparation of anti-AMH specific antibodies, can solve the problems of not providing further antibody evaluation results, underestimation of AMH detection concentration, and low sensitivity of detection methods. , to achieve the effect of reliable clinical diagnosis and treatment, simple preparation method and high specificity

Inactive Publication Date: 2019-09-10
ZHENGZHOU IMMUNO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in clinical practice, many studies (Xan et al., 2014; Lukaszuk et al., 2014) have shown that the performance of kits targeting mature region antibodies does not achieve the effect claimed by the manufacturer. According to the patent document US 7897350 The reported method, the detection concentration of AMH is underestimated, and the sensitivity of the detection method is low
Patent document WO 2014074835 reported multiple antibodies against the linear site of AMH proregion or mature region, and provided different antibody combinations for the determination of AMH concentration, but there is a lack of data to prove that the antibody pairing in this patent document can be applied to clinical test
Patent document WO 2017216334 reported the use of antibodies against the non-conformational epitope of the AMH proregion 157-255 interval, and the antibody sandwich method against the linear site of the mature region to detect the concentration of AMH in serum, and the detection results were not affected by the sample storage conditions. , but did not further provide the evaluation results of the application of antibodies in clinical tests

Method used

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  • Preparation method of anti-AMH specific antibody and application of antibody in AMH detection kit
  • Preparation method of anti-AMH specific antibody and application of antibody in AMH detection kit
  • Preparation method of anti-AMH specific antibody and application of antibody in AMH detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation of AMH full-length protein and mature region fragments

[0025] 1. Whole gene synthesis of AMH coding sequence

[0026] The mRNA sequence (NM_000479.4) of AMH was queried from NCBI (National Center for Biotechnology Information), and its expressed sequence was (SEQ ID NO.1).

[0027] Entrusted Shanghai Sangon Bioengineering Co., Ltd. to carry out the whole gene synthesis.

[0028] 2. Construction and identification of recombinant human AMH expression vector

[0029] Using the synthetic AMH gene as a template, the amplification primers containing KpnI and XbaI restriction enzyme cleavage sites were designed respectively, and a His tag sequence was introduced into the upstream primers for later purification. The target gene was amplified using amplification primers, and a fusion fragment with the structure of "KpnI restriction site-His tag-AMH sequence-XbaI restriction site" was amplified. Subsequently, KpnI and XbaI were used to perform double en...

Embodiment 2

[0035] Example 2 Preparation of mouse monoclonal antibody against AMH

[0036] 1. Immunization of mice

[0037] After fully emulsifying the AMH recombinant protein obtained in Example 1 with Freund's complete adjuvant, intraperitoneal immunization of 5-week-old female Balb / c mice, the initial dose is 100ug / mouse; the interval after the first immunization is 21 days The second and third immunizations were carried out in 42 days respectively, and the immunization dose was 50ug / mouse. About 10 days after the third immunization, the blood was collected from the tail, and the serum titer was detected by the indirect method using a 96-well plate coated with AMH.

[0038] 2. Preparation of hybridoma cells

[0039] Select the indirect method to detect serum titer greater than 10 4 Intrasplenic booster immunization was carried out in the mice, and the immunization dose was 100ug / mouse. Three days after the booster immunization, the mouse spleen was fused with mouse myeloma cell NS1...

Embodiment 3

[0049] Example 3 Establishment of the ELISA method for the detection of AMH by the double-antibody sandwich method

[0050] 1. Magnetic particle coating

[0051] Take 30 μl of the mixed magnetic particle stock solution and wash it 5 times with 300 μl of PBS buffer solution, then activate the magnetic particles with 10% glutaraldehyde for 1 hour, and then use pH 7-8 PBS buffer solution to activate the magnetic particles. Microparticles were washed 2 times. Anti-AMH mouse monoclonal antibody AMH-2# was added to the magnetic beads at an amount of 0.3 μg / person, coated at 4°C for 2 hours, and finally blocked with BSA-containing blocking solution for 2 hours.

[0052] 2. HRP-labeled anti-AMH mouse monoclonal antibody AMH-1#

[0053] The anti-AMH mouse monoclonal antibody AMH-1# was labeled with HRP (Roche) at a mass ratio of 1.5:1. The HRP-labeled anti-AMH mouse monoclonal antibody AMH-1# was diluted to 1:4000 with diluent.

[0054] 3. Clinical sample testing

[0055] Collect ...

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Abstract

The invention discloses a preparation method of an anti-AMH specific antibody. The method comprises the steps that firstly, a pCMV3 expression plasmid containing an AMH encoding sequence is constructed, a CHO cell is transfected, a cell culture supernatant is collected and purified through a Ni column, and purified AMH recombinant protein is obtained for standby use; the prepared AMH recombinant protein is used as an immunogen for immunizing a Balb / c mouse, after it is detected that the serum titer of the mouse exceeds 1 / 8K through an ELISA indirect method, the spleen of the immunized mouse isfused with a myeloma cell NS1 of the mouse, a hybridoma cell is subjected to subcloning screening three times through the ELISA indirect method, and a monoclonal hybridoma cell secreting anti-AMH isobtained; the enterocoelia of the mouse is injected with the hybridoma cell, ascitic fluid is collected and purified, and the anti-AMH specific monoclonal antibody is obtained. The preparation methodis simple, the prepared antibody has high specificity and is applied to a kit for detecting the content of AMH in humans, the detection result is hardly influenced by animal serum, and the reliable basis can be provided for clinical diagnosis and treatment by doctors.

Description

technical field [0001] The invention relates to the field of in vitro diagnosis, in particular to a method for preparing an anti-AMH specific antibody, and also relates to the application of the prepared antibody in a kit for detecting AMH. Background technique [0002] Anti-Mullerian hormone (AMH) is one of the members of the transforming growth factor-β (TGF-β) superfamily, which was first discovered by Professor Alfred Jost in 1947. AMH is composed of two identical 72kD subunits connected by disulfide bonds to form a dimeric glycoprotein with a molecular weight of 144kD, which will be hydrolyzed into two N-terminal propeptides of 55kD and two 12.5 C-terminal mature hormone of kD. Male AMH hormone is produced by testicular Sertoli cells, and it is involved in inhibiting the development of Mullerian ducts at the beginning of life, causing the degeneration of Mullerian ducts, and forming a normal male reproductive tract; female AMH is produced by growing small follicular gr...

Claims

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Application Information

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IPC IPC(8): C07K16/26G01N33/74G01N33/577
CPCC07K16/26G01N33/74G01N33/577
Inventor 代春迎侯鹏云胡晓彤张跃峰张创赵巧辉李桂林付光宇吴学炜杨增利
Owner ZHENGZHOU IMMUNO BIOTECH
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