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Immune plasmid, monoclonal antibody for detecting antimullerian hormone, hybridoma and preparation method and application thereof

A hybridoma cell and plasmid technology, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as errors, impact of test results, and complex operations

Active Publication Date: 2017-12-29
GUANGZHOU WONDFO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the detection of original AMH, Ansh Company uses a proAMH monomer and a Mature AMH monoclonal antibody paired together with the kit technology to determine the original AMH concentration in the sample, but due to the presence of two enzyme-cleaved fragments of the AMH protein, the two fragments can be passed through non-cooperative The valence bond is reconnected to form a complex, which is detected by the paired antibody, therefore, it will cause erroneous results
Adding surfactants to the reagents can dissociate the complex and improve the detection accuracy, but the operation is complicated, and different detection platforms have different tolerances to high concentrations of surfactants, and surfactants may also cause serious problems to the detection results. big impact

Method used

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  • Immune plasmid, monoclonal antibody for detecting antimullerian hormone, hybridoma and preparation method and application thereof
  • Immune plasmid, monoclonal antibody for detecting antimullerian hormone, hybridoma and preparation method and application thereof
  • Immune plasmid, monoclonal antibody for detecting antimullerian hormone, hybridoma and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1A

[0045] The DNA plasmid of each antigen of embodiment 1AMH and the preparation of protein

[0046] 1. Acquisition of genes

[0047] The gene synthesis company synthesized the template for PCR amplification according to the exon sequence of Genbank (Gene ID: 268; NC_000019).

[0048] 2. Plasmid construction:

[0049] 2.1 Design of primers

[0050] A, proAMH, according to proAMH (1~451aar) sequence (amino acid sequence as shown in SEQ ID NO:1, corresponding DNA sequence as shown in SEQ ID NO:2), design a pair of base sequence complementary random primers:

[0051] proAMH-F: 5'-TATGATGCGTGATTTACCTTTGACTTCT-3' (SEQ ID NO: 3);

[0052] proAMH-R: 5'-AATTCTTATCAACGCTGCGCCCTTC-3' (SEQ ID NO: 4).

[0053] B, Mature AMH, according to the Mature AMH (452~560aar) sequence (amino acid sequence as shown in SEQ ID NO:5, corresponding DNA sequence as shown in SEQ ID NO:6), design a pair of base sequence complementary random primers:

[0054] Mature AMH-F: 5'-TATGTCTGCTGGTGCCACTGC-3' (SEQ ...

Embodiment 2

[0110] Embodiment 2 The establishment of hybridoma cells and the preparation of monoclonal antibodies

[0111] 1. Immunotolerant mouse cells

[0112] Using gene recombination technology, the proAMH (1-451aar) and Mature AMH (452-560aar) recombinant proteins synthesized in Example 1 above were used as tolerogens to immunize 10 mice at a dose of 50 μg / mouse. Cyclophosphamide was injected at 10min, 24h, 48h and 6d after immunization for subtractive immunization, and then the titer of mouse tail blood was measured after immunization, and the mouse with the best subtractive effect was selected as the mouse model of successful tolerance, and the DNA immunity.

[0113] 2. Obtain mice with high immune response through DNA immunization.

[0114] Mice muscle gene injection: the mice were anesthetized with 0.75% pentobarbital sodium intraperitoneally, the abdomen was taken, and the leg posture was adjusted to expose the tibialis anterior muscle. After disinfection, use a 1mL syringe w...

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Abstract

The invention discloses an immune plasmid, a monoclonal antibody for detecting antimullerian hormone, a hybridoma and a preparation method and application thereof. A toleragen which consists of proAMH and Mature AMH is creatively used for immunizing rats to prepare immune tolerance rats, high-immune-response rats are obtained through suitable immunogen and by a DNA immunizing mode, finally, the hybridoma capable of secreting a monoclonal antibody for detecting antimullerian hormone can be obtained by screening, the monoclonal antibody secreted by the hybridoma or the monoclonal antibody obtained by the immunogen by the other mode can be directly used for detecting a connection part of proAMH and Mature AMH in original AMH, and the circumstance that ingredients such as a surfactant are added to carry out pretreatment such as dissociation on samples is avoided. Operation is simple and convenient, the monoclonal antibody can adapt to different immunological detection platforms, and can rapidly and accurately detect original AMH protein in samples.

Description

technical field [0001] The invention relates to the field of biochemical detection, in particular to an immune plasmid, a monoclonal antibody for detecting anti-Müllerian hormone, a hybridoma cell, a preparation method and application thereof. Background technique [0002] Anti-Mullerian hormone (AMH) is a member of the transforming growth factor β (TGF-β) superfamily. AMH is a dimeric glycoprotein composed of two identical 70kd subunits linked by disulfide bonds, with a relative molecular mass of 140kd. The human AMH-encoding gene is located on the short arm of chromosome 19. AMH is secreted by the immature Sertoli cells of the testis and the granulosa cells of the preantral and small antral follicles of the ovary. AMH is a regulator of follicle growth and development, and participates in two important recruitments in the process of physiological follicle formation: primordial follicle recruitment and dominant follicle recruitment. AMH is not affected by the menstrual cy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/16C12N5/20C07K16/26G01N33/577G01N33/74
CPCC07K14/575C07K16/26G01N33/577G01N33/74
Inventor 吴培钿袁江海王羽刘锋王涛王继华
Owner GUANGZHOU WONDFO BIOTECH
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