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Humanized antibody of dermatomyositis specific antigen peptide, preparation method and application

An antibody and antigen technology, applied in the field of biomedicine, can solve the problems of inability to quantitatively detect the level of anti-MDA5 antibodies in patients and poor consistency of test results, and achieve the effect of good stability and high antibody activity

Inactive Publication Date: 2022-08-02
SUZHOU FANGKE BIOTECHNOLOGY CO LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Domestic anti-MDA5 antibody detection methods are qualitative western blotting and immunomembrane strip method, the consistency of the test results is poor, and the level of anti-MDA5 antibody in the patient's serum cannot be quantitatively detected

Method used

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  • Humanized antibody of dermatomyositis specific antigen peptide, preparation method and application
  • Humanized antibody of dermatomyositis specific antigen peptide, preparation method and application
  • Humanized antibody of dermatomyositis specific antigen peptide, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0128] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given for illustrative purposes only, since after reading this detailed description, Various changes and modifications will become apparent to those skilled in the art.

[0129] All reagents used in the examples, unless otherwise noted, are commercially available.

Embodiment 1

[0130] Example 1: Construction method of human ScFv phage display library

[0131] Table 1 Main reagents used in this example

[0132]

[0133] 1. Library construction

[0134] 1.1 Assembly of variable heavy (VH) and variable light (VL) domains

[0135] Table 2 PCR reaction conditions and steps

[0136]

[0137]

[0138] Among them, the three steps of denaturation, annealing, and extension (1) were repeated 30 times

[0139] Primer sequence:

[0140] Forward(F):

[0141] 5'L-VH1: ACAGGTGCCCACTCCCAGGTGCAG (SEQ ID NO: 23)

[0142] 5'L-VH3: AAGGTGTCCAGTGTGARGTGCAG (SEQ ID NO: 24)

[0143] 5'L-VH 4 / 6: CCCAGATGGGTCCTGTCCCCAGGTGCAG (SEQ ID NO: 25)

[0144] 5'L-VH 5 / 7: CAAGGAGTCTGTTCCGAGGTGCAG (SEQ ID NO: 26)

[0145] 5'L VK 1 / 2: ATGAGGSTCCCYGCTCAGCTGCTGG (SEQ ID NO: 27)

[0146] 5'L VK3: CTCTTCCTCCTGCTACTCTGGCTCCCAG (SEQ ID NO: 28)

[0147] 5'L VK 4 / 5: ATTTCTCTGTTGCTCTGGATCTCTG (SEQ ID NO: 29)

[0148] 5'L Vλ1: GGTCCTGGGCCCAGTCTGTGCTG (SEQ ID NO: 30)

[0149] ...

Embodiment 2

[0204] Example 2: Preparation of monoclonal antibody that specifically binds to MDA5

[0205] Table 8 Main reagents used in this example

[0206] reagent Numbering manufacturer 96-well plate 42592 Costar Tween 20 P2287 Sigma Tris RES3098T-B7 Sigma Glycine G8200 Solarbio PEG 181986 Sigma PBS C10010500BT Life BSA A104912-100g aladdin Skim milk 6342932 BD

[0207] 1. The first round

[0208] 1.1 Biopanning

[0209] 1.1.1 Coating: Coat the immunotube and incubate at 4°C overnight. Antigen group: 1 mL of MDA5 transfection solution (50 μg / mL), control group: 500 μL of transfection solution (0 μg / mL).

[0210] 1.1.2 Washing: Discard the liquid in the immunotube and wash three times with 5 mL of 0.05% PBST.

[0211] 1.1.3 Blocking: add 5 mL of 5% nonfat milk (dissolved in PBST) to the tube and incubate at 37°C for 2 hours.

[0212] 1.1.4 Washing: Discard the liquid in the immune tube and wash i...

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Abstract

The invention relates to a human antibody of a dermatomyositis specific antigen peptide, a preparation method and application. Specifically, the invention relates to a monoclonal antibody, the antibody is specifically combined with MDA5 antigen protein, a preparation method of the monoclonal antibody and application of the monoclonal antibody in preparation of drugs for diagnosis, prevention or treatment of inflammatory myopathy or complications thereof. The anti-MDA5 antibody or antigen binding fragment screened by the invention is specifically bound with an MDA5 antigen, can be used as a reference standard for qualitative detection of MDA5 positive, realizes qualitative or quantitative detection of the anti-MDA5 autoantibody level in patients with inflammatory myopathy or complications thereof, and is of great significance in clinical diagnosis, disease monitoring and treatment of patients with DM, CAMD, DM-ILD, CADM-ILD and the like.

Description

technical field [0001] The present disclosure belongs to the field of biomedicine, and relates to a humanized antibody of a dermatomyositis-specific antigen peptide, a preparation method and an application thereof. Specifically, the present disclosure relates to a monoclonal antibody that specifically binds to the MDA5 antigen protein, a preparation method of the monoclonal antibody and the preparation of the aforementioned monoclonal antibody in the diagnosis, prevention or treatment of inflammatory myopathy or its complications. use in medicines. Background technique [0002] Inflammatory myopathy (IM) is a group of heterogeneous diseases characterized by skeletal muscle inflammatory cell infiltration and muscle fiber necrosis. Dermatomyositis (DM) is the most common subtype of inflammatory myopathy, mainly manifesting as different degrees of skin, muscle and lung involvement. The incidence of DM in adults is (1-6) / 100,000. A common complication of DM is interstitial lun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N15/13G01N33/564G01N33/577A61K39/395A61P21/00A61P29/00A61P11/00
CPCA61P21/00C07K2317/56C07K2317/565G01N2800/10C07K16/18C12N15/70G01N33/6893A61P17/00G01N2333/47A61K2039/505C07K2317/51C07K2317/515C12R2001/19
Inventor 王雅楠杨衡王迁段利华曾小峰
Owner SUZHOU FANGKE BIOTECHNOLOGY CO LTD
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