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Hybridoma cell strain capable of secreting anti-human AB blood type IgM type monoclonal antibodies, monoclonal antibodies, and application

A hybridoma cell line and monoclonal antibody technology, applied in anti-blood group antigen immunoglobulin, anti-animal/human immunoglobulin, analytical materials, etc.

Active Publication Date: 2020-03-24
苏州国科思倍达生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no real anti-AB monoclonal antibody has been obtained in the world. Although some monoclonal antibodies are named anti-A, (B) monoclonal antibodies, they strongly agglutinate human A (or B) red blood cells, but they are not effective for human B (or A) red blood cells. Restricted agglutination, it must be mixed with at least one anti-B (or anti-A) monoclonal antibody reagent to be used as an anti-AB antibody detection reagent

Method used

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  • Hybridoma cell strain capable of secreting anti-human AB blood type IgM type monoclonal antibodies, monoclonal antibodies, and application
  • Hybridoma cell strain capable of secreting anti-human AB blood type IgM type monoclonal antibodies, monoclonal antibodies, and application
  • Hybridoma cell strain capable of secreting anti-human AB blood type IgM type monoclonal antibodies, monoclonal antibodies, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Obtaining hybridoma cells

[0025] 1. Preparation of immunogen and detection antigen

[0026] (1) The immunogen is fresh human A and B anticoagulant erythrocytes, which are washed four times with normal saline, and prepared into 5% erythrocyte suspension with normal saline.

[0027] (2) The detection antigen is fresh human A, B, O anticoagulated erythrocytes, which are washed four times with normal saline, and prepared into a suspension of erythrocytes of required concentration with normal saline.

[0028] (3) Immunogens and detection antigens are ready-to-use and ready-to-use to ensure fresh red blood cells.

[0029] 2. Immunization of Animals

[0030] (1) BALB / c female mice (albino laboratory mice) with a weight of about 20 g at the age of four weeks were immunized with the immunogen, and 500ul of the immunogen was directly injected intraperitoneally.

[0031] (2) Immunize once every other week.

[0032] (3) After three immunizations, 50ul immunogen was injected i...

Embodiment 2

[0044] Preparation of Monoclonal Antibody Reagent Against Human AB Blood Type

[0045](1) The cell line 2D12 obtained in Example 1 was acclimatized and cultured in phenol red-free gibco-1640 medium containing 5% bovine serum, and expanded.

[0046] (2) When the titer of the cell culture supernatant reaches 64, collect the cell culture supernatant and add a stabilizer.

[0047] (3) The cell culture supernatant was frozen at -20°C, then concentrated by freeze-thawing and ultrafiltration, and the titer could reach 512 / 1024.

[0048] (4) According to production requirements, the concentrated anti-human AB blood type monoclonal antibody is properly diluted, sterile filtered, and stabilizers and color indicators are added according to requirements. It can be used as a monoclonal antibody reagent against human AB blood type to identify human ABO blood type or as a raw material for human ABO blood type identification kit.

Embodiment 3

[0050] Specific identification of secreted monoclonal antibody against human AB blood type

[0051] 1. The monoclonal antibody against human AB blood type prepared in Example 2 was specifically identified by the saline test tube method. Specific steps are as follows:

[0052] (1) Take A 1 , A 2 , B, O blood group 3 cases each, wash 3 times with normal saline, centrifuge at 2000rpm for 5 minutes for the last time, and prepare 2% erythrocyte suspension with normal saline.

[0053] (2) 4 test tubes per group, 3 groups in total, each adding 0.1ml of the antibody to be tested (the anti-human AB blood type monoclonal antibody prepared in Example 2), and then adding 2% A to each test tube 1 , A 2 , B, O blood group red blood cell suspension 0.1ml, react at room temperature for 15 minutes, centrifuge at 1000r / min for 1min, observe with the naked eye whether there is agglutination, and make red blood cell suspension control at the same time (the antibody to be tested is changed to ...

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Abstract

The invention provides a hybridoma cell strain capable of secreting anti-human AB blood type IgM type monoclonal antibodies. The preservation number of the hybridoma cell strain is CGMCC NO. 18535. The invention also provides the monoclonal antibody secreted by the hybridoma cell strain capable of secreting anti-human AB blood type IgM type monoclonal antibodies, and application of the monoclonalantibody. A preparation method comprises following steps: taking a human AB type erythrocyte suspension as an antigen to immunize BALB / c mice; carrying out immunization, cell fusion, screening and cloning to obtain a hybridoma cell strain 2D12 capable of stably subculturing and secreting anti-human AB blood type IgM type monoclonal antibodies; the secreted 2D12 antibody can agglutinate with humanA-type red blood cells, B-type red blood cells and AB-type red blood cells, no agglutination reaction with human O-type red blood cells is caused, the secreted 2D12 antibody is an anti-AB-type blood type monoclonal antibody, and has antibody titer of 512.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a hybridoma cell line secreting IgM monoclonal antibody against human AB blood type, monoclonal antibody and application thereof. Background technique [0002] Since Voak (1980) and Sacks (1981) successively established hybridoma cell lines secreting anti-A and anti-B monoclonal antibodies with lymphocyte hybridization technology, many anti-A and anti-B cells with good characteristics have been developed so far. Strains, anti-A, anti-B monoclonal antibody reagents instead of human serum for ABO system blood group identification, the application has been very extensive and reliable. However, no real anti-AB monoclonal antibody has been obtained in the world. Although some monoclonal antibodies are named anti-A, (B) monoclonal antibodies, they strongly agglutinate human A (or B) red blood cells, but they are not effective for human B (or A) red blood cells. For restrictive agglutinati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/34C12N5/20G01N33/80G01N33/577
CPCC07K16/34G01N33/577G01N33/80
Inventor 刘欢王强王现华李勇
Owner 苏州国科思倍达生物技术有限公司
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