A method for screening monoclonal antibodies with different antigen binding sites

A monoclonal antibody and binding site technology, applied in the biological field, can solve problems such as a lot of workload and time, and achieve the effect of shortening time and simplifying screening steps.

Active Publication Date: 2015-09-30
NINGBO ACCUTECH BIOSCI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the case of a large number of hybridoma cell lines with positive clones of monoclonal antibodies, the detection of antigen-binding sites between cloned antibodies using traditional methods requires a lot of work and time

Method used

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  • A method for screening monoclonal antibodies with different antigen binding sites

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0025] Example 1 Preparation of ST2 Single Clone Antibody Hybrid Tumor Cell

[0026] Mix the complete blessing agent and human ST2 solution as a proportion of 1: 1 to make emulsions.Multi-point injection of immune to 6-8 weeks of age female Balb / C mice (200 micro rises per mouse, about 40 micro rising per point).Two weeks later, a lotion was mixed with an incomplete prefecture agent and human ST2 solution at a ratio of 1: 1.After two weeks, the blood measurement of the antibody was measured.After the end vein injection, the ST2 of the immune mice was strengthened. Four days later, the mice were executed, spleen cells were separated, and the separated spleen cells were merged with the mouse myeloma cell SP2 / 0 cells with 50% PEG.Then choose to train at the medium containing HAT.Only fusion cells can survive in the HAT medium.

Embodiment 2

[0027] Example 2 Anti -person ST2 monoclonal antibody hybrid tumor cell screening

[0028] The ST2 solution with a ST2 solution of 100 micro -ascending in the PBS buffer concentration is 1 micrograms per milliliter, respectively, and placed 4 degrees for one night at 4 degrees.The next day, washed the 96 -hole enzyme standard plate of the ST2 package (PBS buffer containing 0.05% TWEEN) twice.Then use a closed solution (1%BSA / washing liquid) room temperature to close the enzyme standard plate for two hours.Pour the closed liquid, then add 100 microcytopenia cells to 96 pores obtained by the hybrid tumor culture liquid obtained by the Example 1, and the room temperature is incubated for 2 hours.0.05% TWEEN PBS buffer) washing the board twice, and then added 100 microline alkalinase marked sheep antibody IgG antibodies to add 96 -hole enzyme standard plates, room temperature for 1 hour, washed with a lotion with a lotionSecond, add 100 micro-lift color reaction substrates (4-nitrate ...

Embodiment 3

[0029] Example 3 Hybrid Tumor Cell Cultivation Cultivation

[0030] Single cloned hybrid tumor cells can be cultivated by limited dilution method.The specific method is as follows. The hybrid tumor cell counting of the positive anti -human ST2 antibody obtained by the Example 2 is a single -cell suspension with a series of cells / 200 micro -rising single cells.Add 200 microcytes of single -cell suspension to a small hole (1 cell / 0.2ml / hole) of the 96 -hole cell culture plate.At 37 degrees, 5%carbon dioxide cell culture box culture.Choose a monoclonal hole for about 10 days to detect antibodies, such as positive, and then cloned. Generally, cloning 3 times.Choose a cloning of strong antibody positive and cellular growth, expand the cultivation, build, and save.

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Abstract

The invention discloses a method for screening monoclonal antibodies on different antigen binding sites. The method comprises the following steps: preparing an antigen specific monoclonal antibody by an antigen immune animal with two or more antigenic determinants through a hybridoma monoclonal technology; carrying out in-vitro cell culture on positive clone cell strains to obtain cell culture liquid supernatant containing the antigen specific monoclonal antibody; adding the hybridoma cell culture liquid supernatant into a solution of rubber latex grains which are covalently cross-linked with protein A or G in a two-and-two combined manner; and incubating and adding antigens to obtain a monoclonal antibody set with the increased light absorbance, namely obtain the monoclonal antibody with the different antigen binding sites.

Description

Technical field [0001] The present invention involves the field of biotechnology, and specifically involves a monoclonal antibody with different binding sites of antigen. Background technique [0002] In 1975, German scientist Kohler and British scientist Milstein used hybridoma technology to combine the antibody B lymphocytes with osteoma cells, and successfully established a monoclonal anti -system preparation technology.The basic principles of monoclonal antibody technology are: mice can induce immune response after being stimulated by external antigen to produce corresponding antibodies. This function is borne by B lymphocytes;It is a permanent cell.The mice's osteoma cells are fused under the media such as polyethylene glycol with the immune spleen cells of mice.On the other hand, it also has the ability to infinitely proliferation of tumor cells. It can be transplanted in in vitro or transplanted to infinite proliferation in the body, thereby secreting a large number of mon...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577C07K16/28
CPCC07K16/2866C07K2317/21G01N33/577G01N33/6869G01N2333/7155
Inventor 赖增祖叶欣徐健赖晓娟杨奎东
Owner NINGBO ACCUTECH BIOSCI LTD
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