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Method for screening monoclonal antibodies on different binding sites of antigen

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

Active Publication Date: 2014-09-03
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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  • Method for screening monoclonal antibodies on different binding sites of antigen

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0026] Example 1 The preparation of anti-human ST2 monoclonal antibody hybridoma cells

[0027] Mix complete Freund's adjuvant and human ST2 solution at a ratio of 1:1 to make an emulsion. Immunize female Balb / c mice aged 6-8 weeks by subcutaneous injection in multiple points (200 microliters per mouse, about 40 microliters per point). Two weeks later, incomplete Freund's adjuvant and human ST2 solution were mixed at a ratio of 1:1 to make an emulsion, and the mice were strongly immunized with human ST2 subcutaneously. Two weeks later, blood was taken to determine the titer of antibodies. Human ST2 was injected into the tail vein to boost the immunization of the mice. Four days later, the mice were sacrificed, the spleen cells were isolated, and the isolated spleen cells were fused with mouse myeloma cells SP2 / 0 cells with 50% PEG. Then selection culture was carried out in HAT-containing medium. Only confluent cells survive in HAT medium.

Embodiment 2

[0028] Example 2 Screening of anti-human ST2 monoclonal antibody hybridoma cells

[0029] Add 100 microliters of human ST2 solution dissolved in PBS buffer solution with a concentration of 1 microgram per milliliter to the small wells of the 96-well microtiter plate, and place it overnight at 4 degrees. The next day, the human ST2-coated 96-well ELISA plate was washed twice with washing solution (PBS buffer containing 0.05% Tween). Then use blocking solution (1%BSA / washing solution) to block the plate for two hours at room temperature. Pour off the blocking solution, then add 100 microliters of cell culture supernatant to the hybridoma culture solution obtained in Example 1 and add it to a 96-well plate, incubate at room temperature for 2 hours, pour off the supernatant, wash with washing solution (containing 0.05% Tween in PBS buffer) to wash the plate twice, then add 100 microliters of alkaline phosphatase-labeled goat anti-mouse IgG antibody to the wells of the 96-well EL...

Embodiment 3

[0030] Embodiment 3 Cloning culture of hybridoma cells

[0031] Monoclonal hybridoma cells can be cultured by limiting dilution. The specific method is as follows. The positive anti-human ST2 antibody hybridoma cells obtained in Example 2 were counted and serially diluted to prepare a single cell suspension with a concentration of 1 cell / 200 microliters. Add 200 microliters of single cell suspension to the wells of a 96-well cell culture plate (1 cell / 0.2ml / well). Culture in a cell incubator with 5% carbon dioxide at 37°C. About 10 days or so select the monoclonal well, detect the antibody, if positive, clone again, generally clone 3 times. Select clones with strong antibody positive and good cell growth, expand culture, establish lines, and save them.

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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 invention relates to the field of biotechnology, in particular to a method for screening monoclonal antibodies with different binding sites of antigens. Background technique [0002] In 1975, German scientist Kohler and British scientist Milstein used hybridoma technology to fuse antibody-producing B lymphocytes with myeloma cells, and successfully established a monoclonal antibody preparation technology. The basic principle of monoclonal antibody technology is: after being stimulated by external antigens, mice can induce an immune response and produce corresponding antibodies. This function is undertaken by B lymphocytes; tumor cells can be cultured in vitro indefinitely. are permanent cells. The myeloma cells of the mouse and the splenocytes of the immunized mouse are fused under the mediation of polyethylene glycol, etc., and the fused hybridoma cells have the characteristics of the two parental cells. On the one hand, they can secrete specific...

Claims

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Application Information

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Patent Type & Authority Applications(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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