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Method for humanizing recombinant phages antibody library

A phage antibody library and humanized technology, which is applied in chemical libraries, organic compound libraries, peptide libraries, etc., can solve problems that limit the development of scientific research, and achieve the effects of simple preparation, large screening capacity, and strong screening ability

Inactive Publication Date: 2008-10-29
侯明 +2
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At present, there is no experimental technology that can express fully h

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  • Method for humanizing recombinant phages antibody library

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specific Embodiment approach

[0012] Detection method step of the present invention is as follows:

[0013] 1. Isolation and purification of peripheral blood mononuclear cells

[0014] (1) Aseptically collect 20ml of venous blood from the target population and inject it into a sterile vial containing 2ml of heparin, cover and shake well to anticoagulate.

[0015] (2) Add an equal volume of PBS buffer solution to the sterile pipette to dilute the blood and improve the separation effect.

[0016] (3) Draw 10ml of lymphocyte stratification solution into a 50ml conical bottom centrifuge tube, then tilt the centrifuge tube at an angle of 45°, and slowly add the diluted blood along the wall of the test tube at a distance of 1 cm from the interface of the stratification solution to the point of separation. layer on top of the liquid to make the interface between the two clear and prevent blood from mixing into the layered liquid.

[0017] (4) Place the centrifuge tube in a horizontal centrifuge and centrifuge a...

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Abstract

The invention belongs to a humanized phage antibody library method for synthesizing fully humanized antibody. The method adopts such technical principle that an entire set of heavy-chain variable region (VH) and light-chain variable region (VL) genes of an antibody are amplified from human immune cells by polymerase chain reaction (PCR) technology, cloned to a phage vector, and expressed on the surface of coat protein in a format of fusion protein. The desired antibody is then screened by antigen-antibody specific binding and is subjected to clonal amplification, so that the antibody gene is expressed by a host in a secretion manner to obtain soluble antibody fragment. The technology adopted by the invention obviates cell fusion in the conventional preparation of monoclonal antibody and can directly screen fully humanized specific antibody from the antibody library by using the antigen without immunization.

Description

Technical field: [0001] The invention belongs to a novel experimental method for synthesizing fully humanized phage antibody. Background technique: [0002] The development of antibody technology has gone through three important stages: the first generation is polyclonal antibody, which is characterized by the polyclonal antibody against mixed antigens obtained by immunizing animals with antigens; the second generation is produced by hybridoma technology against a Monoclonal antibodies to epitopes. The first and second-generation antibody technologies have made important contributions to the development of immunology, but because they are animal-derived antibodies, they are highly immunogenic to humans and are prone to human anti-mouse antibody reactions; in addition, hybridoma technology produces The low yield, high production cost, and poor stability and affinity of monoclonal antibodies inhibit clinical applications. So the third generation of antibodies - genetically e...

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Application Information

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IPC IPC(8): C40B40/10C40B50/06
Inventor 侯明冀学斌彭军张丽萍石艳秦平张爱军刘新光
Owner 侯明
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