Method and screening kit for screening and validating anti-rabies virus neutralizing antibody from phage antibody library

Abstract

The invention relates to the technical field of biomedical engineering, and discloses a method for screening and verifying neutralizing antibodies against rabies virus from a phage antibody library, including A, culturing monoclonal phage antibodies, and taking the culture supernatant; B, qualitative analysis : The culture supernatant in step A was placed in a 96-well plate, followed by DMEM medium and neutralizing virus neutralization, BSR cell suspension culture, pre-cooled acetone fixation, and diluted FITC-labeled anti- Co-incubation with rabies virus nucleoprotein antibody, select clones that can significantly inhibit virus infection and sequence them, eliminate repetitive sequences, and initially obtain anti-rabies virus antibody sequences with different neutralizing activities; C. Quantitative analysis: select different sequences with neutralizing activities Each clone of the phage antibody particles was re-prepared, purified and diluted to a certain number of times, and the in vitro neutralization activity was determined by RFFIT method; and D, the transient expression and activity analysis of the whole molecule antibody.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to a method for screening and verifying anti-rabies virus neutralizing antibodies from a phage antibody library and an antibody screening kit. Background technique [0002] Rabies is a serious and fatal disease of which humans and all warm-blooded animals are susceptible. Rabies belongs to the Rhabdoviridae family and is mainly divided into 7 genotypes. Rabies virus is an RNA virus, which encodes five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), RNA-dependent RNA polymerase (L) and glycoprotein (G). , G) is a transmembrane protein, which constitutes the protrusion on the surface of the virus and is the main protective antigen, which can induce the production of protective antibodies. The glycoprotein is mainly composed of 3 major antigenic epitopes and other minor epitopes. Epitope I is located at AA226-331, which is a linear epito...

AI Technical Summary

Problems solved by technology

Since some proteins are expressed in the form of inclusion bodies in Escherichia coli, and inclusion body renaturation is difficult, the usual strategy is to discard the antibody sequences expressed in the form of inclusion bodies, which may cause some antibodies with high neutralizing activity to be lost in this step. At the same time, due to the large number of sequences, the verification process needs to invest a lot of resources and time

[0008] Therefore, the traditional initial screening method of phage antibody library has low screening efficiency and high investment. Only a small amount of antibodies can enter the next analysis in each step, and there are many positive clones lost due to the failure of scFv expression or precipitation, and the final obtained full It is difficult to guarantee the neutralizing activity of molecular antibodies, and it is urgent to find another way to find an efficient anti-rabies virus neutralizing antibody screening method

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