A method for detecting the affinity of transmembrane protein monoclonal antibody

Abstract

The invention discloses a method for detecting the affinity of monoclonal antibodies of transmembrane proteins. The method mainly includes steps of 1), constructing stable cell strains with membrane protein high expression to guarantee the maximum right shift of positive combination peaks in FACS (fluorescence-activated cell sorter) assay; 2), labeling antibodies by the aid of fluorecein; 3), carrying out saturation binding assay; 4), carrying out competitive binding assay; 5), processing data. The method has the advantages that the affinity of the monoclonal antibodies of the transmembrane proteins can be effectively measured; radioactive isotopes can be omitted, accordingly, the method is high in safety and sensitivity and good in repeatability, stable and reliable detection results can be obtained, and the like.

Description

technical field [0001] The invention relates to a method for detecting antibody affinity, in particular to a method for detecting the affinity of transmembrane protein monoclonal antibody. Background technique [0002] Antibody affinity is one of the important parameters to determine the properties of antibodies. It indicates the degree of firmness between the antibody binding site and the antigen epitope. It is often expressed by the dissociation equilibrium constant KD, which refers to the time required to bind half of the antigen molecules after reaching equilibrium. Antibody concentration. The binding affinity of antibody to antigen can provide the basis for the selection of monoclonal antibodies for different purposes. Antibodies with high affinity are useful for quantitative detection, diagnosis and targeted treatment of diseases. It can increase the positive detection rate or improve the efficacy of therapeutic antibodies. Antibodies with comparable affinity are used...

AI Technical Summary

Problems solved by technology

However, for multiple transmembrane proteins, such as GPCR or ion channel proteins, soluble extracellular domains without natural conformation or soluble expressed multiple transmembrane proteins with few natural conformations are used as antigens, and the biosensor method is used to detect antibodies and expressed membranes. Cell-to-cell affinity constants for protein antigens are also immature

However, radioimmunoassays for the affinity between antibodies and transmembrane proteins require that the antibodies be radioactively labeled. After the reaction is complete, the antigen-antibody complex needs to be separated from the free antigen and antibody. The experimental process is cumbersome and complicated, and it is not easy to carry out widely. RIA has high sensitivity, but it will cause radioactive pollution and hazards, and there are many shortcomings such as the short half-life of commonly used radionuclides and the inability to automate analysis.

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