Method for detecting affinity of monoclonal antibodies of transmembrane proteins

A monoclonal antibody and transmembrane protein technology, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems that are not easy to be widely used, cannot be analyzed automatically, and radioactive pollution, so as to avoid radioactive isotopes, stable and reliable detection results, and high sensitivity. high effect

Active Publication Date: 2017-02-22
GMAX BIOPHARM +1
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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 p...

Method used

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  • Method for detecting affinity of monoclonal antibodies of transmembrane proteins
  • Method for detecting affinity of monoclonal antibodies of transmembrane proteins
  • Method for detecting affinity of monoclonal antibodies of transmembrane proteins

Examples

Experimental program
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Embodiment

[0027] 1. Construct a stable cell line with high expression of membrane protein

[0028] Inoculate 5×10 5CHO-DHFR-cells were transferred to 6-well plates and transfected with hET after 24 hours of culture A The R gene carrier (modified pIRES, Clontech, commercially available), the culture medium (DMEM / F12+10% dialyzed FBS) was replaced before transfection, and the transfection was carried out according to the transfection conditions recommended by Invitrogen Company Lipofectamine 2000. After 48 hours, change to the complete medium containing 10nM MTX, and change the medium every 3 days. After about two weeks, stable growth clones appear, digest and disperse cell colonies, and gradually increase MTX when the growth reaches 50% healing concentration up to 10 μM. The constructed stable cell lines were tested by FACS respectively, using polyclonal ET A R antibody (Abcam, commercially available) identifies the cell population after pressurization, CHO-DHFR-hET after screening A...

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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...

Claims

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Application Information

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IPC IPC(8): G01N33/533G01N33/577
CPCG01N33/533G01N33/577
Inventor 景书谦汪笑峰张成
Owner GMAX BIOPHARM
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