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Preparation of monoclonal antibody against human m3 receptor
1Department of Pharmacology, Sun Yat-Sen University of Medical Sciences, Guangzhou 510089, China. Tawaipeng@163.net
Aim:
To raise monoclonal antibody against human m3 receptor.
Methods:
The m3 receptor selective peptide segments deduced from its gene were chemically synthesized, coupled to keyhole limpet hemocyanin carrier protein, and injected to Balb/c mice to raise monoclonal antibody. Antibody was purified by a combination of two-step precipitation methods and ion-exchange chromatography. The specificity of monoclonal antibody was tested by enzyme-linked immunosorbent assay, immunohistochemistry, and radioligand binding assay of receptors.
Results:
The monoclonal antibody specifically bound to the protein of rat salivary gland and m3 peptide, but not m4 peptide. In radioligand binding assay of receptors, monoclonal antibody inhibited the binding of 3H-QNB to muscarinic receptor in rat salivary gland, but not in rat heart, and could not inhibit the binding of 3H-PZ to rat brain cerebral cortex membrane protein. Immunohistochemical study showed that the human salivary cell surface was strongly stained, whereas the human heart cell surface was not.
Conclusion:
Highly purified (96.3%) monoclonal antibody against the m3 receptor peptide recognized the m3 receptor.