Title: Preparation and binding activity of monoclonal antibody against P53 protein
Abstract: OBJECTIVE To prepare monoclonal antibodies specially recognized P53 protein.METHODS Recombinant human wild-type P53 protein expressed in E.coli.BL21 was used as antigen.Monoclonal antibodies were prepared and identified via the classic protocol of monoclonal antibody preparation.Identified monoclonal antibodies were purified by affinity chromatography.Antibody titer was determined by enzyme linked immunosorbent assay(ELISA).The specific binding activity of antibody was detected by Westen blotting and immunohistochemistry.RESULTSThree antibodies,1P15,2P37 and 3P40,against P53 protein were prepared and purified by affinity chromatography.The purity of antibodies was higher than 90% shown by SDS-PAGE.The titers of antibodies were more than 1∶6000 determined by ELISA.All antibodies could recognize P53 protein by Western blotting,in which 3P40 had the sensitivity of 5 ng for P53.Two tumor cell lines with different p53 gene type(MDA-MB-231,p53 mutant;H1299,p53 null) were used to determine the binding activity of antibody 3P40 by Western blotting and immunohistochemistry.The result indicated that endogenous P53 protein in tumor cells could be identified by 3P40.CONCLUSIONThree monoclonal antibodies against P53 are prepared.Antibody 3P40 is the most sensitive and specific one to P53.
Publication Year: 2011
Publication Date: 2011-01-01
Language: en
Type: article
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