Characterization of NSE monoclonal antibodies and establishment of a double-antibody sandwich ELISA assay

Objective:Preparation and characterization of monoclonal antibodies against NSE protein,and establishment of a double-antibody sandwich ELISA assay.Methods:BALB/c mice were immunized by using purified recombinant NSE,and monoclonal antibodies were generated by hydridoma technique.These antibodies were characterized with ELISA,Western blot,Immunofluorescent and Immunohistochemical staining.The isotypes of these antibodies were determined with an antibody isotyping kit.With Horseradish Peroxidase labelled NSE monoclonal antibody,we were able to establish a double-antibody sandwich ELISA to detect NSE protein.Results:Two positive hybridoma cell lines were selected for test,the titers of these two monoclonal antibodies could reach 4.2×107-6.5×107,and their isotypes were IgG2b.Our NSE antibodies could detect not only endogernous NSE protein from cells,but also secreted NSE protein from cells in culture medium by Western blot,in addition,they could be used for immunofluorescent and immunohistochemical staining.The minimum amount of NSE protein could be detected by this double-antibody sandwich ELISA was 8.85 ng/ml.Conclusion:Our NSE monoclonal antibodies achieved good sensitivity and specificity with high titers,and we established a double-antibody sandwich ELISA assay which could be used for clinical test in future.