| Background Developing from polyclonal antibodies to monoclonal antibody drugs, antibodies had been widely applied in biological science and medical technological products. Compared with mouse monoclonal antibodies, rabbit monoclonal antibodies (RabMAbs) had the advantages of higher binding affinity and specificity, and was easier to be humanized. Transient receptor potential channels melastatin-2 (TRPM2) was a nonselective Ca2+-permeable cation channel. It was regulated by ADPR(Adenosine diphosphate ribose) and H2O2, when TRPM2 was activated by ADPR and H2O2, it could regulate Ca2+ channel, it was related to the pathological disease by energy metabolism repair and oxidative stress. But there were mainly TRPM2 polyclonal antibodies on the market. Objective To prepare TRPM2 RabMAb. Methods In our experiment, we synthesized three peptides of TRPM2, YCA-10987, YCA-10988 and YCA-10989, immuned three experimental rabbits for each peptide. After preliminary screening by serum blot experiment, the splenocytes were fused with rabbit myeloma-like cell line of 240E (Epitomics Inc., USA), then we acquired rabbit-rabbit hybridoma cells which secreted TRPM2 antibodies. The fused cells were maintained in HAT selection medium. To acquire positive clones, ELISA and WB assays were used for screening. The one with the strongest signal of WB was chosen to plasmid recombinant phase. After further screening, the best plasmid pair was used for large-scale transfection (production). Results YCA-10988 immune failed in immunization. YCA-10987 and YCA-10989 displayed different effects in WB application, in which YCA-10987 was detected with a strong signal in over-expression lysate but a weak one in cell lysate, while YCA-10989 was opposite.Conclusion In our experiment, we synthesized three peptides of TRPM2 by hybridoma and recombinant technology. YCA-10987 and YCA-10989 displayed different effects in WB application, but can meet the different test requirements and have potential application prospects. |
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