| Background Antibodies developed from the polyclonal antibodies to monoclonal antibody drugs. And had been widely applied to biological science and medical technological products. Compared with mouse monoclonal antibodies, rabbit monoclonal antibodies (RabMAbs) had the advantages of higher binding affinity and specificity, also easier to humanized. The number and subunit composition variation of synaptic N-methyl-D-aspartate receptors (NMDARs) play critical roles in synaptic plasticity, learning and memory, and neurological disorders. Protein tyrosine phosphorylation provides a powerful means of regulating NMDAR function.Objective To prepare NMDAR2B RabMAb. Methods In our experiment, we synthesized one peptide of NMDAR2B, YCA-13436, immuned three experimental rabbits. After preliminary screening by serum blot experiment, fused rabbit spleen cells that were probably immunized by NMDAR2B with rabbit myeloma-like cell line of 240E (Epitomics Inc., USA), acquired rabbit-rabbit hybridoma cells, secreted NMDAR2B antibodies. The fused cells were maintained in HAT selection medium. To acquire positive clones, ELISA and WB assays were used for screening. The strongest signal one of the WB was chosen to plasmid recombinant phase. After further screening, then the most strongly secreting pair for large scale production.Results The rabbit H1332 of YCA-13436 enter into cell fusion with good antiserum results. And the clone 37-d recognized specific Phosphorylated protein while showed negative to nonphosphorylated protein. Conclusion In our experiment, we synthesized one peptides of NMDAR2B, by hybridoma and recombinant technology, YCA-13436-37 works well in WB application and have potential application prospect. |
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