Development Of Monoclonal Antibody Specific For Tyrosine Nitriationin Succinyl CoA: 3-oxoacid CoA Transferase

Nitration is a major modification caused by reactive nitrogen species in the condition of oxidative stress, one of the major product is 3-nitrotyrosine (3NT). Succinyl-CoA:3-oxoacid CoA transferase (SCOT) which is an important extra-hepatic mitochondrial rate-limiting enzyme involved in ketones metabolism has been found nitrated in diabetes. Two priority nitration residues Tyr4 and Tyr7 in recombinant protein SCOT were detected by LC-ESI-MS/MS. Because the protein abundance or peptide ionization ability was not satisfied, though decrease of SCOT activity had been reported in diabetes, the nitrated sites of SCOT in vivo was still not clear by mass spectrum. Development of a specific antibody will facilitate the study of SCOT nitration.In order to explore the nitrotyrosine site of SCOT in diabetes mouse model, in this study, we generated monoclonal antibodies special for nitration Tyr4 or Try76 residue of SCOT by two synthetic peptides that contained nitrated Tyr4 or Try76 conjugated with KLH and immunize Balb/c mouse. Spleen cell was fused with myeloma cells SP2/0 through the traditional PEG method to prepare hybridoma. After cloning and ELISA step screening, four monoclonal antibodies (CE-9, CE-14, CN-10, and CN-11) were isolated and regarded as specific monoclone antibody have reaction with respective antigen. The subclasses of antibodies were IgG1, titers were 1:128000, 1:32000,1:512000 and 1:4096000 respectively. A further characterization indicate that these four antibodies only recognized the two nitration residues severally and exhibited no cross-reaction with non-nitrated SCOT nor other possible nitration sites of SCOT and other three nitrated proteins by indirect ELISA and Western blot. Antibody CE-9 and CN-10 have a better specificity than CE-14 and CN-11 respectively. These antibodies lay the groundwork for the further nitration modification research in vivo.