CHARACT OF SPECIFICITY OF ANTIBODIES TO TRYPSINOGEN ACTIVATION PEPTIDE LE

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Antibodies to trypsinogen activation peptide (TAP) are potentially of great value as a marker for studying the intracellular processing of trypsinogen (TG) to trypsin in the early stages of acute pancreatitis. However this requires that these antibodies have a very high selectivity for TAP compared to the much more plentiful TG in the acinar cell. Antibodies to TAP were generated by immunizing rabbits with a synthetic peptide containing the N-terminal of rat TG (LPLDDDDDK) coupled to thyroglobulin. Antibodies were affinity purified employing either the peptide used for immunization or its C-terminal 5 amino acid (DDDDK). The resulting antibody preparations were designated AB9 and AB5 respectively. On immunoblot, AB5 recognized TG at a level only about 5% of that observed with AB9. Because of the potential limitations of ELISA resulting from breakdown of TG in aqueous solution with exposure of the TAP epitope, the specificities of the antibodies were confirmed using mass spectrometry to precisely analyze the molecular masses of the immunoprecipitated products involving AB9, AB5, or commercially available rabbit anti-TG antibodies, following binding of these products to the protein A agarose beads. Compared to results of immunoprecipitation of TG with rabbit anti-TG antibody, binding of TG by AB9 was substantially less; and AB5 binding to TG was markedly reduced further compared to that with AB9. In competitive binding experiments with equal molar amounts of TG and a peptide containing the TAP epitope, no binding of AB5 to TG was detected. Using the immunoprecipitation/mass spectrometry cont... approach to establish antibody selectivity, we have determined that generation of antibodies that demonstrate considerable selectivity for TAP over TG requires affinity purification with a short peptide immediately adjacent to the TG activation site to reduce recognition of the far N-terminal of TG. S.M. Chepilko, T. Otani, F.S. Gorelick, L. Mazzrrelli, R. Wang, and J.H. Grendell (1997), Pancreas 15(4), 431.