Development of radioimmunoassays for free tetra-L-aspartyl-L-lysine trypsinogen activation peptides (TAP)

Tetra-L-aspartyl-L-lysine (D/sub 4/K) containing trypsinogen activation peptides were synthesised on solid-phase supports. Synthetic D/sub 4/K peptides were N-terminally haptenised and used to generate specific C-terminally directed anti-D/sub 4/K antibodies. Affinity purification of antisera using Sepharose-immobilised synthetic D/sub 4/K segregated two highly purifed populations of anti-D/sub 4/K antibodies, one eluting with EDTA recognising the calcium chelate and the other eluting with propionic acid recognising an alternative epitope on the anionic oligopeptide. Both specific anti-d/sub 4/K antibodies were C-terminally directed and did not bind trypsinogen. Specific antisera and calcium-independent antibodies were used to develop and characterise solution and solid-phase immunoassays specific for free trypsinogen activation peptides (TAP assay), with a detection limit of 10/sup -11/ M and between assay CV of 10.7% for the solution-phase system. The release of D/sub 4/K peptides by enteropeptidase activation of trypsinogen and dog pancreatic secretion is demonstrated. TAP assays specifically indicate trypsinogen activation and may contribute to the recognition and understanding of disease states such as pancreatitis