Production of Anti-Glucagon Sera with A C-Terminal Fragment of Pancreatic Glucagon

The C-terminal region-specific anti-glucagon sera were raised in rabbits using as immunogen, a conjugate of BSA and a C-terminal fragment of pancreatic glucagon. The hapten was prepared by trypsin digestion of the glucagon, which was proved to be a 1: 3 mixture of glucagon (18-29) and (19-29). Six rabbits were immunized by subcutaneous injection of an emulsion of the conjugate with complete Freund's adjuvant and five of the rabbits produced antibodies to the glucagon (GC-1, GC-2, GC-3, GC-5 and GC-6). For comparison, rabbit antisera were also produced against glucagon polymer (GA-10) and syrupy glucagon fibrils (PGA-2). All these antisera as well as the pancreatic glucagon-specific antiserum 30K were characterized with dog gut-extract (gut-GLI) and glucagon-related peptide fragments in the radioimmunoassay systems. The assay systems utilized 125I-monosubstituted pancreatic glucagon as tracer and human mono-component glucagon as standard. All sera of the GCseries crossreacted with the dog gut-extract very weakly and antisera GC-5 and GC-6 exhibited the lowest crossreactivities with the extract, which were shown to be as low as that of 30k. Characterization of the antiserum GC-5 with purified glucagonrelated fragments indicated that the major antigenic determinant located exactly in the C-terminal region of glucagon. The present results clearly showed high efficiency of the use of the glucagon C-terminal fragment as haptenic immunogen in obtaining the C-terminal region-specific, i.e., pancreatic glucagon-specific antisera.