Abstract
Anti-insulin monoclonal antibody (MoAb) is considered to be useful in terms of its specificity and stable supply for insulin assay when produced by hybridoma method. The spleen cells of male BALB/c mice immunized with monocomponent porcine insulin were hybridized with mouse myeloma cells (P3-X63-Ag8-U1). The resulting anti-insuln antibdy (Ab) was purified and characterized with radioimmunoassay (RIA) using 125I-porcine insulin and enzyme immunoassay (EIA) of sandwich method using 125I-Ab-conjugated beads and β-galactosidase. As reference, anti-insulin polyclonal antibody raised in guineapig (PoAb) was used.
The subclass of this Ab was identified to be IgG 1 by Ouchterlony technique. The EIA using this Ab revealed no enzyme reaction with porcine insulin, in the range of 0 and 12.5 ng/ml, suggesting that the present Ab was MoAb, while dose-dependent increase of the enzyme reaction was obtained using PoAb. In the RIA, this Ab did not cross-react with glucagon, somatostatin, and pancreatic polypeptide. The suitable concentration of this MoAb for RIA was 1: 1,500,000 and the minimum detection level of porcine insulin in RIA using this Ab was 0.5 ng/ml. They were similar to those obtained by application of the PoAb. The binding activity of this MoAb to human insulin was quite similar to porcine insulin. The insulin determined by RIA using this MoAb and PoAb was in good correlation.
