Production of anti-insulin monoclonal antibody and its clinical application.
Hybridoma-produced monoclonal antibody (MoAb) against insulin is useful for insulin assays because of its specificity and plentiful supply. The spleen cells of male BALB/c mice immunized against monocomponent porcine insulin were hybridized with mouse myeloma cells (P3-X63-Ag8-U1). The resulting anti-insulin antibody (Ab) was purified and characterized by radioimmunoassay (RIA) using 125I-porcine insulin and sandwich-method enzyme immunoassay (EIA) using Ab-conjugated beads and beta-galactosidase. For reference, we used anti-insulin polyclonal antibody raised in guinea pigs (PoAb). Using the Ouchterlony technique, we identified the antibody as being of subclass IgG 1. We judged this antibody to be MoAb because it did not react at all with porcine insulin during EIA, in concentrations between 0 and 12.5 ng/ml; in contrast, PoAb reacted dose-dependently. During RIA, this Ab did not cross-react with glucagon, somatostatin or pancreatic polypeptide. It did cross-react with human and bovine insulins but not with rat insulin. The proper concentration of this MoAb for RIA proved to be 1:1,500,000 and the smallest detectable level of porcine insulin by RIA using this Ab was 0.5 ng/ml. These levels were similar to those obtained with PoAb. The binding activity of this MoAb to human insulin was quite similar to that of porcine insulin. RIA insulin determinations using our MoAb correlated well with those employing PoAb.
Ishihara
K
K
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
Taniguchi
H
H
Hara
Y
Y
Ejiri
K
K
Baba
S
S
Shiroza
S
S
D000818:Animals; D000911:Antibodies, Monoclonal; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007124:Immunoenzyme Techniques; D007328:Insulin / Q000032:analysis* / Q000276:immunology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D011863:Radioimmunoassay; D013552:Swine
D000911:Antibodies, Monoclonal; D007328:Insulin