Following optimization of the reaction conditions, e.g. concentration of oxidizing agents, reaction time, volume of reaction mixture, and pH, chloramine T and the new iodination reagent, Iodogen, were compared for their effectiveness in radioiodination of insulin, glucagon, human growth hormone (hGH), and rabbit anti-mouse IgG. The radioactive peptide hormones prepared were analyzed for the presence of aggregate and breakdown products by polyacrylamide gel electrophoresis (PAGE) at pH 8.9, the rabbit anti-mouse IgG was tested for the presence of low molecular weight damage products by gel filtration on Sephadex G-50. The results demonstrate that with respect to iodine incorporation, specific activity, and immunological reactivity either method can be used to prepare under carefully controlled conditions a wide range of tracers with high specific activity at minimal oxidation damage. These tracers are shown to be highly suitable in radioimmunoassays after previous purification by PAGE and gel filtration, respectively.
A sensitive radioimmunoassay (RIA) for canine C-peptide (CCP) was established using synthetic CCP, a specific antiserum, and rabbit anti-guinea pig serum. Radioiodination was performed according to a modified chloramine-T method. Tracer preparations were used for long as 6 weeks after iodination. The standard curve ranges from 0.028 to 3.0 nmol/l. The intra-assay coefficient of variation (CV) was 3-5% and the inter-assay CV was 6-9% in the optimal range between 0.3 and 0.8 nmol/l. The average recovery of CCP added to plasma samples was 100.6% (n = 9). Canine insulin, porcine proinsulin, bovine proinsulin, and human C-peptide exhibited no cross-reactivity. The mean fasting plasma CCP concentration was 0.089 +/- 0.021 nmol/l in normal dogs and -0.005 +/- 0.007 nmol/l (mean +/- SEM) in diabetic dogs, respectively.
To investigate whether insulin antibody (IAB) formation is associated with the appearance of islet cell cytoplasmic antibodies (ICA), islet cell surface antibodies (ICSA) and insulitis Balb/c Bln mice were immunized with porcine insulin in combination with or without Freund's adjuvant. The animals received 8 i.p. injections and were followed up to 150 days for the development of antibodies and insulitis. Mice immunized with insulin in CFA developed IAB as well as ICSA. Mice only receiving Freund's adjuvant emulsified in saline also developed ICSA. ICA were not detectable. Inflammatory infiltrates were found in the exocrine pancreatic parenchyma but not in islets. The results show that nonspecific stimulation of the immune system and the application of insulin as antigen leads to both the formation of ICSA and IAB, while insulitis was not detectable.
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