A Quantitative Enzyme-Linked Immunosorbent Assay for Rat Insulin

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Abstract
A simple, quantitative micro-ELISA (Enzyme-Linked Immunosorbent Assay) has been developed for rat insulin. The micro-ELISA is a solid phase, indirect, competitive immunoassay. The useful range of the micro-ELISA was superior to that of a commercial RIA for rat insulin (e.g. 0.4 to 46.0 ng/ml for the ELISA; 0.2–8.6 ng/ml for the RIA). The ELISA's sensitivity (the lower limit of detection) was 1.0 ng/ml ± 0.13ng/ml, (mean, ± SEm; 9 assays) or 20 ± 2.6 pg/determination, and compared favorably with the sensitivity of a radioimmunoassay (RIA) for rat insulin (0.38 ± 0.10 ng/ml: 4 assays). The ELISA measured pure rat insulin standards accurately. Correlation experiments showed that the results of the ELISA agreed with those of the RIA (r=0.91), when rat insulin was assayed in crude extracts of isolated pancreatic Islets of Langerhans. When the standard curve was plotted as a log of the dose response curve, a sigmoidally shaped curve was obtained which could be transformed into a straight line relationship with a logit-log program. The goodness of fit of the transformed standard curve to a straight line relationship was excellent (r=0.97 to 0.99: n=4 ELISAs). The transform facilitated dose interpolation, tests of parallelism, and quality control. Tests of parallelism showed that the ELISA was specific for rat insulin. The precision of the ELISA was superior to the precision of the rat insulin RIA tested. The intraassay precision of the ELISA was always <10% (CV%), and its interassay precision was always ± 15% (CV%). The micro-ELISA is versatile, since it can be used to measure human, porcine, rat, and probably mouse insulin.