A sensitive chemiluminescent enzyme immunoassay for the bioanalysis of carboxyl-terminal B-chain analogues of human insulin

Quantification of analogues of human insulin in biological matrices is comp licated by differences in their immunoreactivity and the presence of both t he analogue and endogenous concentrations of insulin in test samples. To fa cilitate pharmacokinetic comparisons of carboxyl-terminal B-chain analogues of human insulin. we undertook development of a sensitive ELISA. The ELISA detection method was optimized systematically to permit routine analysis o f 10-mul serum samples. Accordingly. a noncompetitive 'sandwich' chemilumin escent ELISA was validated for the quantification of carboxyl-terminal B-ch ain insulin analogues in human serum over a concentration range from 5 to 3 125 pM. The mean bias (RE%) within the validated range varied from -10.3 to 4.3%, with an intermediate precision (inter-assay CV%) from 4.2 to 11.5%.. The two-sided 90% expectation tolerance interval for total measurement err or was within +/- 25%, of the nominal concentration for all levels of valid ation samples. Insulin lispro, human insulin. proinsulin, despentapeptide i nsulin (DPI) and porcine insulin displayed comparable crossreactivity in th e ELISA. Potential utility of the new assay for insulin bioanalysis in nonh uman species was investigated by assessing the pharmacokinetic profile of D PI in rats following administration of a single subcutaneous dose. The sens itive chemiluminescent detection method is simple to perform and should be readily adaptable for ELISAs of other therapeutic proteins. (C) 2001 Elsevi er Science B.V. All rights reserved.