DEVELOPMENT AND VALIDATION OF 2 SOLID-PHASE ENZYME IMMUNOASSAYS (ELISA) FOR QUANTITATION OF HUMAN EPIDERMAL GROWTH-FACTORS (HEGFS)

Purpose. The purpose of the present investigation was to develop and v alidate two separate enzyme-linked immunosorbent assays (ELISA) for qu antitation of exogenous human epidermal growth factor (hEGF1-53) and i ts truncated fragment (hEGF1-48) in rat plasma. Methods. The present a ssay systems were based on the sandwiching of the antigen between a mo noclonal mouse anti-hEGF1-53 antibody, pre-coated on a 96-well polysty rene plate, and a polyclonal rabbit anti-hEGF1-48 antibody, which is t hen detected with a peroxidase-labeled goat anti-rabbit antibody. Resu lts. The calibration curves for hEGF1-48 and hEGF1-53 in plasma were v alidated over a concentration range of 7.8-250 and 62.5-1000 pg/ml, re spectively. Determined from replicate assays of hEGF1-48 quality contr ol samples, the intra-assay precision and accuracy were less than or e qual to 8.8% RSD and within +/-9.8%; and the inter-assay precision and accuracy were less than or equal to 14.8% RSD and within +/-9.7% RE, respectively. Determined from replicate assays of hEGF1-53 quality con trol samples, the intra-assay precision and accuracy were less than or equal to 10.0% RSD and within +/-8.5%; and the inter-assay precision and accuracy were less than or equal to 10.0% RSD and within +/-5.7% R E, respectively. The limit of quantitation of the hEGF1-48 and hEGF1-5 3 assay using 200 mu l plasma per well is 7.8 and 62.5 pg/ml, respecti vely. These two ELISA methods are specific to hEGFs and do not cross-r eact with mouse EGF or other growth factors (TGF(alpha), TGF(beta), PD GF, and FGF) or lymphokines (IL(1 beta) and TNFalpha). These validated methods have been routinely applied to assay of plasma samples from v arious pharmacokinetic studies in rats receiving intravenous hEGFs. Bo th assay methods were also adapted to assay endogenous hEGFs in biolog ical fluids of different animal species. Conclusions. Two sensitive EL ISA methods have been validated for quantitation of hEGF1-53 and hEGF1 -48 in rat plasma. Their utility has been demonstrated in the applicat ion of assaying immunoreactive concentrations of exogenous and endogen ous epidermal growth factors.