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Development and validation of two solid-phase enzyme immunoassays (ELISA) for quantitation of human epidermal growth
N Sizemore1, R C Dudeck, C M Barksdale
1Department of Oncology, Vanderbilt University, Nashville, Tennessee 37232, USA.
Purpose:
The purpose of the present investigation was to develop and validate two separate enzyme-linked immunosorbent assays (ELISA) for quantitation of exogenous human epidermal growth factor (hEGF1-53) and its truncated fragment (hEGF1-48) in rat plasma.
Methods:
The present assay systems were based on the sandwiching of the antigen between a monoclonal mouse anti-hEGF1-53 antibody, pre-coated on a 96-well polystyrene plate, and a polyclonal rabbit anti-hEGF1-48 antibody, which is then detected with a peroxidase-labeled goat anti-rabbit antibody.
Results:
The calibration curves for hEGF1-48 and hEGF1-53 in plasma were validated over a concentration range of 7.8-250 and 62.5-1000 pg/ml, respectively. Determined from replicate assays of hEGF1-48 quality control samples, the intra-assay precision and accuracy were < or = 8.8% RSD and within +/- 9.8%; and the inter-assay precision and accuracy were < or = 14.8% RSD and within +/- 9.7% RE, respectively. Determined from replicate assays of hEGF1-53 quality control samples, the intra-assay precision and accuracy were < or = 10.0% RSD and within +/- 8.5%; and the inter-assay precision and accuracy were < or = 10.0% RSD and within +/- 5.7% RE, respectively. The limit of quantitation of the hEGF1-48 and hEGF1-53 assay using 200 microliters plasma per well is 7.8 and 62.5 pg/ml, respectively. These two ELISA methods are specific to hEGFs and do not cross-react with mouse EGF or other growth factors (TGF alpha, TGF beta, PDGF, and FGF) or lymphokines (IL1 beta and TNF alpha). These validated methods have been routinely applied to assay of plasma samples from various pharmacokinetic studies in rats receiving intravenous hEGFs. Both assay methods were also adapted to assay endogenous hEGFs in biological fluids of different animal species.
Conclusions:
Two sensitive ELISA methods have been validated for quantitation of hEGF1-53 and hEGF1-48 in rat plasma. Their utility has been demonstrated in the application of assaying immunoreactive concentration of exogenous and endogenous epidermal growth factors.