An enzyme immunoassay for determining 17α-hydroxyprogesterone in dried blood spots on filter paper using an ultramicroanalytical system

Abstract

Background 17α-hydroxyprogesterone has been used for the diagnosis of congenital adrenal hyperplasia (CAH) in the newborn period. Methods A simple and rapid competitive ultramicro ELISA assay based on competition between 17-OHP-alkaline phosphatase conjugate and 17-OHP in blood specimens for a limited number of binding sites on specific polyclonal rabbit anti-17-OHP antibodies, has been developed for the measurement of 17-OHP in dried blood spots on filter paper. The assay buffer contains danazol to displace 17-OHP from steroid-binding proteins. Results The 17-OHP assay was completed in 3 h, with a measuring range of 10–250 nmol/l. The intra- and inter-assay CV were 5.5–8.2% and 6.4–9.1%, respectively, depending on the 17-OHP concentrations. The recovery ranged from 98–103%. Of 3750 newborn samples collected on filter paper, 903 from the national neonatal screening program were analyzed, and the mean 17-OHP concentration was 12.2 nmol/l. Our assay showed high Pearson and concordance correlations with the commercially available ICN Neoscreen ELISA 17α-hydroxyprogesterone kit. Conclusions The analytical performance characteristics of our 17-OHP Neonatal UMELISA suggest that it can be used for the neonatal screening of CAH.