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

• Published in: Clinica chimica acta 2008-08, Vol.394 (1-2), p.63-66
• Main Authors: González, Ernesto Carlos, Marrero, Neivis, Pérez, Pedro Lucio, Frómeta, Amarilys, Zulueta, Orlando, Herrera, Darlenis, Martínez, Luisa, Castells, Elisa
• Format: Article
• Language: English
• Subjects: 17-alpha-Hydroxyprogesterone - blood; 17-alpha-Hydroxyprogesterone - immunology; Antibody Specificity - immunology; Cross Reactions - immunology; Humans; Immunoenzyme Techniques - instrumentation; Immunoenzyme Techniques - methods; Microchemistry - instrumentation; Microchemistry - methods; Micropore Filters
• ISSN: 0009-8981
• DOI: 10.1016/j.cca.2008.04.004

17alpha-hydroxyprogesterone has been used for the diagnosis of congenital adrenal hyperplasia (CAH) in the newborn period. 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. 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 17alpha-hydroxyprogesterone kit. The analytical performance characteristics of our 17-OHP Neonatal UMELISA suggest that it can be used for the neonatal screening of CAH.