Fluorescence enzyme immunoassay of 21-deoxycortisol in plasma and dried blood sample on filter paper

An enzyme immunoassay of 21-deoxycortisol (21-DOF) in plasma and dried blood spotted on filter paper has been developed. 21-DOF was conjugated to horseradish peroxidase by the mixed anhydride method. Separation of free and bound fractions was done by the use of insolubilized antibody, prepared by coating polyacetal beads with purified IgG of goat anti-rabbit IgG serum. The enzyme activity was measured by the fluorophotometric method using 3-(p-hydroxyphenyl) propionic acid and H2O2 as substrates. The sensitivity of the present method was 0.5 pg/tube for 21-DOF. The intra- and interassay coefficients of variation were 3.2 and 8.2, and 7.9 and 9.2% respectively. The present enzyme immunoassay could be applied to mass-screening for congenital adrenal hyperplasia.