A direct enzyme immunoassay of 6.BETA.-hydroxycortisol in human urine.

Abstract

A sensitive enzyme immunoassay for urine 6.beta.-hydroxycortisol was developed. Enzyme labeling of 6.beta.-hydroxycortisol was accomplished by the N-succinimidyl ester method. The use of N-succinimidyl ester of 6.beta.-hydroxycortisol 21-hemisuccinate and .beta.-galactosidase in an appropriate molar ratio provided a conjugate suitable for enzyme immunoassay. Antiserum was prepared by immunization with the 6.beta.-hydroxycortisol 21-hemisuccinate-bovine serum albumin conjugate in the rabbit. Sufficient sensitivity and improved specificity of the assay system could be obtained by the selective blocking of less specific antibodies. The quantitation limit of 6.beta.-hydroxycortisol was .apprx. 10 pg, which is comparable to that of radioimmunoassay. The intra- and inter-assay coefficients of variation for 6.beta.-hydroxycortisol in human urine were 5.9-8.1% and 2.8-12.3%, respectively.