ENZYME IMMUNOASSAY FOR CONJUGATED 7α-HYDROXY-3-OXO-4-CHOLENOIC ACID IN HUMAN URINE

Abstract

A microplate enzyme immunoassay (EIA) was developed for the measurement of glycine- and taurine-conjugated 7α-hydroxy-3-oxo-4-cholenoic acids (CDCA-Δ4-3-one) in human urine. The antiserum was prepared by immunizing rabbits with N-(7α-hydroxy-3-oxo-4-cholen-24-oyl)-3-aminopropionic acid - bovine serum albumin conjugate. A colorimetric EIA was established using horseradish peroxidase-labeled antigen having a shorter bridge length than that of the immunogen, and 3, 3', 5, 5'-tetramethylbenzidine/hydrogen peroxide for the measurement of the enzyme activity. The reactivities of the antiserum for glycine and taurine conjugates of CDCA-Δ4-3-one was almost the same. The specificity of the antiserum was investigated by determining the cross-reactivities of various bile acids and related compounds. An appropriate dose-response curve for conjugated CDCA-Δ4−3-one was obtained in the range of 0.05–10 ng/well. This method was used for direct analysis of conjugated CDCA-Δ4−3-one in urine of healthy infants and patients with liver diseases.