A monoclonal antibody-based enzyme-linked immunosorbent assay of ursodeoxycholic acid 3-sulfates in human urine

Sulfation of the 3-hydroxy group is assumed to be a major metabolic route o f ursodeoxycholic acid (UDCA) which is used for treating various hepatobili ary diseases. We have developed a sensitive enzyme-linked immunosorbent ass ay (ELISA) for determining the total amount of nonamidated, glycine- and ta urine-amidated ursodeoxychoric acid 3-sulfates (UDCA 3-Suls) using a newly established monoclonal antibody. In this study, 12 kinds of antibody-secret ing hybridoma crones were generated by a fusion experiment between P3/NS1/1 -Ag4-1 myeloma cells and the spleen cells from a BALB/c or an A/J mouse whi ch had been immunized with a conjugate of nonamidated UDCA 3-Sul and bovine serum albumin. One of the monoclonal antibodies, Ba-IO (gamma 2a, kappa), had suitable binding properties for clinical application, which was group-s pecific to the UDCA 3-Suls, and showed negligible cross-reactivities with v arious related bile acids including potentially interfering compounds, name ly, the unconjugated UDCA, UDCA 7-N-acetylglucosaminide, the 3-sulfates of cholic acid, chenodeoxycholic acid and deoxycholic acid. The antibody Ba-10 allowed us to develop a sensitive competitive ELISA system whose measurabl e range was approximately 2-200 pg per assay. A serial dilution study indic ated that the ELISA enables the direct measurement of the UDCA 3-Suls in hu man urine before and after the administration of exogenous UDCA. The daily urinary excretion rate of UDCA 3-Suls from healthy male volunteers (n = 5) was determined to be a mean of 131 +/- 61.2 (SD) mu g as the nonamidated UD CA 3-Sul equivalent. (C) 2000 Elsevier Science Ltd. All rights reserved.