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 of ursodeoxycholic acid (UDCA) which is used for treating various hepatobiliary diseases. We have developed a sensitive enzyme-linked immunosorbent assay (ELISA) for determining the total amount of nonamidated, glycine- and taurine-amidated ursodeoxycholic acid 3-sulfates (UDCA 3-Suls) using a newly established monoclonal antibody. In this study, 12 kinds of antibody-secreting hybridoma clones 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 which had been immunized with a conjugate of nonamidated UDCA 3-Sul and bovine serum albumin. One of the monoclonal antibodies, Ba-10 (gamma2a, kappa), had suitable binding properties for clinical application, which was group-specific to the UDCA 3-Suls, and showed negligible cross-reactivities with various related bile acids including potentially interfering compounds, namely, 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 measurable range was approximately 2-200 pg per assay. A serial dilution study indicated that the ELISA enables the direct measurement of the UDCA 3-Suls in human 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) microgram as the nonamidated UDCA 3-Sul equivalent.
Kobayashi
N
N
Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai 980-8578, Japan.
Katsumata
H
H
Katayama
H
H
Oiwa
H
H
Goto
J
J
Takeuchi
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity; D002459:Cell Fusion; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008815:Mice, Inbred Strains; D012016:Reference Values; D012680:Sensitivity and Specificity; D013431:Sulfates / Q000276:immunology / Q000652:urine*; D014580:Ursodeoxycholic Acid / Q000276:immunology / Q000494:pharmacology / Q000652:urine*
D000911:Antibodies, Monoclonal; D013431:Sulfates; D014580:Ursodeoxycholic Acid