Production and characterization of group-specific monoclonal antibodies recognizing nonamidated, glycine- and taurine-amidated ursodeoxycholic acid 7-N-acetylglucosaminides.

Ursodeoxycholic acid 7-N-acetylglucosaminides (UDCA 7-NAGs) are novel conjugated metabolites whose urine levels are expected to be a specific diagnostic index for primary biliary cirrhosis. To obtain a specific antibody which is useful for developing immunochemical analytical methods of UDCA 7-NAGs, a variety of monoclonal antibodies have been generated. Spleen cells from an A/J mouse, which had been immunized with a conjugate of nonamidated UDCA 7-NAG and bovine serum albumin, were fused with P3/NS1/1-Ag4-1 myeloma cells. After screening by an enzyme-linked immunosorbent assay (ELISA) using a beta-galactosidase-labeled antigen, thirteen kinds of antibody-secreting hybridoma clones were established. Binding properties of these monoclonal antibodies were investigated in detail by ELISA. One of these antibodies, Ab-#8 (gamma1, kappa) had the most favorable characteristics for clinical application, which was group-specific to the 7-NAG conjugates of nonamidated, glycine- and taurine-amidated UDCAs providing a highly sensitive dose-response curve for each conjugate (midpoint 17 pg per assay for nonamidated UDCA 7-NAG). Cross-reactivities with eleven kinds of bile acids, including some potential interfering metabolites as UDCA 3-sulfate, were negligibly low. By using direct ELISA based on Ab-#8, daily urinary excretion rates of UDCA 7-NAGs of two healthy subjects were determined to be 1030 and 469 microg as GUDCA 7-NAG equivalent.