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

• Published in: The Journal of steroid biochemistry and molecular biology 1998-02, Vol.64 (3), p.171-177
• Main Authors: Kobayashi, Norihiro, Oiwa, Hiroyuki, Goto, Junichi
• Format: Article
• Language: English
• Subjects: Acetylglucosamine - analogs & derivatives; Acetylglucosamine - immunology; Animals; Antibodies, Monoclonal - metabolism; Bile Acids and Salts - urine; Biological and medical sciences; Enzyme-Linked Immunosorbent Assay; Female; Hybridomas - immunology; Immunological methods for diagnosis and exploration; Immunopathology; Liver Cirrhosis, Biliary - diagnosis; Medical sciences; Mice; Mice, Inbred BALB C; Molecular Structure; Other methods; Protein Binding - immunology; Ursodeoxycholic Acid - immunology; Ursodeoxycholic Acid - urine
• ISSN: 0960-0760; 1879-1220
• DOI: 10.1016/S0960-0760(97)00162-3

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 β-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 ( γ 1, κ) 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 μg as GUDCA 7-NAG equivalent.