Production of a Monoclonal Antibody for Sensitive Monitoring of Deoxycholic Acid Residues Anchored on Endogenous Proteins

Abstract

Bile acid carboxy-linked glucuronides and adenylates have active ester groups, and thus may easily react with amino groups on endogenous tissue proteins resulting in physiological disorders. To identify such tissue-bound bile acids in vivo, we generated a monoclonal antibody which recognizes deoxycholic acid (DCA) residues anchored on endogenous proteins. The spleen cells from a BALB/c mouse, which was immunized with a conjugate of DCA and bovine serum albumin (BSA), were fused with P3/NS1/1-Ag4-1 myeloma to generate antibody-secreting hybridoma clones. The resulting monoclonal antibody (Ab#88; isotype γ1, λ) was specific to N-ε-deoxycholyl lysine as well as the nonamidated DCA, and enabled sensitive detection of DCA residues (270 femtograms) anchored on BSA molecules, introduced by the reaction of the DCA adenylate. The immunoaffinity column immobilizing the Ab#88 allowed selective extraction of the DCA-introduced substance P. The antibody will be useful for monitoring the formation and localization of tissue-bound DCA, which may be concern with the activity of DCA as a colon cancer promoter.