Analytical Sciences

Production and Characterization of a Monoclonal Antibody to Capture Proteins Tagged with Lithocholic Acid

Shigeo IKEGAWA,* Tetsushi YAMAMOTO,* Takahiro MIYASHITA,* Rika OKIHARA,* Shunji ISHIWATA,* Toshihiro SAKAI,* Rung-Hwa CHONG,* Masako MAEDA,** Alan F. HOFMANN,*** and Kuniko MITAMURA*

*Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-osaka 577-8502, Japan
**School of Pharmaceutical Sciences of Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan
***Department of Medicine, University of California, San Diego, La Jolla, CA 92093-0063, USA

Reactive metabolic-modified proteins have been proposed to play an important role in the mechanism(s) of the hepatotoxicity and colon cancer of lithocholic acid (LCA). To identify cellular proteins chemically modified with LCA, we have generated a monoclonal antibody that recognizes the 3α-hydroxy-5β-steroid moiety of LCA. The spleen cells from a BALB/c mouse, which was immunized with an immunogen in which the side chain of LCA was coupled to bovine serum albumin (BSA) via a succinic acid spacer, was fused with SP2/0 myeloma cells to generate antibody-secreting hybridoma clones. The resulting monoclonal antibody (γ2b, κ) was specific to LCA-N^α-BOC-lysine as well as the amidated and nonamidated forms of LCA. The immunoblot enabled the detection of LCA residues anchored on BSA and lysozyme. The antibody will be useful for monitoring the generation, localization, and capture of proteins tagged with LCA, which may be the cause of LCA-induced toxicity.

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