Production and characterization of a monoclonal antibody to capture proteins tagged with lithocholic acid.
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 3alpha-hydroxy-5beta-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 (gamma2b, kappa) was specific to LCA-N(alpha)-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.
Ikegawa
Shigeo
S
Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-osaka 577-8502, Japan. ikegawa@phar.kindai.ac.jp
Yamamoto
Tetsushi
T
Miyashita
Takahiro
T
Okihara
Rika
R
Ishiwata
Shunji
S
Sakai
Toshihiro
T
Chong
Rung-Hwa
RH
Maeda
Masako
M
Hofmann
Alan F
AF
Mitamura
Kuniko
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D006825:Hybridomas; D007118:Immunoassay; D008095:Lithocholic Acid / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D015336:Molecular Probe Techniques; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D011506:Proteins; D008095:Lithocholic Acid