Generation of polyclonal antibody against mu-conotoxin GIIIA using an immunogen of [Cys(5)]mu-conotoxin GIIIA site-specifically conjugated with bovine serum albumin.
mu-Conotoxin GIIIA, one of the strong peptide toxins in the cone shell, preferentially blocks the skeletal muscle-type sodium channels in vertebrates. The toxicity of mu-conotoxin GIIIA is nearly equal to that of tetrodotoxin. The generation of an antibody for the native toxins is analytically useful, but practically difficult due to its high toxicity to animals. In this study, we generated the polyclonal antibody for mu-conotoxin GIIIA using a specific conjugation method in which the immunogen was detoxified while retaining the active-site structure for the sodium channels. ELISA analysis showed that the generated antibody recognized the native toxin folded with three disulfide bridges, but not the linear one. Furthermore, the physiologically active mutants of GIIIA were recognized while the inactive mutants were not, suggesting that the newly generated antibody can selectively recognize the physiologically active toxins. These methods for generating an antibody against peptide toxins will be applicable to other peptide toxins.
Nakamura
Mitsuhiro
M
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8601, Japan.
Oba
Yuichi
Y
Mori
Tatsuya
T
Sato
Kazuki
K
Ishida
Yukisato
Y
Matsuda
Tsukasa
T
Nakamura
Hideshi
H
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001667:Binding, Competitive; D002417:Cattle; D020916:Conotoxins / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D007928:Lethal Dose 50; D051379:Mice; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects; D009154:Mutation; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012710:Serum Albumin, Bovine / Q000737:chemistry*
D000906:Antibodies; D020916:Conotoxins; C046101:conotoxin GIII; D012710:Serum Albumin, Bovine; D003545:Cysteine