Antigen selectivity characteristic of polyclonal antibodies against omega-conotoxin GVIA and N-type voltage-dependent calcium channels |
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Authors: | Ichida S Abe J Sugimoto W Uematsu M Komoike K Imoto K Mori M Wada T Minami T Kakutani K |
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Affiliation: | (1) Department of Biological Chemistry, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashiosaka, 577-8502, Japan;(2) Department of Informational Physiology, National Institute for Physiological Sciences, Okazaki, 444-8585, Japan;(3) Laboratory of Environmental Biology, Department of Life Science, School of Science and Engineering, Kinki University, Kowakae 3-4-1, Higashiosaka, 577-8502, Japan;(4) Pharmaceutical Research and Technology Institute, Kinki University, Kowakae 3-4-1, Higashiosaka, 577-8502, Japan |
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Abstract: | The antibodies against omega-conotoxin GVIA (-CTX GVIA; N-type voltage-dependent calcium channel [VDCC] blocker) and B1Nt (N-terminal segment [residues 1–13] of BI 1 subunits of VDCCs) were prepared, and the selectivity for each antigen -CTX GVIA and B1Nt was investigated. For the antigen selectivity of anti–-CTX GVIA antibody against -CTX GVIA, ELISA, and immunoprecipitation were used. The reactions for ELISA and immunoprecipitation were observed except when antibody IgG purified by Protein A–Sepharose CL-4B from nonimmunized serum (purified NI-Ab) was used. The specific reactions were inhibited by 10 nM -CTX GVIA, but not by -CTX SVIB (N-type VDCC blocker), -CTX MVIIC (N- and P-type VDCC blocker), or -Aga IVA (P-type VDCC blocker). For the antigen selectivity of the anti-B1Nt antibody, analyses by ELISA, immunoprecipitation, and Western blotting were conducted. The reactions were observed except when NI-Ab was used. The ELISA and immunoprecipitation reactions were inhibited by the antigen peptide B1Nt, and the IC50 values were about 1.2 × 1028 and 1.3 × 1028 M, respectively. The bands of 210 and 190 kD by Western blotting of crude membranes from chick brain were also inhibited by 1 M B1Nt. These results suggest that the antibodies prepared against -CTX GVIA and B1Nt in this work have high selectivity for their antigen. Therefore we assume that the antibodies against -CTX GVIA and B1Nt are useful tools for the analyses of the function and distribution of N-type VDCCs. The anti -CTX GVIA antibody must also be useful for the radioimmunoassay of -CTX GVIA. |
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Keywords: | Polyclonal antibody omega-conotoxin GVIA N-type calcium channels |
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