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Molecular Recognition of Antigen Determinant by Monoclonal Antibody Specific to Neurotoxins from Snake Venoms

蛇毒神经毒素特异性单克隆抗体对抗原决定簇的分子识别

基本信息

批准号：
62580140
负责人：
INAGAKI Fuyuhiko
金额：
$ 1.02万
依托单位：
Tokyo Metropolitan Institute of Medical Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

alpha-bungarotoxin was purified from the venom of Bungarus multicinctus. Immunization of mice was achieved by injecting subcutaneously native toxin at two week intervals using the incomplete Freunds' adjuvant into BALB c mice. After the conventional methods, nine hybridomas producing monoclonal antibodies specific to alpha-bungarotoxin were obtained. Large quantity of monoclonal antibody MaB-2311 produced by one of these hybridoma was purified from the ascites fluid of BALB c mice. MaB-2311 inhibited the lethal effect of alpha-bungarotoxin in vivo assay, suggesting that this monoclonal antibody binds to the region specific to neurotoxic activity. Cross reactivity of MaB-2311 was studied using several long neurotoxins, including Toxin B, Toxin III and LS III. Compared with the cross reactivity of these toxins and MaB-2311, and homology of the primary sequences, an antigenic determinat for MaB-2311 was determind to be Ser34-Ser35-Arg36-Gly37-Lys38. The peptide fragment containing this sequence also reacts with MaB-2311, supporting again that the region involving this sequence is an antigenic determinat. Since Arg36 is an essential residue for neurotoxicity, it is reasonable that this monoclonal antibody neutralizes the neurotoxicity of alpha-bungarotoxin. The MaB-2311 producing hybridoma was subsequently cultured in serum free medium so that deuterium labelled amino acids were incorporated into MaB-2311. NMR studies to investigate the molecular recognition of alpha-bungarotoxin and MaB-2311 are now in progress using the deuterium labelled monoclonal antibody and the peptide fragment.

α-银环蛇毒素是从银环蛇（Bungarusmulticinctus）的毒液中纯化的。通过将天然毒素使用不完全弗氏佐剂以两周间隔皮下注射到BALB c小鼠中来实现小鼠的免疫。在常规方法后，获得了9个产生特异性抗α-银环蛇毒素的单克隆抗体的杂交瘤。其中一株杂交瘤从BALB c小鼠腹水中纯化出大量单克隆抗体MaB-2311。MaB-2311在体内测定中抑制α-银环蛇毒素的致死作用，表明该单克隆抗体结合至对神经毒性活性特异的区域。使用几种长神经毒素（包括毒素B、毒素III和LS III）研究了MaB-2311的交叉反应性。通过比较这些毒素与MaB-2311的交叉反应性和一级序列的同源性，确定MaB-2311的抗原决定簇为Ser 34-Ser 35-Arg 36-Gly 37-Lys 38。含有该序列的肽片段也与MaB-2311反应，再次支持涉及该序列的区域是抗原决定簇。由于Arg 36是神经毒性的必需残基，因此该单克隆抗体可中和α-银环蛇毒素的神经毒性。随后在无血清培养基中培养产生MaB-2311的杂交瘤，使得氘标记的氨基酸掺入MaB-2311中。目前正在使用氘标记的单克隆抗体和肽片段进行NMR研究，以研究α-银环蛇毒素和MaB-2311的分子识别。