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

从多刺黄颡鱼毒液中分离纯化了α -黄颡鱼毒素。用不完全弗氏佐剂每隔两周向BALB - c小鼠皮下注射天然毒素，实现小鼠免疫。在常规方法的基础上，获得了9个产生α -班加罗毒素特异性单克隆抗体的杂交瘤。其中一种杂交瘤从BALB - c小鼠的腹水中纯化出大量单克隆抗体MaB-2311。单克隆抗体（MaB-2311）在体内抑制了α -班加罗毒素的致死作用，表明该单克隆抗体与神经毒性活性特异性区域结合。研究了MaB-2311与毒素B、毒素III和长链神经毒素的交叉反应性。将这些毒素与MaB-2311的交叉反应性和初级序列的同源性进行比较，确定MaB-2311的抗原决定蛋白为Ser34-Ser35-Arg36-Gly37-Lys38。含有该序列的肽片段也与MaB-2311发生反应，再次支持涉及该序列的区域是抗原决定因子。由于Arg36是神经毒性必需的残基，因此该单克隆抗体可以中和α -班加罗毒素的神经毒性。随后在无血清培养基中培养产生MaB-2311的杂交瘤，使氘标记的氨基酸掺入MaB-2311中。目前正在利用氘标记单克隆抗体和肽片段进行核磁共振研究，以研究α -班加罗毒素和单克隆抗体-2311的分子识别。

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.