Chang L S, Lin J, Kuo K W, Lin S R, Chang C C
Department of Biochemistry, Kaohsiung Medical College, Taiwan.
J Biochem. 1995 Oct;118(4):686-92. doi: 10.1093/oxfordjournals.jbchem.a124966.
Rabbits hyperimmunized with cobrotoxin from Taiwan cobra venom produced non-precipitating as well as precipitating antibodies. Both antibody preparations exhibited higher affinity for native cobrotoxin than for reduced and S-carboxymethylated (RCM) cobrotoxin. This indicated that the epitope structures in cobrotoxin are mostly conformation-dependent. In order to identify the conformational epitopes, native cobrotoxin was hydrolyzed with acid protease A, and 12 peptides were obtained on HPLC. Three peptide fragments, AP-10, AP-11, and AP-12, showed pronounced antigenicities toward precipitating as well as non-precipitating antibodies. AP-10, AP-11, and AP-12 contained a common segment in the C-terminal region of cobrotoxin, residues 43 to 62, with intact disulfide linkages. Complete removal of the C-terminal antibodies from antisera and precipitating antibodies on a C-terminal segment-Sepharose affinity column resulted in the loss of their precipitability with cobrotoxin, whilst restoration of precipitability was observed on the addition of the C-terminal antibodies to the C-terminal antibody-depleted antisera and precipitating antibodies. Studies on the antigenic structures of RCM-cobrotoxin revealed that RCM-cobrotoxin contains an immunodominant epitope at positions 22-38. The N-terminal and C-terminal regions of RCM-cobrotoxin encompass other epitopes which exhibit low reactivities toward anti-RCM-cobrotoxin antibodies. However, no precipitated antigen-antibody complexes were observed with the mixture of anti-RCM-cobrotoxin antibodies and RCM-cobrotoxin. These results suggest that the inherently different immunogenicities with different segments might affect the precipitabilities of the resulting antibodies, and that the notable immunogenecity of the C-terminal region is related to the production of precipitating and non-precipitating antibodies against cobrotoxin.
用台湾眼镜蛇毒的眼镜蛇毒素对兔子进行超免疫后，产生了非沉淀性抗体和沉淀性抗体。两种抗体制剂对天然眼镜蛇毒素的亲和力均高于对还原型和S-羧甲基化（RCM）眼镜蛇毒素的亲和力。这表明眼镜蛇毒素中的表位结构大多依赖于构象。为了鉴定构象表位，用酸性蛋白酶A水解天然眼镜蛇毒素，并通过高效液相色谱法获得了12个肽段。三个肽片段AP-10、AP-11和AP-12对沉淀性抗体和非沉淀性抗体均表现出明显的抗原性。AP-10、AP-11和AP-12在眼镜蛇毒素的C末端区域包含一个共同片段，即第43至62位氨基酸残基，具有完整的二硫键。在C末端片段-琼脂糖亲和柱上从抗血清和沉淀性抗体中完全去除C末端抗体，导致它们与眼镜蛇毒素的沉淀性丧失，而向去除了C末端抗体的抗血清和沉淀性抗体中添加C末端抗体时，观察到沉淀性得以恢复。对RCM-眼镜蛇毒素抗原结构的研究表明，RCM-眼镜蛇毒素在第22 - 38位氨基酸处含有一个免疫显性表位。RCM-眼镜蛇毒素的N末端和C末端区域包含其他表位，这些表位对抗RCM-眼镜蛇毒素抗体的反应性较低。然而，抗RCM-眼镜蛇毒素抗体与RCM-眼镜蛇毒素的混合物未观察到沉淀的抗原-抗体复合物。这些结果表明，不同片段固有的不同免疫原性可能会影响所产生抗体的沉淀性，并且C末端区域显著的免疫原性与针对眼镜蛇毒素产生沉淀性和非沉淀性抗体有关。
