中国大虎头蜂蜂毒毒性及其F(ab)2抗体的制备研究

 目的测定中国大虎头蜂蜂毒毒性,制备能有效中和中国大虎头蜂蜂毒的F(ab)₂抗体。方法小鼠腹腔注射测定胡蜂毒LD₅₀,以胡蜂毒免疫日本大耳白兔获得免疫血浆,经盐析、酶解、加热等步骤获得F(ab)₂抗体,并以免疫扩散、ELISA和小鼠体外中和法分别测定超免血浆、纯化的IgG及制备的F(ab)₂抗体对中国大虎头蜂及多种蛇毒的抗体效价。结果小鼠腹腔注射中国大虎头蜂蜂毒的LD₅₀为0.697mg·kg^-1,制备的F(ab)₂抗体SDS-PAGE电泳纯度为94.3%;ELISA检测结果表明纯化的IgG质量浓度为1×10^-9mg·mL^-1时对胡蜂毒素为阳性;免疫扩散结果表明制备的F(ab)₂抗体与中国大虎头蜂毒素（1∶1）时出现明显沉淀线,该抗体与胡蜂毒质量比（3.5∶1）时可保护小鼠100%存活。结论首次利用胡蜂毒免疫动物制备高纯度F(ab)₂抗体,该抗体可有效中和抗中国大虎头蜂毒蜂毒,并对银环蛇毒具有交叉中和作用。

Toxicity of Vespa manderinia Venom and Preparation of Its F(ab)2 antibody

OBJECTIVE To determine the LD₅₀ of Vespa manderinia venom and prepare Vespa manderinia F(ab)₂ antibody. METHODS The LD₅₀ for mice was determined by intraperitoneal injection. Immune plasma was prepared by immunizing rabbits with Vespa manderinia toxin, and the F(ab)₂ antibody was obtained by enzymolysis. sulfate precipitation and heating. The specificity titer of the hyperimmune plasma and purified IgG and F(ab)₂ antibody were determined by ELISA and immunodiffusion test as well as protective test in mice. RESULTS The LD₅₀ of venom was 0.697 mg g^-1, and the purity of F(ab)₂ antibody was 94.3%. ELISA analysis showed that the purified IgG was active to venom at 1?10^-9 mg L^-1. The immunodiffusion test revealed that immune precipitation lines clearly appeared when the ratio of F(ab)₂ antibody to venom was 1;1. The obtained antivenin can neutralize the lyophilized Vespa manderinia venom efficiently when their mass ratio was 3.5;1. CONCLUSION The F(ab)₂ antibody with high specificity and high potency to Vespa manderinia venom was prepared for the first time, and it shows cross neutralization effect for bungarotoxin.