中国大虎头蜂蜂毒毒性及其F(ab)_2抗体的制备研究

Toxicity of Vespa manderinia Venom and Preparation of Its F(ab)_2 antibody

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0.697 mg·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。

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） 2antibody were determined by ELISA and immunodiffusion test as well as pro- tective test in mice. RESULTS The LDs0 of venom was 0. 697 mg · kg ^-1 , and the purity of F （ab） 2 antibody was 94. 3 %. ELISA a- nalysis showed that the purified IgG was active to venom at 1 ×10^-9mg·mL^-1. The immunodiffusion test revealed that immune precipi- tation lines clearly appeared when the ratio of F（ab） 2antibody 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.