东亚钳蝎蝎毒的毒性及其F(ab’)_2抗体的制备  

Toxicity of Buthus martensii Karsch venom and preparation of its F(ab')_2 antibody

作　　者：李妍[1,2] 范泉水[3] 张志晓[3] 郭平[3] 叶锋平[3] 姜晓梅[3] 邱薇[3] 张富强[3] 郑颖[3] 敖琳[1] 

机构地区：[1]第三军医大学预防医学院毒理学研究所,重庆400038 [2]成都军区昆明总医院呼吸内科,云南昆明650118 [3]成都军区疾病预防控制中心军事医学研究所,云南昆明650118

出　　处：《中国生物制品学杂志》2014年第5期647-651,共5页Chinese Journal of Biologicals

基　　金：全军"十二五"医学科研面上项目(CWS11J280);成都军区"十二五"医学科研重点项目(B12026)

摘　　要：目的测定东亚钳蝎(Buthus martensii Karsch,BMK)蝎毒的毒性,并制备能有效中和BMK蝎毒的F(ab’)2抗体。方法分别经腹腔和肌肉注射小鼠,测定不同产地(山西、山东、河南、辽宁)BMK蝎毒对小鼠的半数致死量(LD50);以山西产BMK蝎毒免疫云南矮种马,获得免疫血浆,经盐析、酶解、加热等步骤制备F(ab’)2抗体,采用ELISA、免疫扩散和小鼠体外中和试验分别测定超免血浆、纯化的IgG及F(ab’)2抗体对各地产BMK蝎毒的抗体效价。结果山西产BMK蝎毒的LD50最小,小鼠腹腔注射的LD50为1.67 mg/kg,肌肉注射LD50为2.06 mg/kg。制备的F(ab’)2抗体的纯度为86.5%;纯化的IgG稀释至1×10-9 mg/ml,抗体ELISA效价呈阳性,较纯化前(1×10-7 mg/ml)提高了100倍;纯化的IgG和制备的F(ab’)2抗体与山西产BMK蝎毒比例为0.4∶1时,免疫扩散试验出现清晰的沉淀线,与其他产地BMK蝎毒比例均为0.1∶1时,出现清晰的沉淀线;F(ab’)2抗体与山西产BMK蝎毒质量比为2∶1时,可保护小鼠100%存活。结论成功利用蝎毒免疫动物制备了高纯度的F(ab’)2抗体,该抗体可有效中和多个产地的BMK蝎毒。Objective To determine the toxicity of Buthus martensii Karsch（BMK）venom and prepare the F（ab＇）2antibody neutralizing the venom effectively. Methods Mice were immunized i.p. and i.m. with BMK venom from various producing areas（Shanxi,Shandong,Henan and Liaoning Provinces）,of which the LD50values were determined. Immune plasma was obtained by immunizing Yunnan ponies with BMK venom from Shanxi Province,from which F（ab＇）2antibody was prepared by salting out,enzyme digestion and heating. The titers of hyperimmune plasma,purified IgG and F（ab＇）2antibody against BMK venom were determined by ELISA,immunodiffusion test and in vitro neutralizing test in mice. Results The LD50of BMK venom produced in Shanxi Province were the lowest,which were 1. 67 mg / kg by intraperitoneal injection and 2. 06 mg / kg by intramuscular injection. The purity of prepared F（ab＇）2antibody was 86. 5%. The ELISA titer of purified IgG was positive after dilution to a concentration of 1 × 10^-9mg / ml,which increased by 100 folds as compared with that before purification（1 × 10^-7 mg / ml）. Clear precipitation lines were observed in immunodiffusion test when both the ratios of purified IgG and prepared F（ab＇）2antibody to venom from Shanxi Province were 0. 4 ∶ 1,while when the ratios were 0. 1 ∶ 1 to the venoms from other producing areas. The survival rate of mice was 100% when the mass ratio of F（ab＇）2antibody to the venom from Shanxi Province was 2 ∶ 1. Conclusion Highly purified F（ab＇）2antibody was successfully prepared by immunizing animals with BMK venom,which neutralized the venom from various producing areas effectively.

关 键 词：东亚钳蝎 毒性 F(ab’)2抗体 

分 类 号：R996.3[医药卫生—毒理学] R392-33[医药卫生—药学]