免疫亲和层析法制备马破伤风免疫球蛋白F(ab')_(2)  

Preparation of equine tetanus immunoglobulin F(ab')_(2) by immunoaffinity chromatography

作　　者：包正琦 段丽娟 戴于栋 孙伟 杨凯 高建军 BAO Zheng-qi;DUAN Li-juan;DAI Yu-dong;SUN Wei;YANG Kai;GAO Jian-jun(Lanzhou Institute of Biological Products Co.,Ltd.,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司,甘肃兰州730046

出　　处：《中国生物制品学杂志》2022年第5期598-602,共5页Chinese Journal of Biologicals

摘　　要：目的采用免疫亲和层析法纯化制备马破伤风免疫球蛋白F(ab')_(2)。方法破伤风类毒素(tetanus toxoid,TT)免疫马血浆经胃酶消化、加温变性、硫酸铵盐析、明矾吸附、超滤,获得破伤风抗毒素;再经CNBr activated Sepharose 4FF介质偶联TT制成的亲和层析柱纯化,获得马破伤风免疫球蛋白F(ab')_(2)。检测其蛋白质含量、分子大小分布、纯度、效价、比活性,并与破伤风抗毒素进行比较。将马破伤风免疫球蛋白F(ab')_(2)置(25±2)℃分别保存1、2、3、6个月,测定效价。结果与破伤风抗毒素比较,马破伤风免疫球蛋白F(ab')_(2)的蛋白质含量平均值由45.6 mg/mL下降至20.03 mg/mL,降低了56%;F(ab')2含量变化较小,聚合物含量由2.92%下降至1.22%,降低了59%;纯度略有降低;效价平均值由2533 IU/mL升高至3733 IU/mL,提高了47%;比活性平均值由55563 IU/g升高至186277 IU/g,提高了335%。加速稳定性试验6个月,马破伤风免疫球蛋白F(ab')_(2)效价及F(ab')2含量均在合格范围之内。结论采用免疫亲和层析方法制备了高效价及高比活性的马破伤风免疫球蛋白F(ab')_(2),且稳定性良好。Objective To prepare equine tetanus immunoglobulin F(ab')_(2) by immunoaffinity chromatography.Methods The equine plasma immunized with tetanus toxoid(TT)was digested with pepsin,denaturalized by heating,salted out with ammonium sulphate,adsorbed with alum and ultrafiltrated.The obtained tetanus antitoxin(TAT)was purified by affinity chromatography with cyanogen bromide activated Sepharose 4FF medium coupled with TT.The prepared equine tetanus immunoglobulin F(ab')2was determined for protein content,molecular size distribution,purity,titer and specific activity,and the results were compared with those of TAT.The equine tetanus immunoglobulin F(ab')_(2)was stored at(25±2)℃ for 1,2,3 and 6 months,and determined for titer.Results Compared with those of TAT,the mean protein content of equine tetanus immunoglobulin F(ab')_(2)decreased by 56% from 45.6 to 20.03 mg/mL,while the F(ab')_(2)showed no significant change,the polymer content decreased by 59% from 2.92% to 1.22%,the purity showed a slight decrease,the mean titer increased by 47% from 2533 to 3733 IU/mL,and the mean specific activity increased by 335% from 55563 to 186277 IU/g.The accelerated stability test result after storage for 6 months showed that both the titer and F(ab')_(2)content of equine tetanus immunoglobulin F(ab')_(2)were within the range of qualification.Conclusion Equine tetanus immunoglobulin F(ab')_(2)with high titer,high specific activity and high stability was successfully prepared by immun-oaffinity chromatography.

关 键 词：免疫亲和层析 马破伤风免疫球蛋白F(ab')_(2) 破伤风抗毒素 效价 比活性 

分 类 号：R392-33[医药卫生—免疫学]