摘要
目的制备分泌抗旋毛虫肌幼虫排泄分泌(ES)抗原单克隆抗体(McAb)杂交瘤细胞株,并对其及分泌的McAb进行鉴定。方法用旋毛虫肌幼虫ES抗原免疫BALB/c小鼠,取其脾细胞与SP2/0细胞融合,筛选分泌特异性、高滴度McAb的杂交瘤细胞株,制备腹水,进行纯化。ELISA间接法、夹心法和竞争法分别测定McAb滴度、相对亲和力和相加效应,琼脂双扩散试验鉴定免疫球蛋白类型,检测McAb与其他寄生虫抗原的交叉反应。电镜观察杂交瘤细胞超微结构,计数染色体数目及观察杂交瘤细胞分泌McAb的稳定性。结果筛选出2株(B2C12和E11F11)分泌抗旋毛虫肌幼虫ES抗原McAb杂交瘤细胞株。电镜显示,杂交瘤细胞具有肿瘤细胞和浆细胞的特征,胞浆内可见大量分泌颗粒;杂交瘤细胞染色体数目平均为98.6条,均能稳定分泌McAb(属IgM)。B2C12株培养上清液和腹水McAb滴度分别为1∶1280和1∶2.048×104,E11F11株为1∶640和1∶1.024×104,其中前者的相对亲和力较后者稍高。2株McAb识别ES抗原不同的抗原决定簇;且不与其他寄生虫抗原发生交叉反应。结论获得2株抗旋毛虫肌幼虫ES抗原杂交瘤细胞株,均能稳定分泌高滴度、特异性的IgM类McAb,并识别不同的抗原决定簇,为研制旋毛虫病诊断试剂盒和制备基因工程抗体奠定了基础。
Objective To prepare and identificate the monoclonal antibodies （McAb） against excretory secretory （ES） antigen of muscle larvae of Trichinella spiralis. Methods BALB/c mice were immunized by ES antigen of muscle larvae of T. spiralis. The spleen lymph cell of the immunized BALB/c mice were fused with SP2/0 cells of mice, anc[ then select hybridoma cell strain secreting McAbs and extract and purify ascites. Indirect ELISA, indirect sandwich ELISA and competitive ELISA were used to determine the activity, relative affinity and additivity effect, respectively. Class or subclass of immunoglobulin was examined by agarose double immuodiffusion test. Cross reaction of McAbs were tested with adult worm antigen and egg antigen of Schistosoma japonicum, tachyzoites antigen of To3-oplasrna gondii RH strain, cyst liquid antigen of Cysticercus cellulosae, cystic fluid antigen of Echinococcus granulosus, surface antigen, somatic antigen and ES antigen of adult worm of T. spiralis, soluble antigen, 48 h ES antigen, 72 h ES antigen of muscle larvae. The ultrastructure of hybridoma cell was observed under electronic microscope. The number of chromosome was counted. The stability of hybridoma cells secreting McAbs was evaluated. Results Two strains hybridoma cells （B2C12 and E11F11） were acquired. B cell hybridoma characterizing tumour cell and plasma cell was seen through electronic microscope, which included secreting granule of McAb. The mean number of chromosome was 98.6. Two strains hybridoma cells could stably secrete McAb during 2 months and belong to IgM. The titer of B2C12 strain culture medium and ascites were 1： 1 280, 1 ： 2. 048×10^4 respectively and that of E11F11 were 1 ： 640 and 1 ： 1. 024×10^4 respectively. The former had much higher relative affinity than that of the latter. Two McAbs could recognize the different ES epitopes. The McAbs had no cross reaction with all the mentioned antigens. Conclusion The obtained two hybridoma cell strains can secrete high titer and specific McAbs belonging to IgM. They can recognize the different ES epitopes and will form the basis of the diagnosis kit for detecting circulating antigen （CAg） and producing gene engineering antibodies.
出处
《中国寄生虫病防治杂志》
CSCD
2005年第4期255-258,共4页
Chinese Journal of Parasitic Disease Control
