己烯雌酚单克隆抗体细胞株的筛选及间接ELISA法的建立

Study on preparation of monoclonal antibodies against diethylstilbestrol and development of indirect competitive inhibition ELISA

以自行合成的己烯雌酚 (DES)免疫原免疫BALB/c小鼠 ,采用细胞融合、显微克隆等单克隆抗体技术 ,制备了 5株抗DES单克隆抗体分泌杂交瘤细胞株 (7B6、4B10、1G10、4E4、2C7) ;选择其中的 7B6株抗体 ,建立了间接竞争ELISA法 (indirectcompetitiveinhibitionELISA) ,通过初步优化 ,其检测限为 10 pg/孔 ,与同类二苯乙烯类雌激素的交叉反应高 ,而与天然雌激素雌二醇几乎无交叉反应性。这项研究为DES残留检测试剂盒的开发打下了基础。

Four female BALB/c mice aged 4-weeks-old were immunized by intraperitoneal injection with emulsion of DES-MCPE-BSA and Freund’s adjuvant. Splenocytes from the immunized mouse which exhibited the highest titer (1∶12 800) were fused with SP2/0 myeloma cells. The hybridoma cells secreting antibodies against DES were chosen and cloned. Ultimately, five kinds of hybridoma cells(7B6,1G10,4B10,4E4 and 2C7) were cultured. A method of indirect competitive inhibition ELISA was developed by using the supernatant of 7B6 hybridoma cells . The calibration curve in the method was y= -31.67x+95.892(r=0.983)in 1-250 ng/mL. The half maximal inhibition to Ag-Ab reaction was 500 pg per well or so,LOD= 10 pg per well. Cross reactivityof 7B6 antibody with HEX,DIEN,DES-MG was rather high(1-50 ng/mL) respectively, but with E 2 was considerablyfew.