摘要
目的制备呋喃唑酮代谢物AOZ单克隆抗体及其胶体金与荧光淬灭免疫层析试纸条。方法将AOZ衍生成CPAOZ后,与载体蛋白BSA偶联,制备完全抗原CPAOZ-BSA,经皮下多点注射免疫BALB/c小鼠,共5次,取小鼠脾细胞与SP2/0细胞融合,获得稳定分泌特异性抗体的杂交瘤细胞株。利用获得的抗CPAOZ单克隆抗体制备胶体金及荧光淬灭免疫层析试纸条,并对试纸条的灵敏度及特异性进行验证。结果制备的完全抗原经紫外扫描鉴定偶联成功。共获得3株能稳定分泌抗CPAOZ单克隆抗体的杂交瘤细胞株,其中效价和特异性最好的1株2H11针对CPAOZ的50%抑制质量浓度(IC50)为0.88 ppb。以该抗体制备的胶体金及荧光淬灭免疫层析试纸条的最低检测限分别为7.5和0.062 5 ppb;两种试纸条与其他3种硝基呋喃类代谢物CPAMOZ、CPAHD、CPSEM均不存在交叉反应。结论成功制备了抗CPAOZ单抗及其胶体金免疫层析和荧光淬灭免疫层析试纸条,两种试纸条均具有较高的灵敏度和较强的特异性,其中荧光淬灭免疫层析试纸条的灵敏度较胶体金免疫层析试纸条高约100倍,具有良好的应用前景。
Objective To prepare the monoclonal antibody against furazolidone metabolite（AOZ）and the colloidal gold and fluorescence quenching immunochromatographic strips. Methods AOZ was derived to CPAOZ and coupled with bovine serum albumin（BSA）as a carrier. BALB / c mice were immunized with the prepared complete antigen CPAOZBSA by multiple subcutaneous injection for 5 times,of which splenocytes were fused with SP2 / 0 cells to obtain hybridoma cell strains stably secreting specific monoclonal antibody（McAb）. Colloidal gold and fluorescence quenching immunochromatographic strips were prepared with the obtained McAb,and verified for sensitivity and specificity. Results UV spectroscopy proved that complete antigen CPAOZ-BSA was successfully coupled. Three hybridoma cell strains stably secreting McAb against CPAOZ were obtained,of which strain 2H11,with the highest titer and specificity,showed a median inhibitory concentration（IC50）of 0. 88 ppb. The minimum detection limits（MDLs）of colloidal gold and fluorescence quenching immunochromatographic strips prepared with McAb 2H11were 7. 5 and 0. 062 5 ppb respectively. The two strips showed no cross reactivity with CPAHD,CPAMOZ or CPSEM. Conclusion The McAb against CPAOZ and the relevant colloidal gold and fluorescence quenching immunochromatographic strips were prepared successfully. Both the strips showed high sensitivities and specificities. However,with sensitivity 100 times higher than that of colloidal gold strip,fluorescence quenching immunochromatographic strip was of a good prospect in application.
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第7期927-931,共5页
Chinese Journal of Biologicals
基金
第47批教育部留学归国人员科研启动基金
关键词
呋喃唑酮
代谢物
单克隆抗体
胶体金
荧光
免疫层析
试纸条
Furazolidone（AOZ） Metabolite Monoclonal antibody Colloidal gold Fluorescence Immunochromatography Strip
