玉米赤霉烯酮抗独特型单抗的制备及特异性分析

目的制备并鉴定玉米赤霉烯酮(Zearalenone,ZEN)抗独特型单抗Ab2β-1D5。方法将ZEN单抗1G4与载体蛋白KLH偶联作为免疫原,免疫BALB/c小鼠,取免疫小鼠脾细胞,与小鼠骨髓瘤细胞SP2/0融合后,以ZEN单抗Fab片段作为包被抗原,间接ELISA法筛选阳性杂交瘤细胞。经小鼠腹腔注射杂交瘤细胞,制备腹水型单抗,并经Protein G亲和层析柱进行纯化。间接ELISA法检测ZEN抗独特型单抗的抗体效价及特异性;间接竞争ELISA法检测ZEN抗独特型单抗类型、灵敏度及其与ZEN毒素间的相关性。结果共获得6株稳定分泌ZEN抗独特型单抗的杂交瘤细胞株,腹水型抗体效价为1∶1.2×105～1∶2.0×105;6株单抗均为β型抗独特型抗体(Ab2β),其中Ab2β-1D5抗体灵敏度最高,对ZEN的IC50值达10.09 ng/ml,其与ZEN毒素呈线性相关(r=0.990);ZEN抗独特型单抗与莱克多巴胺、重金属铅、铬及虾过敏原单抗均无交叉反应,特异性良好。结论已成功制备玉米赤霉烯酮抗独特型单抗,该抗体与ZEN间存在"内影像"关系,可以替代ZEN毒素标准品,用于建立ZEN的无毒免疫学检测技术。

Preparation and characterization of monoclonal anti-idiotype antibody to zearalenone

Objective To prepare and identify the monoclonal anti-idiotype antibody Ab2β-1D5 to zearalenone(ZEN). Methods Anti-ZEN monoclonal antibody(mAb)1G4 was coupled with carrier protein KLH and used as an immunogen to immunize BALB/c mice.The spleen cells of immunized mice were fused with murine myeloma SP2/0 cells,based on which positive hybridoma cells were screened by indirect ELISA using the Fab fragment of anti-ZEN mAb as a coating antigen.Mice were injected i.p.with the hybridoma cells,of which the ascites were collect and purified by protein G affinity column chromatography,then determined for titer and specificity by indirect ELISA,and for type,sensitivity and correlation to ZEN toxin by indirect competitive ELISA.Results Six hybridoma strains stably secreting monoclonal antiidiotype antibody to ZEN were obtained,and the titers of ascites were 1:1.2×10 5～1:2.0×10 5.All the mAbs secreted by the six hybridoma cell strains were anti-idiotype antibody type β(Ab2β).Of the mAbs,Ab2β-1D5 showed the highest sensitivity,of which the IC 50 to ZEN was 10.09 ng/ml.There was a linear correlation between the concentrations of Ab2β-1D5 and ZEN(r = 0.990).The mAbs showed no cross reactions with monoclonal antibodies against Ractopamine,Pb 2+,Cr 3+ or the allergens in shrimp,indicating high specificity.Conclusion The monoclonal anti-idiotype antibodies against ZEN were successfully prepared,which showed"inner image"relationship to ZEN,and might be used for development of avirulent immune detection method for ZEN instead of standard ZEN toxin.