抗α-玉米赤霉醇单克隆抗体的研制及初步应用

Preparation and preliminary application of monoclonal antibody against α-zearalanol

目的:制备抗α-玉米赤霉醇(α-ZER)的单克隆抗体(mAb),建立对动物源性食品中残留α-ZER及其同系物的检测方法。方法:结合O-羧甲基羟胺法和EDC法制备α-ZER-BSA偶联物,免疫BALB/c小鼠,采用杂交瘤技术制备mAb。用夹心ELISA测定mAbIg亚类;按Beatty法计算亲和常数;间接ELISA法测定效价;用直接竞争ELISA检测mAb与α-ZER的同系物、己烯雌酚、19-去甲睾酮、链霉素及氯霉素等的交叉反应;绘制α-ZER标准品竞争抑制曲线,检定抗体的灵敏度。用该抗体建立的直接竞争ELISA对37份动物肝组织样品进行检测,并与HPLC检测结果比较。结果:紫外线扫描证明,α-ZER-BSA偶联成功。实验获得8株可稳定分泌抗α-ZERmAb杂交瘤细胞株,其中1株(4E5)分泌的mAb效价较高,为5.142×107,其Ig亚型为IgG1。该mAb能特异识别α-ZER及其同系物,而与其它常用兽药无交叉反应。建立了α-ZER直接竞争ELISA,从HPLC确认为阴性的37份样品中,筛出8份阳性样品。结论:利用mAb建立的直接竞争ELISA检测方法,适合动物源性食品中残留α-ZER及其同系物的快速筛选。

AIM： To prepare monoclonal antibody （mAb） against α-zearalanol （α-ZER） and develop an immunoassay for the detection of α-ZER and its analogues residues in food derived from animal tissues. METHODS： α-ZER was conjugated to BSA as immunogen to immunize BALB/c mice and mAb were prepared by hybridoma technique, mAb＇s characteristics （titer, Ig subclass, specificity and relative affinity） were identified by ELISA. Standard inhibitive cure was made and sensitivity of the mAb was identified. 37 samples derived from animal liver were detected for α-ZER residues by competitive ELISA established in the study. RESULTS： 8 hybridoma cell lines stably secreting anti-α-ZER mAb were obtained. The titer of one of them （4E5）was 5.142 × 10^7. The Ig subclass was IgG1. The mAb was specific for α-ZER and its analogues and had no crossreactivity with other compounds. 8 positive results were found from the 37 samples derived from animal liver which were negative detected by HPLC. CONCLUSION： Anti-α- ZER mAb has been prepared successfully. A rapid method using the mAb for detecting α-ZER and its analogues residues in animal tissues has been established.