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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。
OBJECTIVE: To prepare monoclonal antibody (mAb) against α-ZER and to establish a method for the determination of α-ZER and its homologues in animal-derived foods. Methods: The α-ZER-BSA conjugate was prepared by the combination of O-carboxymethylhydroxylamine and EDC. The BALB / c mice were immunized and the hybridomas were used to prepare mAbs. The mAbIg subtype was determined by Sandwich ELISA. The affinity constants were calculated by the Beatty method. The titer was determined by indirect ELISA. The mAb was detected by direct competitive ELISA and the homolog of α-ZER, diethylstilbestrol, 19-nortestosterone, streptomycin, Etc. cross-reaction; draw α-ZER standard competitive inhibition curve, test the sensitivity of the antibody. Using the direct competitive ELISA established by the antibody, 37 animal liver samples were tested and compared with the HPLC test results. Results: UV-scan demonstrated that the coupling of α-ZER-BSA was successful. Eight strains of monoclonal antibodies that can stably secrete anti-α-ZER mAb were obtained experimentally. One of the 4 strains (4E5) secreted mAb titer was 5.142 × 107, and its Ig subtype was IgG1. The mAb specifically recognizes α-ZER and its homologues without cross-reactivity with other commonly used veterinary drugs. An α-ZER direct competitive ELISA was established, out of 37 samples identified as negative by HPLC, 8 positive samples were screened. Conclusion: The direct competitive ELISA assay established by mAb is suitable for the rapid screening of residual α-ZER and its homologue in animal-derived foods.