目的: 制备可特异性识别金雀异黄酮（Gen）的单克隆抗体,建立特异、快速定量Gen的间接竞争酶联免疫吸附分析（ELISA）方法。方法: 通过曼尼希法将Gen和载体蛋白偶联,合成人工抗原。免疫6~8周龄的雌性Balb/c小鼠,冲击免疫后取小鼠脾细胞与SP2/0骨髓瘤细胞,在50% PEG的作用下进行细胞融合。采用有限稀释法筛选阳性亚克隆,获取特异性单克隆抗体,建立间接竞争ELISA法。结果: 试验成功筛选出C1和C3 2株抗Gen单克隆抗体,效价分别为1 ∶ 32 000、1 ∶ 40 000。利用C3抗体优化间接竞争ELISA反应条件,检测抗体的灵敏度IC50为16.15 ng · mL-1,线性范围（IC20~IC80）为3.78~55.85 ng · mL-1。交叉反应结果显示,Gen单克隆抗体与鹰嘴豆芽素A、尼泊尔鸢尾异黄酮的交叉反应率分别为4.96%和2.40%,而与芹菜素、4-苯并吡喃酮2-羧酸的交叉反应率均<0.1%。回收率在85%~110%,批内和批间RSD均<10%。结论: 本研究制备了Gen特异性的单克隆抗体,用其建立了Gen间接竞争ELISA检测方法,灵敏度高,符合免疫分析的基本要求。
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