糖皮质激素ELISA检测方法的建立及群选性抗体的筛选

Development of an ELISA for Detection of Dexamethasone and Screening of Polyclonal Antibody

设计间接竞争ELISA方法,并优化二抗稀释比、竞争时间、PBS溶液的pH值3个条件参数。优化后的地塞米松、倍他米松、双氟米松3种抗体的间接竞争ELISA曲线的IC50分别为2.66、7.36、6.12ng/ml。通过3种抗体的交叉反应来评估其群选特性。结果表明,地塞米松抗体的群选性最强,对双氟米松的交叉反应率高达120%、倍他米松73%、曲安息龙37%、泼尼松龙21%,而对其他3种内源性类糖皮质激素交叉反应率均小于0.1%,然后地塞米松以0.375、0.75、1.5、3.0ng/g分别添加到鸡肉样本中测定回收率,所得回收率在61.3%～80.3%之间,变异系数小于15%。在实际样本检测中,将ELISA方法的测定结果与LC-MS的结果进行比较,两者具有很好的相关性。

Indirect competitive ELISA methods were developed based on the high-titer antibody and the dilution ratio of goat anti-rabbit-HRP, competition time and the pH of PBS solution were optimized. The IC50 of optimized indirect compete ELISA for dexamethasone, betamethasone and flumethasone were 2.66, 7.36, 6.12 ng/ml, respectively. The group selection of three antibodies was accessed by detecting their cross-reactivities. The results showed that the anti-dexamethasone antiboidy was the best suitable for group selection and the cross-reactivity toward flumethasone, betamethasone, riacinolone and predinisolon was up to 120%, 73%, 37%, predinisolone 21% while the cross activity towards several natural glucocorticoids was lower than 0.1%. The recoveries were detected by spiking four concentrations （0.375, 0.75, 1.5 ng/ml and 3.0 ng/ml） of DEX in chicken muscle tissue and the results indicated that the recoveries were between 61.3% and 80.3% and the coefficients of variation were lower than 15% The ELISA method and LC-MS method were applied to analyze the real samples, and high correlation was observed between the two methods.