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建立了Eu~(3+)标记的间接竞争时间分辨荧光免疫分析法(Indirect competitive time-resolved fluoroimmunoassay,ic-TRFIA)用于鱼肉样品中呋喃它酮代谢物AMOZ的检测。通过单因素实验考察了包被抗原浓度、抗体稀释倍数、竞争反应时间等参数对方法灵敏度的影响。结果表明,ic-TRFIA的最佳反应条件为:包被抗原浓度为0.25μg/m L,抗体稀释5×10~4倍,最佳竞争时间为50 min。在优化的条件下,方法的检出限(LOD,IC_(10))为0.01 ng/m L,半抑制浓度(IC_(50))为0.26 ng/m L,线性范围(IC_(20)~IC_(80))为0.025~2.83 ng/m L,对鱼样中AMOZ回收率在78.0%~86.0%之间,各样品变异系数均小于15%,与HPLC-MS/MS对比检测结果显示相关性良好。本方法灵敏度高、特异性好,能够满足实际样品的检测需求。
An Indirect competitive time-resolved fluoroimmunoassay (ic-TRFIA) with Eu ~ (3+) labeling was established for the detection of furosemide metabolite AMOZ in fish samples. Through the single factor experiment, the influence of the antigen concentration, antibody dilution and competition reaction time on the sensitivity of the method was investigated. The results showed that the best reaction conditions of ic-TRFIA were as follows: the antigen concentration of coating was 0.25μg / ml, the dilution of antibody was 5 × 10-4 times, the best competition time was 50 min. The detection limit (LOD, IC_ (10)) was 0.01 ng / m L and the IC 50 (IC 50) was 0.26 ng / m L under the optimum conditions. The linear range The IC_ (80)) was 0.025-2.83 ng / m L, and the recoveries of AMOZ in fish samples were between 78.0% and 86.0%. The coefficients of variation of all the samples were less than 15%, which were correlated with the results of HPLC-MS / MS Good sex. The method has high sensitivity and good specificity and can meet the detection requirements of actual samples.