呕吐毒素时间分辨荧光免疫层析法的建立与评价

构建并评价了呕吐毒素(Deoxynivalenol,DON)时间分辨荧光免疫层析法(TRFIA)。以Eu3+螯合物的纳米微球为荧光探针标记抗DON单抗,采用竞争抑制建立免疫层析定量方法,优化了微球与抗体标记量,检测的环境温度、反应时间、加样体积及缓冲体系;研究了方法的灵敏度、精密度及准确度。结果表明,每100 μL荧光微球结合纯单抗50 μg,最佳划膜浓度为1.0 mg/mL,最佳测定条件为室温(25±2)℃,10 min,加样体积100 μL,PBS (0.01 mol/L,pH7.2)的缓冲体系,方法的灵敏度为0.25 ng/mL,线性范围为0.5~25.0 ng/mL,玉米、小麦阴性样本(加标浓度200、500、1000、2000 μg/kg)平均加标回收率为91.4%~109.3%,6种典型样本经TRFIA与免疫亲和净化-高效液相色谱法(IAC-HPLC)同时测定DON含量,相关系数r达0.9754。TRFIA具有灵敏度高、速度快、定量准等技术特点,适用于谷物及制品中DON的快速定量。

Establishment and Evaluation of Time-Resolved Fluorescence Immunochromatographic Assay for Detecting Deoxynivalenol

The time-resolved fluorescence immunochromatographic assay (TRFIA) was established by competitive inhibition for determination of deoxynivalenol (DON).Nanometer microspheres with Eu3+ chelate were chosen as fluorescent probes using for tagging DON monoclonal antibody.This quantitative method was studied in the application of mycotoxins detection to determine the sensitivity, precision and accuracy.The results showed that for each 100 μL fluorescence microspheres combined with pure single antibody 50 μg, the best film concentration was 1.0 mg/mL, the optimal measurement conditions were room temperature (25 ±2)℃, 10 min, loading volume 100 μL, buffer solution of PBS (0.01 mol/L, pH7.2), the detection limit was 0.25 ng/mL, the linearity range was 0.5~25.0 ng/mL, and the average recovery rate was 91.4%~109.3% respectively when the concentration of standard addition was 200, 500, 1000 and 2000 μg/kg.The content of DON was detected in different samples such as corn and wheat by TRFIA and immune affinity-high performance liquid chromatography (IAC-HPLC) method simultaneously.The correlation coefficient (r) was 0.9754 comparing these two methods.Therefore, TRFIA was characterized by high sensitivity, fast speed and quantitative accuracy, which could be applied to the quick determination of DON in cereals and derived products.