基于免疫磁珠前处理的荧光免疫层析法定量检测牦牛肉中金刚烷胺

Quantitative Detection of Amantadine in Yak Beef by Fluorescence Immunochromatography Based on Pretreatment of Immunomagnetic Beads

研究旨在提供一种基于免疫磁珠分离、富集和净化前处理,检测牦牛肉中金刚烷胺残留的荧光免疫层析定量分析方法。通过对免疫磁珠偶联液、抗体偶联量、偶联时间、微球的抗体标记量、金刚烷胺抗原和羊抗鼠二抗包被浓度等进行优化,初步建立了基于免疫磁珠前处理的时间分辨荧光免疫层析方法(TRIFA),并对其方法的灵敏度、准确度和精密度进行了评价。结果表明,经过优化后荧光微球抗体标记量为6μL,抗原和羊抗鼠二抗的包被浓度均为0.52μg/mL;免疫磁珠偶联液为0.02 mol/L PB、磁珠与抗体最佳偶联量为100μg、捕获时间为60 min;所建立方法的检出限为0.46μg/kg,IC50为2.327μg/kg,线性范围为0.87~7.32μg/kg,回收率为82.05%~109.95%,批内变异系数为4.77%~8.86%,批间变异系数为9.13%~11.92%。经验证,牦牛肉样本检测结果与液相色谱-串联质谱法(LC-MS/MS)结果一致。结论:该研究建立的基于免疫磁珠前处理的时间分辨荧光免疫层析方法适用于牦牛肉样品中金刚烷胺类药物残留的检测,方法的灵敏度高、稳定性好,说明免疫磁珠-荧光免疫层析法可作为牦牛肉中金刚烷胺快速检测的有效手段。

This study aims to provide a fluorescence immunochromatographic quantitative analysis method for detecting amantadine residues in yak meat based on the separation,enrichment and purification pretreatment of immunomagnetic beads.By optimizing the immunomagnetic bead coupling solution,antibody coupling amount,coupling time,as well as the amount of microsphere antibody labeling,amantadine antigen and goat anti-mouse secondary antibody coating concentration,etc,a preliminary establishment based on immunomagnetic beads.The pre-processing time-resolved fluorescence immunochromatography method,and the sensitivity,accuracy and precision of the method were evaluated.The results show that the optimized fluorescent microsphere antibody labeling volume is 6μL,the coating concentration of antigen and goat anti-mouse secondary antibody are both 0.52μg/mL;the immunomagnetic bead coupling solution is 0.02 mol/L PB,magnetic beads and The optimal coupling amount of antibody is 100μg,and the capture time is 60 min;the detection limit of the established method is 0.46μg/kg,the linear range is 0.87~7.32μg/kg,IC50 is 2.327μg/kg,and the recovery rate is 82.05%~109.95%.The coefficient of variation is 4.77%~8.86%,and the coefficient of variation between batches is 9.13%~11.92%.It has been verified that the test results of yak meat samples are consistent with the results of the LC-MS/MS method.Conclusion:The time-resolved fluorescence immunochromatography method based on the pretreatment of immunomagnetic beads established in this study is suitable for the detection of amantadine residues in yak meat samples.The method has high sensitivity and good stability,indicating that immunomagnetic-fluorescence immunoassay chromatography can be used as an effective method for rapid detection of amantadine in yak meat.