流式微球一步法快速免疫检测马铃薯A病毒

One-step cytometric bead array for rapid detection of Potato Virus A

【目的】建立流式微球一步法快速免疫检测马铃薯A病毒(PVA)的新方法。【方法】以荧光微球为反应载体,通过在微球表面进行双抗夹心免疫反应形成微球-捕获抗体-PVA-标记FITC检测抗体的复合物,利用流式细胞仪荧光检测系统收集荧光信号。【结果】通过实验优化检测条件,最佳捕获抗体工作浓度为4μg/mL、最佳检测抗体工作浓度为1:25倍稀释、最佳反应时间为2h;与马铃薯Y病毒、莴苣花叶病毒、番茄环斑病毒等均未出现交叉反应;阳性样品经64倍稀释后依然可检出,检测灵敏度是传统微孔板ELISA的4倍。【结论】流式微球一步法能灵敏、快速、简便的检测马铃薯A病毒。

[Objective] Based on immunoassay, we developed a method to detect Potato Virus A （PVA） using one-step cytometric bead array （CBA）. [Methods] Using fluorescent beads as carriers, we formed the complexes of bead-capture antibody-PVA-fluoresceinisothiocyanate （FITC） labeled detection antibody by immunoreaction of double antibody sandwich on bead surface. We measured the fluorescent signals produced from beads and from FITC with a multiparameter flow cytometer. [Results] By optimization test, capture antibody and detection antibody （from PVA detection kit, Agdia, USA） were finally diluted at 1：50 （4ug/mL） and 1：25 to be used in one step CBA, respectively. A total CBA reaction time of 2 h was needed. No cross reactivity with other plant virus similar to potato virus Y, lettuce mosaic virus and tomato ring spot virus were found. Detection sensitivity for positive control sample in one-step CBA was 4 folds higher than that in normal plate double antibodies sandwich enzyme-linked immunosorbent assay （DAS-ELISA）. [Conclusion] A one-step CBA method for rapid and sensitive detection of PVA is developed.