JWA蛋白时间分辨荧光免疫层析检测方法的建立

Establishment of Time-resolving Fluorescence Immunochromatographic Assay for JWA Protein

目的:建立时间分辨荧光免疫层析法检测JWA蛋白的体系,并对该法进行评价。方法:运用EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)-NHS(N-羟基琥珀酰亚胺)活化时间分辨荧光微球,活化的时间分辨荧光微球按100μg Ab∶1 mg微球标记JWA单克隆抗体7C3;将荧光标记的抗体运用于免疫层析检测。结果:时间分辨荧光微球成功标记JWA单克隆抗体,荧光标记的7C3可以准确识别JWA多肽;测量范围可从20 ng·mL^(-1)至1500 ng·mL^(-1),线性范围可达100 ng·mL^(-1)～1000 ng·mL^(-1),回收率在95%以上,高、中、低浓度的重复性检测离散度(CV)均小于10%,加速稳定性亦较好,同时可以检测出胃癌细胞中的JWA蛋白。结论:本研究建立的时间分辨免疫荧光层析法测定JWA蛋白,各项分析性能良好,且便捷准确、快速经济,为检测JWA蛋白提供了一种高效的新途径。

Objective:Using time-resolving fluorescent microspheres to label the JWA monoclonal antibodies and to establish the immunofluorescence chromatography strip.Methods:The EDC-NHS activated time-resolving fluorescent microspheres were used to label JWA monoclonal antibody 7C3 with 100μg Ab:1 mg beads,and the fluorescently labeled antibodies were used for immunochromatographic detection.Results:The time-resolving fluorescent microspheres labeled JWA monoclonal antibody 7C3 could accurately recognize JWA polypeptides,with a measuring range of 20-1500 ng·mL-1.The linear response range of the method was 100-1000 ng·mL-1.The values for coefficient of variation at high,medium and low concentrations were all less than 10%with average recovery rates over 95%.The JWA protein in gastric cancer cells can be detected simultaneously.Conclusion:The time-resolving immunofluorescence chromatographic method for assaying JWA protein in cells provided a convenient,rapid and economical option.It is a highly efficient new way for the detection of JWA protein.