近红外荧光标记-免疫磁珠偶联法定量检测TB-LAM抗原

Quantitative detection of TB-LAM antigen with near infrared fluorescent labeled-immunomagnetic beads coupling method

目的基于标记免疫分析的原理,建立近红外荧光染料-免疫磁珠偶联法定量检测结核分枝杆菌脂阿拉伯甘露聚糖(LAM)的方法。方法以近红外荧光染料标记靶向于LAM的单克隆抗体,靶向于LAM的多克隆抗体包被在纳米磁珠表面。采用双抗体夹心法,磁性分离结合物和游离物,采用便携式高灵敏度低噪声激发式荧光检测仪检测磁性结合物的荧光强度,从而检测待检样品中LAM含量。结果该方法的检测线性范围为1 ng/ml^625 ng/ml,最低检测限量为0.5 ng/ml;10 ng/ml水平加样回收率为93%,50 ng/ml水平加样回收率为109%;批间CV为6.5%,批内CV为4.1%。临床样本检测LAM特异度为92.9%,灵敏度为90.0%。结论本方法检测LAM是结核感染实验室诊断的有效指标。

Objective Based on the marking immunoassay principles,a near- infrared fluorescent dye- immunomagnetic beads coupling method can be set up for quantitative detection of lipoarabinomannan（ LAM）. Methods Near- infrared fluorescent dye was used to mark LAM- targeting monoclonal antibodies,and LAM- targeting monoclonal antibody was coated on the surface of nano- magnetic beads. Double antibody sandwich method was used for magnetic separation of conjugates and dissociants. Portable high- sensitivity and low- noise excitation fluorescence detector were used to detect the intensity of magnetic combination,so as to test the LAM content in the test samples. Results The detecting linear range of this method was1 ng / ml- 625 ng / ml,and the minimum detection limit was 0. 5 ng / ml; the recovery rate of the added samples of the 10 ng / ml level was 93%,and the recovery rate of the added samples of the 50 ng / ml was 109%; the between- run CV value was 6. 5%,and the within- run CV value was 4. 1%. The TB- LAM specificity was found to be 92. 9% through testing clinical samples and the sensitivity was 90. 0%. Conclusion This method of LAM detection is valid index for laboratory diagnosis of tuberculosis infection.