近红外荧光标记-免疫磁珠偶联法定量检测结核分枝杆菌ESAT-6蛋白

目的建立定量检测结核分枝杆菌早期分泌抗原靶-6(ESAT-6)的近红外荧光标记-免疫磁珠偶联法。方法以近红外荧光染料(Dylight 800)标记靶向ESAT-6的单克隆抗体,靶向ESAT-6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ESAT-6水平。结果该方法的检测线性范围为2.4~750.0ng/mL,最低检测限为0.48ng/mL;10ng/mL水平加样回收率为96%,50ng/mL水平加样回收率为95%;批间变异系数(CV)为5.8%,批内CV为4.3%。该方法检测胸腔积液标本ESAT-6的特异度为80%,灵敏度为95%。结论该方法检测ESAT-6的线性范围广、灵敏度高、稳定性好。

Study on quantitatively detecting Mycobacterium tuberculosis ESAT-6 antigen by using near-infrared fluorescent dye-immunomagnetic beads coupling method

Objective To establish a near‐infrared fluorescent dye‐immunomagnetic beads coupling method for quantitative de‐tection of Mycobacterium tuberculosis early secretory antigenic target‐6(ESAT‐6) .Methods Near‐infrared fluorescent dye ,dylight 800 ,was used to mark ESTA‐6‐targeting monoclonal antibodies ,and the surface of nano‐magnetic beads were coated with ESAT‐6‐targeting polyclonal antibodies .Double antibody sandwich method was used for magnetic separation of conjugates and dissociants . Portable high‐sensitivity and low‐noise excitation fluorescence detector was used to detect the intensity of magnetic combination ,so as to test the ESAT‐6 content in test samples .Results The detecting linear range of this method was 2 .4-750 .0 ng/mL ,and the minimum detection limit was 0 .48 ng/mL .The recovery rate was 96% at a concentration of 10 ng/mL ,and at a concentration of 50 ng/mL the recovery rate was 95% .The between‐run coefficient of variation(CV)value was 5 .8% ,and the within‐run CV value was 4 .3% .The specificity and sensitivity of this method for detecting ESAT‐6 in clinical pleural effusion samples were 80% and 95% , respectively .Conclusion This method might have wide linear range ,high sensitivity and good stability in the detection of ESAT‐6 .