两种量子点标记用于抗核抗体检测的对比研究与评价

Comparative investigation and evaluation of detection for anti-nuclear antibodies labeled by quantum dots with two different types of size

目的对比研究两种不同粒径量子点(QDs)标记抗体在抗核抗体(ANA)检测中的成像效果,评价其在临床样本检测中的应用价值。方法 Hep-2细胞为抗原片,分别以QD655和QD605标记第二抗体(二抗),用间接免疫荧光分析(IIFA)检测114例自身免疫病患者和30例健康对照者血清ANA,以异硫氰酸荧光素(FITC)标记的二抗为对照。单特异性ANA用免疫印迹法(IBT)确认。比较和评价各法应用效果。结果经对114例患者血清研究发现,两种QD标记二抗均可见与FITC标记二抗检测ANA结果相同或类似的荧光模式,但ANA滴度略显不同,对核颗粒型与胞浆颗粒型ANA测定灵敏度以QD655为最高,QD605次之,FITC标记二抗相对较弱。而对核均质型、核仁型与核着丝点型ANA测定灵敏度高低依次为FITC、QD655和QD605标记二抗。相关分析结果显示,两种QD与FITC标记物检测ANA滴度结果均呈高度正相关,r值分别为0.834、0.832(P均<0.01)。两种QD标记二抗对核颗粒型、胞浆颗粒型荧光模式与FITC标记二抗检测结果阳性符合率均为100%;而核均质型与核仁型检测,阳性结果符合率为80.6%～84.4%。30例用FITC标记二抗检测ANA阴性血清中,两种QD标记二抗检测均见1例ANA阳性。两种QD与FITC标记物检测ANA总符合率均超过90.0%,有高度的一致性(Kappa值均>0.75)。结论 QD655和QD605标记二抗用于ANA检测总体结果稳定、灵敏、可行;QDs粒径越大其标记二抗测定ANA灵敏度越高,但对某些低滴度免疫荧光核型(包括核均质型、核仁型与核着丝点型)ANA有漏检可能。

Objective To compare the imaging effect of anti-nuclear antibodies （ANA） detected by indirect immunofluorescence assay （IIFA） based on quantum dots （QDs） labeling with two different types of size, and evaluate their clinical application. Methods U- sing Hep-2 cells as experimental matrix, QD655 and QD605 as the marker of the secondary antibody, the titers and the immunofluores- cence patterns of ANA bound to the intracellular components were detected by IlFA in 114 ANA-positive patients and 30 ANA-negative controls. The results of assay were compared with those of the assay in which fluorescein isothiocyanate （FITC） was used as the marker of the secondary antibody. Single specific ANA was further confirmed by immunoblotting technique. Results The fluorescent patterns of ANA detected by the two QDs in the 114 patients were identical or similar with those detected by FITC markers. However, the sensi- bilities of 3 assay varied with different labeling markers. For the patterns of nuclear speckle and cytoplasmic granule, the highest sensi- bility was visualized in the assay with QD655 as the marker, followed by QD605, and the lowest was FITC. For the patterns of nuclear homogeneity, nucleolus and centromere, the highest sensibility was visualized by FITC marker, followed by QD655, and the lowest was QD605. Correlation analysis showed that ANA titers detected by the both of QDs markers were highly correlated with those of FITC markers, and the correlation coefficient values were 0. 834 for QD 655 and 0. 832 for QD605, respectively （ P 〈 0.01 ）. The coinci- dence rates of detected ANA were 100% between the two QDs and FITC markers for the patterns of nucleus speckle and cytoplasm granule, but for the patterns of nuclear homogeneity and nucleolus, the coincidence rates were from 80.6% to 84.4% between the two QDs and FITC markers. Among 30 ANA-negative controls detected by FITC marker, one was ANA-positive by the two QDs markers. The overall coincidence rate was more than 90.0% for the results of ANA detection between the two QDs and FITC markers （ Kappa values 〉 0.75）. Conclusion The IIFA with both QD655 and QD605 as the marker of secondary antibody was stable, sensitive and feasible for the detection of ANA in serum. The sensitivity of the assay increased with larger size of QDs. However, the ANA of some immunofluorescence patterns with low titer, including nuclear homogeneity, nucleolus and centromere, may be undetectable by the IIFA with QDs markers.