ABSTRACT

Background: Autoantibodies (AAbs) are important biomarkers for the diagnosis of Autoimmune Diseases (ADs). The detection of AAbs performed by current methods (indirect immunofluorescence test (IIFT)/Immunoblot (dot/line)/enzyme-linked immunosorbent assay ELISA) which have limitations in terms of performing multiple assays to arrive at laboratory diagnosis. We validated a novel multiplex bead-based assay (NMBA) that could quantify five common antibodies, simultaneously, on a flow-cytometry platform.

Methods: A total of five recombinant antigens (SS-A Ro60, CENP B, RNP 70, Scl 70 and Histones) were covalently coupled onto beads and tested using known positive sera (positive for AAbs) and analyzed using flow cytometer.

Results: The sensitivity, specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) were obtained for each antigen, analyzed by both assays (NMBA and IIFT). It showed comparable or higher values for the NMBA. The Spearman’s rank correlation coefficient (Rho) were ≥ 0.97, (P < .05), indicating that multiplexing of the five autoantigens did not alter the results obtained when antigens were tested individually. The mean intra-assay precision measured by coefficient of variation (CV) was7.56 ± 1.6% and the mean inter-assay CV was 10.03 ± 1.34%. The time taken from sample receipt to reporting of results was 90 minutes in NMBA as compared to 150 minutes of IIFT

Conclusion: The NMBA could quantitatively measure antibodies against five autoantigens, simultaneously in patient’s sera. The assay is faster, objective, reproducible, requires low sample volume, and stable. Moreover, the flow cytometer in diagnostic laboratory settings for hematological and transplant immunology tests, can also be used for testing AAbs.

摘要

背景：自身抗体（AAbs）是诊断自身免疫性疾病（ADs）的重要生物标志物。通过当前方法（间接免疫荧光测试（IIFT）/免疫印迹（点/线）/酶联免疫吸附测定ELISA）进行的Aab的检测在进行多种测定以达到实验室诊断方面存在局限性。我们验证了一种新颖的基于多重珠的测定法（NMBA），该测定法可以在流式细胞仪平台上同时定量五种常见抗体。

方法：将总共五种重组抗原（SS-A Ro60，CENP B，RNP 70，Scl 70和组蛋白）共价偶联至磁珠上，并使用已知的阳性血清（AAb呈阳性）进行测试，并使用流式细胞仪进行分析。

结果：获得每种抗原的敏感性，特异性，阳性预测值（PPV）和阴性预测值（NPV），并通过两种测定法（NMBA和IIFT）进行分析。它显示出与NMBA相当或更高的价值。Spearman等级相关系数（Rho）≥0.97，（P <0.05），表明五种自身抗原的多重性不会改变单独测试抗原时获得的结果。通过变异系数（CV）测量的平均批内分析准确性为7.56±1.6％，批间平均CV为10.03±1.34％。在NMBA中，从样品接收到报告结果所需的时间为90分钟，而IIFT为150分钟

结论：NMBA可以同时定量检测患者血清中针对五种自身抗原的抗体。该测定更快，更客观，可重复，需要的样品量少且稳定。此外，流式细胞仪在诊断实验室设置中可用于血液学和移植免疫学测试，也可用于测试Abb。