Department of Oral Biology, University of Florida, P,O, Box 100424, 1395 Center Drive, Gainesville, FL 32610-0424, USA.
Arthritis Res Ther. 2012 May 29;14(3):R128. doi: 10.1186/ar3858.
Classic anti-nucleolar antibodies anti-Th/To and U3 ribonucleoprotein (-U3RNP) can help in the diagnosis, prediction of organ involvement and prognosis in systemic sclerosis (SSc); however, no validated commercial assay is available. We aimed at establishing a novel quantitative real time PCR (qPCR) method to detect these antibodies.
Standard immunoprecipitation (IP) was performed using K562 cell extract and RNA components were extracted. cDNA was reverse transcribed from RNA components and Th RNA and U3 RNA were detected by qPCR using custom primers. Cycle threshold (Ct) values were compared in a titration experiment to determine the assay efficacy. The new assay was evaluated by testing 22 anti-Th/To and 12 anti-U3RNP positive samples in addition to 88 controls, and the results were compared with IP as a gold standard.
By testing serial 1:8 dilutions of cell lysate as the substrate in the IP step, RNA extracted after IP, and its derived cDNA, linear dose response curves were noted for both anti-Th/To and -U3RNP. With every dilution, Ct values changed approximately three as expected, reflecting the eight-fold difference of cDNA. The Ct difference between positive and negative samples was 8 to 13, which was similar throughout the dilutions. In the specificity analysis, the Ct values of positive samples were clearly different from the negative groups and the results by qPCR had a near perfect correlation with IP.
Our new method readily detects these two clinically important antibodies in SSc. Making tests for anti-Th/To and -U3RNP antibodies widely available to clinicians should be helpful in the diagnosis and follow-up of SSc patients.
经典的抗核仁抗体抗-Th/To 和 U3 核糖核蛋白（-U3RNP）可帮助诊断、预测系统性硬皮病（SSc）的器官受累和预后；然而，目前尚无经过验证的商业检测方法。我们旨在建立一种新的定量实时 PCR（qPCR）方法来检测这些抗体。
使用 K562 细胞提取物进行标准免疫沉淀（IP），提取 RNA 成分。使用定制引物从 RNA 成分反转录 cDNA，并通过 qPCR 检测 Th RNA 和 U3 RNA。在滴定实验中比较循环阈值（Ct）值以确定检测方法的功效。通过测试 22 种抗-Th/To 和 12 种抗-U3RNP 阳性样本以及 88 个对照，评估新方法，并将结果与 IP 作为金标准进行比较。
通过测试作为 IP 步骤中底物的细胞裂解物的连续 1:8 稀释液、IP 后提取的 RNA 及其衍生的 cDNA，可以得到抗-Th/To 和 -U3RNP 的线性剂量反应曲线。随着每一次稀释，预期 Ct 值变化约三倍，反映了 cDNA 的八倍差异。阳性和阴性样本之间的 Ct 值差异为 8 到 13，在整个稀释过程中相似。在特异性分析中，阳性样本的 Ct 值与阴性组明显不同，qPCR 的结果与 IP 具有近乎完美的相关性。
我们的新方法可轻松检测 SSc 中这两种重要的临床抗体。为临床医生提供抗-Th/To 和 -U3RNP 抗体的检测应该有助于 SSc 患者的诊断和随访。
