A new immunoprecipitation-real time quantitative PCR assay for anti-Th/To and anti-U3RNP antibody detection in systemic sclerosis
A new immunoprecipitation-real time quantitative PCR assay for anti-Th/To and anti-U3RNP antibody detection in systemic sclerosis
复制标题
DOI：
10.1186/ar3858
复制
发表时间：
2012-01-01
影响因子：
4.9
通讯作者：
Chan, Edward K. L.
中科院分区：
文献类型：
--
作者：
Ceribelli, Angela;Satoh, Minoru;Chan, Edward K. L.
简介：经典抗核仁抗体抗Th/To和U3核糖核蛋白（-U3RNP）有助于系统性硬化症（SSc）的诊断、器官受累预测和预后；然而，没有经过验证的商业化验可用。我们的目的是建立一种新的定量实时PCR (qPCR) 方法来检测这些抗体。方法：使用K562 细胞提取物进行标准免疫沉淀(IP)，并提取RNA 成分。从 RNA 成分逆转录 cDNA，并使用定制引物通过 qPCR 检测 Th RNA 和 U3 RNA。在滴定实验中比较循环阈值 (Ct) 以确定测定效果。除了 88 个对照外，还通过测试 22 个抗 Th/To 和 12 个抗 U3RNP 阳性样品来评估新测定，并将结果与作为金标准的 IP 进行比较。结果：通过测试 IP 步骤中作为底物的细胞裂解液的系列 1:8 稀释液、IP 后提取的 RNA 及其衍生的 cDNA，记录了抗 Th/To 和 -U3RNP 的线性剂量反应曲线。每次稀释，Ct 值都会按预期变化大约 3，反映 cDNA 的八倍差异。阳性和阴性样品之间的 Ct 差异为 8 至 13，在整个稀释度中相似。在特异性分析中，阳性样本的Ct值与阴性组明显不同，并且qPCR结果与IP具有近乎完美的相关性。结论：我们的新方法很容易检测SSc中的这两种临床上重要的抗体。临床医生广泛使用抗 Th/To 和 -U3RNP 抗体检测应该有助于 SSc 患者的诊断和随访。
Introduction: 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.Methods: 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.Results: 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.Conclusions: 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.