Validation of a multiplex chip-based assay for the detection of autoantibodies against citrullinated peptides.
Validation of a multiplex chip-based assay for the detection of autoantibodies against citrullinated peptides.
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DOI：
10.1186/ar4039
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发表时间：
2012-10-01
影响因子：
4.9
通讯作者：
Rönnelid J
中科院分区：
文献类型：
--
作者：
Hansson M;Mathsson L;Schlederer T;Israelsson L;Matsson P;Nogueira L;Jakobsson PJ;Lundberg K;Malmström V;Serre G;Holmdahl R;Nystrand M;Klareskog L;Rönnelid J
针对瓜氨酸蛋白/多肽(ACPA)的自身抗体对类风湿关节炎(RA)的发展具有高度的特异性和预测性。不同的类风湿关节炎患者亚组具有不同的自身抗体谱，具有不同的预后，可能需要不同的治疗。本研究的目的是开发一种用于检测多种RA相关自身抗体的微阵列，最初专注于RA候选自身抗原上瓜氨酸表位的反应。该芯片基于Phadia的免疫CAP ISAC系统，通过使用微量血清体积(<；10μL)，可以同时分析对100多种抗原的反应性。12个瓜氨酸多肽和相应的天然含精氨酸的对照多肽以阵列的方式固定在经过化学修饰的玻璃片上，从而形成具有高结合能力的三维层。对血清稀释度和玻片表面进行了优化，对每个抗原的偶联化学、抗原浓度和斑点缓冲液的选择进行了优化。将免疫CAP ISAC系统中各多肽的性能与酶联免疫吸附试验(ELISAs)中的性能进行比较。将927例RA患者和461例健康对照的血清涂抹在玻片上的反应部位，然后用荧光标记的抗人免疫球蛋白G(Ig G)抗体。用激光扫描仪检测荧光强度，用图像分析软件对结果进行分析。对于单独的瓜氨酸多肽，免疫CAP ISAC系统和ELISA结果之间存在很强的相关性(Spearmanρ通常在0.75%到0.9%之间)。RA血清与这些多肽的反应性主要见于抗环瓜氨酸多肽2(CCP2)阳性亚群，但在抗CCP2阴性亚群中也可与单一的瓜氨酸多肽发生反应。对含有精氨酸的对照多肽的反应性进行调整，并不会统一改变针对个别瓜氨酸多肽的抗体的诊断性能。该多重阵列用于检测针对候选RA自身抗原上的多个瓜氨酸表位的自身抗体，将有助于RA发病机制、诊断的研究，并有可能作为个体化治疗的指南。
Autoantibodies directed against citrullinated proteins/peptides (ACPAs) are highly specific and predictive for the development of rheumatoid arthritis (RA). Different subgroups of RA patients, which have different prognoses and may require different treatments, are characterized by different autoantibody profiles. The objective of this study was to develop a microarray for the detection of multiple RA-associated autoantibodies, initially focusing on responses against citrullinated epitopes on candidate autoantigens in RA. The microarray is based on Phadia's ImmunoCAP ISAC system, with which reactivity to more than 100 antigens can be analyzed simultaneously, by using minute serum volumes (< 10 μl). Twelve citrullinated peptides, and the corresponding native arginine-containing control peptides, were immobilized in an arrayed fashion onto a chemically modified glass slide, allowing a three-dimensional layer with high binding capacity. The assay was optimized concerning serum dilution and glass surface, whereas each individual antigen was optimized concerning coupling chemistry, antigen concentration, and selection of spotting buffer. The performance of each peptide in the ImmunoCAP ISAC system was compared with the performance in enzyme-linked immunosorbent assays (ELISAs). Serum from 927 RA patients and 461 healthy controls from a matched case-control study were applied onto reaction sites on glass slides, followed by fluorescent-labeled anti-human immunoglobulin G (IgG) antibody. Fluorescence intensities were detected with a laser scanner, and the results analyzed by using image-analysis software. Strong correlations between the ImmunoCAP ISAC system and ELISA results were found for individual citrullinated peptides (Spearman ρ typically between 0.75 and 0.90). Reactivity of RA sera with the peptides was seen mainly in the anticyclic citrullinated peptide 2 (CCP2)-positive subset, but some additional reactivity with single citrullinated peptides was seen in the anti-CCP2-negative subset. Adjusting for reactivity against arginine-containing control peptides did not uniformly change the diagnostic performance for antibodies against the individual citrullinated peptides. The multiplexed array, for detection of autoantibodies against multiple citrullinated epitopes on candidate RA autoantigens, will be of benefit in studies of RA pathogenesis, diagnosis, and potentially as a guide to individualized treatment.
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影响因子：
4.9
作者：
Mullazehi M;Wick MC;Klareskog L;van Vollenhoven R;Rönnelid J
通讯作者：
Rönnelid J
影响因子：
4.8
作者：
Deinhofer, K;Sevcik, H;Spitzauer, S
通讯作者：
Spitzauer, S
影响因子：
--
作者：
Snir, Omri;Widhe, Mona;Malmstrom, Vivianne
通讯作者：
Malmstrom, Vivianne
影响因子：
5.4
作者：
Sebbag, Mireille;Moinard, Nathalie;Serre, Guy
通讯作者：
Serre, Guy
影响因子：
4.9
作者：
Kinloch, Andrew;Tatzer, Verena;Wait, Robin;Peston, David;Lundberg, Karin;Donatien, Phillipe;Moyes, David;Taylor, Peter C;Venables, Patrick J
通讯作者：
Venables, Patrick J