Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint
Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint
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DOI：
10.1186/ar4683
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发表时间：
2014-01-01
影响因子：
4.9
通讯作者：
Lundberg, Karin
中科院分区：
文献类型：
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作者：
Ossipova, Elena;Cerqueira, Catia Fernandes;Lundberg, Karin
介绍：类风湿性关节炎（RA）患者的一个主要亚群的特征是存在针对瓜氨酸化蛋白/肽（ACPA）的循环自身抗体。这些自身抗体通常通过使用基于合成环瓜氨酸肽（CCP）的酶联免疫吸附测定（ELISA）来检测，可预测临床发作和破坏性病程。在本研究中，我们使用了来自RA患者的血浆和滑液，用于抗CCP 2反应性抗体的亲和纯化和表征，目的是产生可用于体外和体内的分子工具，用于将来研究ACPA反应的病理生物学。具体而言，本研究的目的是证明，替代标记物CCP 2可以捕获ACPA，结合在体内表达的自身抗原在RA的主要炎性病变（即，在类风湿关节）。方法：血浆（n = 16）和滑液（n = 26）样本收集自RA患者的抗CCP 2 IgG水平高于300 Au/mL。在蛋白G柱上分离总IgG，随后应用于CCP 2亲和柱。通过使用CCPlus（R）ELISA、内部肽ELISA、Western印迹和免疫组织化学/免疫细胞化学分析纯化的抗CCP 2 IgG的反应性和特异性。来自不同患者的纯化抗CCP 2反应性抗体显示与CCP 2结合的差异以及与来自α-烯醇化酶、波形蛋白、纤维蛋白原和II型胶原蛋白的瓜氨酸化肽结合的差异，说明不同的ACPA精细特异性特征。此外，纯化的ACPA不仅在体外结合瓜氨酸化蛋白，而且更重要的是，在体内结合RA患者滑液细胞和滑膜组织上产生的表位。我们已经从血浆和滑液中分离出ACPA，并证明了经常用于诊断ELISA的CCP 2肽事实上充当至少四种不同的、充分表征的、基本上非交叉反应的ACPA的替代抗原，ACPA精细特性。此外，我们已经确定了类风湿血浆和滑液中CCP 2反应性IgG分子的浓度和比例，并且我们已经表明，纯化的ACPA可用于通过各种技术检测体外和体内产生的瓜氨酸化表位。我们预计，这些抗体将为我们提供新的机会，调查潜在的致病作用的人ACPA。
Introduction: A major subset of patients with rheumatoid arthritis (RA) is characterized by the presence of circulating autoantibodies directed to citrullinated proteins/peptides (ACPAs). These autoantibodies, which are commonly detected by using an enzyme-linked immunosorbent assay (ELISA) based on synthetic cyclic citrullinated peptides (CCPs), predict clinical onset and a destructive disease course. In the present study, we have used plasma and synovial fluids from patients with RA, for the affinity purification and characterization of anti-CCP2 reactive antibodies, with an aim to generate molecular tools that can be used in vitro and in vivo for future investigations into the pathobiology of the ACPA response. Specifically, this study aims to demonstrate that the surrogate marker CCP2 can capture ACPAs that bind to autoantigens expressed in vivo in the major inflammatory lesions of RA (that is, in the rheumatoid joint).Methods: Plasma (n = 16) and synovial fluid (n = 26) samples were collected from RA patients with anti-CCP2 IgG levels of above 300 AU/mL. Total IgG was isolated on Protein G columns and subsequently applied to CCP2 affinity columns. Purified anti-CCP2 IgG was analyzed for reactivity and specificity by using the CCPlus (R) ELISA, in-house peptide ELISAs, Western blot, and immunohisto-/immunocytochemistry.Results: Approximately 2% of the total IgG pool in both plasma and synovial fluid was CCP2-reactive. Purified anti-CCP2 reactive antibodies from different patients showed differences in binding to CCP2 and differences in binding to citrullinated peptides from a-enolase, vimentin, fibrinogen, and collagen type II, illustrating different ACPA fine-specificity profiles. Furthermore, the purified ACPA bound not only in vitro citrullinated proteins but, more importantly, in vivo-generated epitopes on synovial fluid cells and synovial tissues from patients with RA.Conclusions: We have isolated ACPAs from plasma and synovial fluid and demonstrated that the CCP2 peptides, frequently used in diagnostic ELISAs, de facto act as surrogate antigens for at least four different, well-characterized, largely non-cross-reactive, ACPA fine specificities. Moreover, we have determined the concentration and proportion of CCP2-reactive IgG molecules in rheumatoid plasma and synovial fluid, and we have shown that the purified ACPAs can be used to detect both in vitro-and in vivo-generated citrullinated epitopes by various techniques. We anticipate that these antibodies will provide us with new opportunities to investigate the potential pathogenic effects of human ACPAs.