A protein array platform for anti-citrulline antibodies in Rheumatoid Arthritis

描述(申请人提供)：抗瓜氨酸抗体是类风湿性关节炎(RA)的特异性和预测性抗体。临床上用环瓜氨酸多肽(CCP)酶联免疫吸附试验(EL ISA)检测瓜氨酸抗体阳性。尽管抗CCP阳性是一种很好的诊断替代试验，但它并不能揭示引起免疫反应的实际潜在抗原的任何信息。最近的研究表明，识别针对特定抗原的自身抗体在阐明类风湿性关节炎病因中的价值。此外，特异性瓜氨酸抗原的鉴定有助于提高抗CCP试验的检测性能。不幸的是，在过去的几十年里，只发现了几种瓜氨酸抗原。开发一个识别抗瓜氨酸蛋白抗体的平台不仅有助于了解该病的发病机制，还将提高诊断水平。传统的蛋白质免疫化学鉴定瓜氨酸抗原的方法存在通量低、重复性差、定量不充分、分辨率低等缺点。商业蛋白质阵列价格昂贵，缺乏候选抗原的平等表达，并且与翻译后修饰不兼容，例如要求苛刻条件的瓜氨酸化。我们提出了一种覆盖捕获蛋白质微阵列平台，其中蛋白质在硅微孔中表达，并被覆盖载玻片上的配体捕获为裸露的蛋白质。我们的开发将基于我们创新的NAPPA蛋白质微阵列平台，以规避与商业蛋白质阵列相关的挑战，以发现纯化的重组蛋白质。NAPPA包括在微阵列底物上打印与感兴趣的蛋白质相对应的全长cDNA，然后在检测时原位转录/翻译。Cover Capture NAPPA将在翻译后实现阵列上的即时蛋白质表达和瓜氨酸化，而不受NAPPA打印混合物组件的干扰。捕获的蛋白质将呈现在标准的显微镜载玻片上，高亲和力标签/配体的使用将使阵列上的各种翻译后修饰成为可能，包括瓜氨酸化。然后可以并行地评估对多种瓜氨酸蛋白的血清反应性。我们将建立这个平台来分析 在蛋白质组水平上检测类风湿关节炎中的抗瓜氨酸蛋白抗体，并在阵列上定位抗瓜氨酸自身抗原的免疫优势表位，其主要优点是高通量、多重、定量和低成本，从而可以检测足够的样本以得出统计合理的结论。我们的目标是开发一个高通量的平台，以发现更多的抗原，当瓜氨酸作用时，可以在蛋白质组水平上被RA患者的抗体识别。我们相信，覆盖捕获NAPPA将使这一发现成为可能，并极大地有助于RA研究了解疾病的发病机制，并开发更敏感的诊断方法，使患者能够基于自身抗原进行分层。此外，我们提出的研究可以应用于理解其他翻译后修饰的生物学方面的研究。

DESCRIPTION (provided by applicant): Anti-citrulline antibodies are specific and predictive of rheumatoid arthritis (RA). Seropositivity against citrulline is clinically assayed using cyclic citrullinated peptide (CCP) ELISA. Despite being an excellent proxy assay for diagnosis, anti-CCP positivity does not reveal any information about the actual underlying antigens that elicited the immune response. Recent studies demonstrated the value of identifying autoantibodies to particular antigens in the elucidation of RA etiology. Furthermore, the identification of specific citrullinated antigens might help improve the assay performance of anti-CCP test. Unfortunately, only a few citrullinated antigens have been discovered in the past several decades. Developing a platform that will identify antibodies against citrullinated proteins will not only help understad the disease pathogenesis but also improve diagnosis. Traditional protein immuno-chemistry methods to identify citrullinated antigens suffer drawbacks such as low throughput, poor reproducibility, inadequate quantification and low resolution. Commercial protein arrays are expensive, lack equal representation of candidate antigens and are not compatible with post-translational modifications such as citrullination, which requires harsh conditions. We propose a cover capture protein microarray platform where proteins are expressed in silicon microwells and captured as naked protein by ligands on the cover slides. Our development will be based on our innovative NAPPA protein microarray platform to circumvent challenges associated with commercial protein arrays for spotting purified recombinant proteins. NAPPA involves printing full length cDNAs corresponding to proteins of interest on the microarray substrate and then transcription/translation in situ at the time of assay. Cover capture NAPPA will enable just-in-time proteins expression and citrullination on array post-translationally without the interference of NAPPA printing mixture components. Captured proteins will be presented on standard microscopic slides and the employment of high affinity tag/ligand will enable a variety of post-translational modifications on array including citrullination. Sero- reactivity against multiple citrullinated proteins can then be assessed in parallel. We will establish this platform to profile anti-citrullinated protein antibodies in RA at the proteome level and to map immuno-dominant epitopes of citrullinated autoantigens on array with the key advantages being high-throughput, multiplexed, quantitative, and low-cost so that enough samples can be assayed to draw statistical sound conclusion. Our goal is to develop a high-throughput platform to discover additional antigens, when citrullinated, can be recognized by antibodies in RA patients at the proteome level. We believe cover-capture NAPPA will enable this discovery and greatly benefit RA research to understand disease pathogenesis and develop more sensitive diagnostics enabling patient stratification based on autoantigens. Furthermore, what we propose to develop can be applied to studies in understanding the biology of other post-translational modifications.