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

描述（由申请方提供）：抗瓜氨酸抗体具有特异性，可预测类风湿性关节炎（RA）。临床上使用环瓜氨酸化肽（CCP）ELISA检测抗瓜氨酸血清阳性。尽管抗CCP阳性是一种很好的诊断替代检测，但它并不能揭示引起免疫应答的实际潜在抗原的任何信息。最近的研究表明，确定特定抗原的自身抗体在阐明RA病因的价值。此外，特异性瓜氨酸化抗原的鉴定可能有助于提高抗CCP试验的检测性能。不幸的是，在过去的几十年中，只有少数瓜氨酸化抗原被发现。开发一个能够识别瓜氨酸化蛋白抗体的平台，不仅有助于理解疾病的发病机制，还有助于提高诊断水平。鉴定瓜氨酸化抗原的传统蛋白质免疫化学方法存在诸如低通量、差的再现性、不充分的定量和低分辨率的缺点。商业蛋白质阵列价格昂贵，缺乏候选抗原的同等代表性，并且与需要苛刻条件的翻译后修饰如瓜氨酸不相容。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，并通过载玻片上的配体捕获为裸蛋白。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，用于定位纯化的重组蛋白。NAPPA涉及在微阵列基底上打印对应于感兴趣的蛋白质的全长cDNA，然后在测定时原位转录/翻译。Cover capture NAPPA将使蛋白质表达和瓜氨酸在阵列上的即时表达能够在不受NAPPA打印混合物组分干扰的情况下进行。捕获的蛋白质将呈现在标准显微镜载玻片上，并且高亲和力标签/配体的使用将使包括瓜氨酸在内的阵列上的各种翻译后修饰成为可能。然后可以平行评估针对多种瓜氨酸化蛋白的血清反应性。我们将建立这个平台， 在蛋白质组水平上检测RA患者的抗瓜氨酸蛋白抗体，并在芯片上定位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，以便可以分析足够的样品以得出统计学上合理的结论。我们的目标是开发一个高通量平台，以发现瓜氨酸化的其他抗原，这些抗原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，并大大有利于RA研究，以了解疾病的发病机制，并开发更敏感的诊断，使患者分层的基础上自身抗原。此外，我们所提出的发展可以应用于了解其他翻译后修饰生物学的研究。 公共卫生相关性：风湿性关节炎项目中抗瓜氨酸抗体的蛋白质阵列平台叙述/相关性三百万美国人患有风湿性关节炎。一个特征性特征是针对瓜氨酸修饰的自身蛋白的抗体的发展。我们将开发一个平台，使瓜氨酸修饰的人类蛋白质的整个库的平行筛选和RA特异性抗体的鉴定。疾病特异性抗体的鉴定将大大促进RA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. PUBLIC HEALTH RELEVANCE: A protein array platform for anti-citrulline antibodies in Rheumatoid Arthritis Project Narrative/Relevance Three million Americans suffer Rheumatoid Arthritis. One characterizing feature is the development of antibodies against citrulline modified self proteins. We will develop a platform that enables the screening of the entire repertoire of citrulline modified human proteins in parallel and the identification of RA specific antibodies. Th identification of disease specific antibodies will significantly accelerate RA research.