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

描述（由申请人提供）：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。应用环瓜氨酸肽（CCP）酶联免疫吸附试验检测血清瓜氨酸抗体阳性。尽管抗ccp阳性是一种很好的替代诊断方法，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。最近的研究证明了识别特定抗原的自身抗体在阐明RA病因学中的价值。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，在过去的几十年里，只有少数瓜氨酸化抗原被发现。开发一个平台来识别针对瓜氨酸化蛋白的抗体不仅有助于了解疾病的发病机制，而且有助于提高诊断。传统的蛋白免疫化学方法鉴定瓜氨酸化抗原存在通量低、重复性差、定量不足和分辨率低等缺点。商业蛋白质阵列价格昂贵，缺乏候选抗原的平等代表，并且与翻译后修饰（如瓜氨酸化）不兼容，这需要苛刻的条件。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，并通过覆盖载玻片上的配体作为裸蛋白捕获。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包括在微阵列底物上打印与感兴趣蛋白质对应的全长cdna，然后在检测时原位转录/翻译。覆盖捕获NAPPA将在不受NAPPA打印混合物组分干扰的情况下，在翻译后的阵列上实现蛋白质的及时表达和瓜氨酸化。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/配体的使用将实现阵列上的各种翻译后修饰，包括瓜氨酸化。对多种瓜氨酸化蛋白的血清反应性可以同时评估。我们将建立这个平台来分析

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.