Multiplex In-Solution Protein Array (MISPA) for high throughput, quantitative, early profiling of pathogen-induced head and neck

摘要 分析抗体对疾病相关抗原的反应对癌症研究很重要。相对于 测试对单个蛋白质的反应、筛选和诊断的历史方法越来越依赖于 多重测定以阐明疾病和患者异质性。蛋白质微阵列允许蛋白质组规模 以低样品消耗进行筛选但受到表面结合蛋白质的结合动力学的限制， 非特异性结合、荧光检测的有限动态范围以及在临床上不易获得。 基于肽的方法将测定限制于线性表位。在IMAT R21的支持下， 开发了下一代液相蛋白质微阵列平台“Multiplex In Solution Protein Array” （MISPA），它利用了下一代测序（NGS）的非凡动态范围， 适用于研究和临床实验室。我们定量分析了 使用“条形码化的”人乳头瘤病毒（HPV）检测口咽（OPSCC）患者和对照样品 12种HPV亚型NGS的抗原库。该测定法成功地检测到了 OPSCC样品和表现出更大的信号背景比，再现性和动态范围。 随后，我们将MISPA用于检测针对SARS-CoV-2、季节性 冠状病毒和其他呼吸道病原体在1000多个样本同时作为一部分， NCI SeroNet与临床COVID-19诊断和商业诊断的总体一致率超过90% EUA血清学测定。在R33阶段，我们建议进一步开发MISPA平台，使其完全 自动化研究平台，是定量的，强大的，高度可重复的，高通量的， 用于早期癌症筛查。我们将建立稳健的蛋白质生产、稳定的蛋白质 库存储和最小的试剂批间变化。我们将通过以下方式展示MISPA的多功能性： 通过包括来自HPV的不同亚型的抗原，将条形码化的蛋白质文库大小增加到192， 病毒、细菌、真菌和肿瘤抗原。我们将通过端到端技术提高再现性和吞吐量， MISPA平台的自动化，以支持需要分析数万个样品的大型项目 样品我们将确定空白限、检测限、线性动态范围、精密度等 定量分析的性能测量。我们将分析192种癌症相关抗体， OPSCC和宫颈癌患者以及1，000多名无癌对照， 与目前的金标准ELISA平台性能。我们在开发创新型高... 使用实验室自动化和我们的初步数据质量的通量免疫蛋白质组学平台 说明我们有能力实施我们提出的发展计划。一种定量可重复的测定方法， 在数千个个体中测量数百种针对全长正确折叠蛋白的抗体 同时将大大有利于癌症血清流行病学、风险评估和筛查。

Abstract Profiling antibody response to disease-associated antigens is important to cancer research. In contrast to the historical approach of testing responses to individual proteins, screening and diagnosis increasingly rely on multiplexed assays to elucidate disease and patient heterogeneity. Protein microarrays allow proteome-scale screening with low sample consumption but are constrained by binding kinetics of surface-bound proteins, non-specific binding, limited dynamic range of fluorescence detection and not readily available in clinics. Peptide-based approaches limit the assay to linear epitopes. With support from IMAT R21, we have developed a next-generation, liquid-phase protein microarray platform, “Multiplex In Solution Protein Array” (MISPA), which exploits the extraordinary dynamic range of next generation sequencing (NGS) with wide applicability in both research and clinical labs. We quantitatively profiled the immune responses of oropharyngeal (OPSCC) patient and control samples using a “barcoded” human papillomavirus (HPV) antigen library for 12 HPV subtypes NGS. The assay successfully detected the positive responses in the OPSCC samples and demonstrated greater signal-to-background ratio, reproducibility, and dynamic range. Subsequently, we have advanced MISPA to assay antibody response against SARS-CoV-2, seasonal coronaviruses, and other respiratory pathogens in more than 1000 samples simultaneously as part of the NCI SeroNet with over 90% overall percent agreement with a clinical COVID-19 diagnosis and commercial EUA serological assays. In the R33 phase, we propose to further develop the MISPA platform to a fully automated research platform that is quantitative, robust, highly reproducible, high-throughput, and inexpensive for early cancer screening. We will establish SOPs for robust protein production, stable protein library storage, and minimal reagent lot-to-lot variations. We will demonstrate the versality of MISPA by increasing the barcoded protein library size to 192 by including antigens from different subtype of HPV, other viruses, bacteria, fungi and tumor antigens. We will improve reproducibility and throughput with end-to end automation for the MISPA platform to support large-scale projects requiring assaying tens of thousands samples. We will determine the limit of blank, limit of detection, linear dynamic range, precision, and other performance measures for quantitative assays. We will profile the 192 cancer related antibodies in hundreds of patients with OPSCC and cervical cancer and more than 1,000 cancer free controls and benchmark the performance with the current gold standard ELISA platform. Our experience with developing innovative high- throughput immunoproteomics platforms using laboratory automation and the quality of our preliminary data speak for our competency in implementing our proposed development. A quantitatively reproducible assay to measure hundreds of antibodies against full length properly folded proteins in thousands of individuals simultaneously will greatly benefit cancer sero-epidemiology, risk assessment and screening.