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.

Project Narrative/Relevance Measuring the strength of patient antibody responses in clinical samples is important in cancer biology and diagnostics. We propose to develop an innovative high-throughput platform, in which we will marry the powerful DNA sequencing technology and immunoassay to assess antibody response to many hundreds of cancer related pathogens in many thousands of individuals simultaneously. This will create a fast, sensitive, low cost, robust research tool for early screening of pathogen induced cancers.