A high-throughput screening RT-qPCR assay for quantifying surrogate markers of immunity from PBMCs

Browne, Daniel J., Kelly, Ashton M., Brady, Jamie L., and Doolan, Denise L. (2022) A high-throughput screening RT-qPCR assay for quantifying surrogate markers of immunity from PBMCs. Frontiers in Immunology, 13. 962220.

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Abstract

Immunoassays that quantitate cytokines and other surrogate markers of immunity from peripheral blood mononuclear cells (PBMCs), such as flow cytometry or Enzyme-Linked Immunosorbent Spot (ELIspot), allow highly sensitive measurements of immune effector function. However, those assays consume relatively high numbers of cells and expensive reagents, precluding comprehensive analyses and high-throughput screening (HTS). To address this issue, we developed a sensitive and specific reverse transcription-quantitative PCR (RT-qPCR)-based HTS assay, specifically designed to quantify surrogate markers of immunity from very low numbers of PBMCs. We systematically evaluated the volumes and concentrations of critical reagents within the RT-qPCR protocol, miniaturizing the assay and ultimately reducing the cost by almost 90% compared to current standard practice. We assessed the suitability of this cost-optimized RT-qPCR protocol as an HTS tool and determined the assay exceeds HTS uniformity and signal variance testing standards. Furthermore, we demonstrate this technique can effectively delineate a hierarchy of responses from as little as 50,000 PBMCs stimulated with CD4+ or CD8+ T cell peptide epitopes. Finally, we establish that this HTS-optimized protocol has single-cell analytical sensitivity and a diagnostic sensitivity equivalent to detecting 1:10,000 responding cells (i.e., 100 Spot Forming Cells/106 PBMCs by ELIspot) with over 90% accuracy. We anticipate this assay will have widespread applicability in preclinical and clinical studies, especially when samples are limited, and cost is an important consideration.

Item ID: 76273
Item Type: Article (Research - C1)
ISSN: 1664-3224
Keywords: HTS, PBMC, RT-qPCR, cost-effective, IFN-gamma
Copyright Information: © 2022 Browne, Kelly, Brady and Doolan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Date Deposited: 12 Oct 2022 08:19
FoR Codes: 32 BIOMEDICAL AND CLINICAL SCIENCES > 3204 Immunology > 320404 Cellular immunology @ 100%
SEO Codes: 20 HEALTH > 2001 Clinical health > 200105 Treatment of human diseases and conditions @ 100%
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