期刊
FRONTIERS IN IMMUNOLOGY
卷 13, 期 -, 页码 -出版社
FRONTIERS MEDIA SA
DOI: 10.3389/fimmu.2022.962220
关键词
HTS; PBMC; RT-qPCR; cost-effective; IFN-gamma
类别
This study developed a high-throughput screening assay using reverse transcription-quantitative PCR (RT-qPCR) to quantify surrogate markers of immunity. The assay has low cell consumption and reagent cost, making it suitable for measuring immune effector function with limited PBMCs. It also has single-cell analytical sensitivity.
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/10(6) 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.
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