A High-Throughput Screening (HTS) Assay for Quantifying Surrogate Markers of Immunity from PBMCs

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, these assays consume relatively high numbers of cells and expensive reagents, precluding comprehensive analyses and high-throughput screening (HTS). Herein, we report a sensitive and specific reverse transcription quantitative PCR (RTqPCR)-based HTS assay to quantify surrogate markers of immunity from low numbers of PBMCs without the need for in vitro expansion. We establish that this assay is amendable to miniaturization and automation, exceeds HTS uniformity and signal variance testing standards, and represents a cost saving of almost 90% on standard-practice assays. We demonstrate 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 95% confidence. We anticipate this assay will have widespread applicability in preclinical and clinical studies, especially when samples are limited, and cost is an important consideration. Funding Statement: Work was supported by the National Health and Medical Research Council of Australia (grant #1069466 and 1132975). DLD is supported by an NHMRC Principal Research Fellowship (#1137285). DJB is supported by James Cook University Prestige Research Training Program Stipend (RTPS). Declaration of Interests: The authors declare no competing interest. Ethics Approval Statement: Blood was collected under informed consent from healthy donors provided by the Australian Red Cross Lifeblood, under a protocol approved by the James Cook University Human Research Ethics Committee (#H7886).