Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of advanced antigen-specific immunoassays. Traditionally, such immunoassays, especially antigen-specific T-cell analysis by flow cytometry or enzyme-linked immunosorbent spot assays, often rely on the isolation of peripheral blood mononuclear cells. This process is time-consuming, subject to many pre-analytic confounders, and requires large blood volumes. Whole blood-based assays provide a facile alternative with increased pre-analytic robustness and lower blood volume requirements. Furthermore, whole blood-based assays allow for the preservation of inter-cellular interactions that are not captured by assays using isolated cell subsets. Recently, a refined whole blood immunoassay with dual anti-CD28 and anti-CD49d co-stimulation for comprehensive analysis of both antigen-specific T-cell functions and complex intercellular interactions in response to various fungal and viral antigens has been proposed. This protocol provides guidance for the preparation of stimulation tubes, blood stimulation, and downstream sample processing for flow cytometry, cytokine secretion assays, and transcriptional analyses. This includes a validated and functionally equivalent, previously unpublished, low-volume protocol (250 µL) to make flow cytometric and cytokine-based T-cell monitoring more accessible for studies in pediatric patients or preclinical studies in small animals (e.g., mice). Altogether, these protocols provide a versatile toolbox for complex antigen-specific immune analysis in both clinical and translational research settings.