Extending ImmunoSpot^® Assays' Sensitivity for Detecting Rare Antigen-Specific B Cells to One in a Million-And...

Area of Science:

• Immunology
• Cell Biology

Background:

• Memory B cells (Bmem) are long-lived and crucial for rapid recall responses.
• Detecting rare antigen-specific Bmem is challenging but vital for assessing immune history.
• Traditional serum antibody measurements offer limited insights compared to Bmem detection.

Purpose of the Study:

• To establish reliable methods for detecting rare antigen-specific memory B cells (Bmem) in human peripheral blood mononuclear cells (PBMC).
• To investigate the utility of the ImmunoSpot® assay for quantifying Bmem specific to SARS-CoV-2 antigens.
• To provide guidance for routine immune monitoring of Bmem.

Main Methods:

• Utilized the ImmunoSpot® assay to visualize antibody secretion from individual B cells.
• Employed SARS-CoV-2 Spike (S) and Nucleocapsid (NCAP) antigens to identify specific Bmem.
• Tested large numbers of PBMC (2 million) across multiple wells to enhance detection sensitivity for rare cells.

Main Results:

• Achieved 100% diagnostic accuracy for both SARS-CoV-2 S- and NCAP-specific Bmem detection.
• Optimized assay sensitivity by increasing PBMC input and cumulative secretory footprint analysis, particularly for NCAP-specific Bmem.
• Demonstrated the capability to detect antigen-specific Bmem at varying precursor frequencies.

Conclusions:

• Developed and validated protocols for reliable detection of rare antigen-specific Bmem.
• The optimized ImmunoSpot® assay facilitates immune monitoring beyond traditional antibody titers.
• These methods can be adapted for detecting Bmem specific to a wide range of antigens.