Theoretical and practical considerations for validating antigen-specific B cell ImmunoSpot assays

Area of Science:

• Immunology
• Assay Development

Background:

• B cell ELISPOT/FluoroSpot (B cell ImmunoSpot) assays are valuable for detecting rare antigen-specific B cells in peripheral blood mononuclear cells (PBMC).
• These assays are increasingly used for immune monitoring in regulated environments.
• Currently, no standardized guidelines exist for qualifying and validating B cell ImmunoSpot assay results.

Purpose of the Study:

• To propose guidelines for the qualification and validation of B cell ImmunoSpot assay results.
• To adapt existing T cell ImmunoSpot testing experience for B cell assays within regulatory requirements.
• To address the unique characteristics of B cell assays in the validation process.

Main Methods:

• Development of a streamlined protocol for formal validation of antigen-specific B cell ImmunoSpot assays.
• Utilizing three experiments and 2.2 × 10^7 PBMC per donor for validation.
• Validation of a multiplexed assay requiring only 1-2 × 10^6 PBMC per sample.

Main Results:

• A streamlined protocol enables formal validation of B cell ImmunoSpot assays.
• A validated multiplexed assay accurately quantifies antigen-specific memory B cells secreting various antibodies (IgM, IgG, IgA, IgE).
• The assay requires a minimal sample volume (1-2 mL of blood).

Conclusions:

• The proposed guidelines facilitate the validation of B cell ImmunoSpot assays for regulated immune monitoring.
• Validated multiplexed B cell ImmunoSpot assays provide high-content data with ease of implementation and scalability.
• These assays offer an economical use of PBMC and broad applicability to diverse antigenic systems.