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

Author:

Lehmann Paul V.1,Karulin Alexey Y.1,Becza Noémi1,Yao Lingling1,Liu Zhigang1,Chepke Jack1,Maul-Pavicic Andrea1,Wolf Carla1,Köppert Sebastian1,Valente Alexis V.1,Gorbachev Anton V.1,Tary-Lehmann Magdalena1,Kirchenbaum Greg A.1ORCID

Affiliation:

1. Cellular Technology Limited

Abstract

Abstract Owing to their ability to reliably detect even very rare antigen-specific B cells in cellular isolates such as peripheral blood mononuclear cells (PBMC), and doing so robustly in a high throughput-compatible manner, B cell ELISPOT/FluoroSpot (collectively “B cell ImmunoSpot”) tests have become increasingly attractive for immune monitoring in regulated settings. Presently, there are no guidelines for the qualification and validation of B cell ImmunoSpot assay results. Here, we propose such guidelines, building on the experience acquired from T cell ImmunoSpot testing in an environment adhering to the requirements of regulatory bodies yet taking the unique features of B cell assays into account. A streamlined protocol is proposed that permits the performance of all tests needed for the formal validation of an antigen-specific B cell ImmunoSpot assay in only three experiments, utilizing 2.2 x 107 PBMC per donor. Subsequently, utilizing only 1–2 x 106 PBMC per sample (obtainable from 1–2 mL of blood), a validated multiplexed assay enables accurate quantification of the frequency of antigen-specific memory B cell-derived blasts secreting IgM, IgG, IgA or IgE antibodies. Collectively, such multiplexed B cell ImmunoSpot assays offer immense value for B cell immune monitoring programs due to their ease of implementation, scalability, applicability to essentially any antigenic system, economy of PBMC utilization, and last but not least, the high content information gained.

Publisher

Research Square Platform LLC

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