Tu214 - Optimizing PBMC Utilization and Cryopreservation for B Cell ImmunoSpot® Analysis

Abstract Text: Measuring frequencies of antigen-specific memory B cells (Bmem), their immunoglobulin (Ig) class and subclass usage, cross-reactivity and affinity can provide insights into the efficacy of future antibody responses in case of antigen re-encounter. B cell ImmunoSpot® assays can provide such information; however, like most cell-based tests, they too require considerable amounts of blood to be drawn from the test subject and this has hindered their inclusion into clinical trials, and into routine immune diagnostics. Here, we introduce strategies for reducing the cell numbers required to less than 2 million peripheral blood mononuclear cells (PBMC) per antigen, obtainable from 2-3mL of blood. We establish that testing PBMC in singlet wells, but in serial dilution, enables more reliable and accurate Bmem frequency assessments than testing replicate wells at a single fixed cell number; except when Bmem frequencies are very low. We further show that these assays can be multiplexed for measuring four Ig classes, or IgG subclasses, without any appreciable loss in sensitivity. Lastly, we establish that PBMC can be cryopreserved in smaller numbers than the standard 10 million PBMC per vial and retain indistinguishable functionality. Collectively, the predictable need for and recovery of PBMC facilitates optimal cell utilization in downstream B cell ImmunoSpot® assays.