The reversed B-cell FluoroSpot assay enables the simultaneous study of multiple antigen specificities in the same well. For example, Hadjilaou et al. (J Immunol, 2015) made use of all four dengue virus serotypes and fluorochrome-labeled serotype-specific mAbs to identify to which serotype(s) antibodies from memory B-cells bind.
Memory B cells often have a broader antigen repertoire than plasma cells and are detectable after antibody levels have dropped below detection level. Thus, conventional ELISA assays may underestimate the humoral response as it excludes the memory B-cell pool.
In the brand-new paper by Luque et al. (J Immunol Methods, 2018), the reversed B-cell FluoroSpot assay was utilized in an organ transplantation setting to enumerate HLA-specific antibody secreting cells. Instead of using fluorescent detection mAbs, the authors successfully used HLA multimers which (i) have a high binding affinity and (ii) are already fluorochrome conjugated at purchase. By using two-color FluoroSpot instead of ELISpot, the authors cut the patient sample required in half.