Multiplexing T- and B-Cell FLUOROSPOT Assays: Experimental Validation of the Multi-Color ImmunoSpot Software Based on Center of Mass Distance Algorithm.

Over the past decade, ELISPOT has become a highly implemented mainstream assay in immunological research, immune monitoring, and vaccine development. Unique single cell resolution along with high throughput potential sets ELISPOT apart from flow cytometry, ELISA, microarray- and bead-based multiplex assays. The necessity to unambiguously identify individual T and B cells that do, or do not co-express certain analytes, including polyfunctional cytokine producing T cells has stimulated the development of multi-color ELISPOT assays.

Detecting all Immunoglobulin Classes and Subclasses in a Multiplex 7 Color ImmunoSpot Assay.

Antibody molecules in peripheral blood have a relatively short half-life of roughly 20 days, and therefore their persistence in the serum depends on continuous replenishment by plasma cells. Serum antibody titers are thus indirect and unreliable indicators of immunological memory. In contrast, memory B cells persist in peripheral blood for decades, and enumerating these cells provides direct evidence of having developed an immune response to a given antigen.

B Cells and B Cell Blasts Withstand Cryopreservation While Retaining Their Functionality for Producing Antibody.

In individuals who have once developed humoral immunity to an infectious/foreign antigen, the antibodies present in their body can mediate instant protection when the antigen re-enters. Such antigen-specific antibodies can be readily detected in the serum. Long term humoral immunity is, however, also critically dependent on the ability of memory B cells to engage in a secondary antibody response upon re-exposure to the antigen.

Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify.

It is essential to identify donors who have not been infected with human cytomegalovirus (HCMV) in order to avoid transmission of HCMV to recipients of blood transfusions or organ transplants. In the present study, we tested the reliability of seronegativity as an indicator for the lack of HCMV exposure in healthy human blood donors. Eighty-two HCMV seronegative individuals were identified, and their peripheral blood mononuclear cells (PBMC) were tested in ImmunoSpot® assays for the presence of HCMV-specific T- and B-memory lymphocytes.

Characterization of the HCMV-Specific CD4 T Cell Responses that Are Associated with Protective Immunity.

Most humans become infected with human cytomegalovirus (HCMV). Typically, the immune system controls the infection, but the virus persists and can reactivate in states of immunodeficiency. While substantial information is available on the contribution of CD8 T cells and antibodies to anti-HCMV immunity, studies of the TH1, TH2, and TH17 subsets have been limited by the low frequency of HCMV-specific CD4 T cells in peripheral blood mononuclear cell (PBMC).