Why Elispot?

Elispot Sensitivity Figure 1  

Figure 1: Unique sensitivity of the ELISPOT assay for detecting low-frequency cytokine producing cells. The specified number of IFN-ץ-producing T cells was mixed into one million antigen presenting cells (APC). The number of IFN-ץ-producing cells was measured by ELISPOT (upper panel) and intracytoplasmic staining (middle panel); results of ELISA measurements are shown in the lower panel.

 

The advantages of cytokine ELISPOT assays are:

  • Accurate ex vivo frequency measurements down to the one-in-one-million cell range. This resolution is orders of magnitude higher than that reached with intracellular cytokine or tetramer staining or by ELISA measurements (see Figure 1). Such added sensitivity is critical, since antigen-specific T cells typically occur in low frequencies in vivo.

  • High-throughput T cell analysis becomes feasible. A trained reference laboratory team can test hundreds of samples for reactivity to dozens of antigens.

  • Fewer cells are required compared to other cellular assays. For example, 45 ml of blood is sufficient for testing reactivity to 600 different antigens/peptides.

  • Determinants targeted by CD4 or CD8 cells can be defined. Because few cells are needed and the assay can be performed in high-throughput mode, ELISPOT assays are ideal for screening peptide libraries for determinant mapping.

  • The functional avidity of antigen specific T cells can be established. This is reflected by the 50% maximally stimulating peptide dose.

  • The actual secretory process of pharmacologically untreated cells can be studied. If decreased net production is seen, the distinction can be made as to whether fewer cells produce or there is reduced per cell productivity.

  • Lymphocytes survive the testing in ELISPOT assays. This permits post-assay propagation for further analysis, cloning, or cryopreservation.

  • Cryopreserved human lymphocytes can be tested without loss of function. Pre- and post-treatment samples can be tested side by side and results can be reproduced.