Enzyme linked immuno-spot; a useful tool in the search for elusive immune markers in common pediatric immunological diseases

Abstract

In order to provide better therapy we strive to increase our knowledge of how the immune system behaves and communicates in common pediatric immunological diseases, such as type 1 diabetes, allergic and celiac diseases. However, when dealing with pediatric diseases, where study subjects are almost exclusively children, blood volumes available for immunological studies are limited and as such must be carefully handled and used to their full extent. Single immune markers can easily be detected by a traditional Enzyme Linked Immunosorbent Assay (ELISA), whereas multiple markers can be detected by a fluorochrome (Luminex) or electrochemiluminescence (MSD) technique. These techniques however are sometimes not sensitive enough to detect low levels of secreted immune markers in limited sample sizes. To detect immune markers at the single-cell level, an Enzyme Linked Immuno-spot (ELISPOT) can be used to pin-point elusive immune markers in common pediatric immunological diseases.

Figure 1

Schematic overview of T-helper subsets. The antigen presenting cell (APC) presents a degraded antigen (Ag) bound to the MHC class II. The complex is recognized by the T-cell receptor (TCR) and delivers a first signal for T-cell activation and a secondary signal by interaction between B7 and CD28. Through stimulation, a naïve T-cell can differentiate into different T-helper (Th) cells. Th1-cells, producing IFN- γ and IL-2, are associated with autoimmunity and intracellular pathogens whereas Th2-cells are associated with allergy and asthma and extracellular parasites and produce IL-4, -5 and -13 upon activation. Inducible antigen-specific sub-populations of CD4⁺ T-regulatory (Treg) cells are IL-10-producing T-regulatory cell type 1 (Tr1) cells and transforming growth factor (TGF)-β-secreting Th3 cells. Th17-cells producing IL-17A, -17F, -21 and IL-22 are capable of inducing inflammation and autoimmunity.

Figure 2

Methodological principal of ELISPOT. The wells of the ELISPOT-plate are coated with a primary antibody and thereafter non-specific binding sites are blocked (A). Cells are added in the presence or absence of specific stimulus. During incubation, cells become activated by the stimulus and start to produce and secrete, e.g., cytokine that binds to the capture antibody (B). Cells are removed and a detection antibody, which may be directly conjugated with enzyme or biotinylated, is added. Finally, a substrate will form a colored spot at the location of the secreting cell (C). By counting the number of spots in stimulated cultures and controls without stimulus the frequency of responding cells is determined.