B-cell ELISpot

In spite of the first described use of the ELISpot being that of detecting antibody-secreting B cells, there have so far not been any designated ELISpot kits for this application. To accommodate this need for validated reagents and protocols we have recently developed a series of kits for detection of human, monkey and mouse Ig-secreting cells. 

As schematically illustrated below, the B-cell ELISpot may be used both for determining the number of antigen-specific B cells (A and B) as well as the total number of antibody-secreting cells (C). In the antigen-specific setting, the assay can be performed in either of two ways, one where the antigen is immobilized on the ELISpot plate (A) and one where the same antigen is instead used for detection (B). While the first approach has been the one almost exclusively used, we have found that using antigen for detection may have several advantages including better sensitivity and a lesser need for antigen.

ELISA

The cytokine ELISA (Enzyme-Linked Immuno Sorbent Assay) is a specific and highly sensitive method for quantitative measurements of cytokines or other analytes in solutions. 

A specific monoclonal antibody (mAb) able to capture the cytokine of interest is coated on a microtiterplate. A second mAb, used for detection, binds a different epitope on the cytokine. The detection mAb is labeled with biotin, which allows subsequent binding of a Streptavidin-conjugated enzyme. Any unbound reagents are washed away. 

When substrate is added, a color reaction will develop that is proportional to the amount of cytokine bound. The concentration of cytokine is determined by comparison with a standard curve with known concentrations of cytokine. 

The sensitivity of an ELISA depends mainly on the affinity of the antibodies and on the amplification system used. 

The detection limits for cytokine ELISAs are commonly in the lower picogram/ml range.