ELISAs

We use this technique to:

• confirm the expression of the target protein in transfected or stable cell lines;
• screen a range of samples for the presence of the target of interest;
• compare levels of target antigen expression in treated or untreated tissue;
• characterize the effects of an inhibitor (competitive ELISAs).

In a direct ELISA, we immobilize the target antigen directly to the assay plate, then bind an antibody specific for the target. This antibody is coupled to a reporter enzyme. Substrate is added and a coloured reaction product generated. The absorbance of the wells are read after terminating the reaction.

In an indirect ELISA, we immobilize the target antigen directly to the assay plate, then bind a primary antibody specific for the target antigen. This is followed by binding of a secondary antibody coupled to a reporter enzyme. Substrate is then added resulting in a colorimetric readout.

In a sandwich ELISA, we use matched-antibody pairs (two antibodies that bind to two different epitopes on the target protein). The first antibody (capture antibody) is precoated onto the ELISA plate and immobilises the target antigen present in the sample. Any unbound antigen is then washed off, and the second matched pair antibody is incubated in the wells. This antibody is either conjugated to a reporter enzyme, or after an additional wash to remove unbound antibody, bound by a third antibody conjugated to a reporter enzyme. After a final wash step to remove unbound antibody, the wells are incubated with a substrate. The reaction is terminated, and the absorbance of the wells read.