ELISAs are 96 well plate-based assays allowing detection of a target in a range of sample types including plasma, tissue homogenate and cell lysate. We use this technique to validate protein expression in transfected cells, or to compare levels of protein expression in treated or untreated tissue, providing additional information alongside radioligand binding assays.
There are two main techniques, sandwich and indirect:
In a sandwich ELISA, two antibodies specific to two separate epitopes on the target protein (matched-antibody pairs) are utilized; plates are pre-coated with one of these antibodies (capture antibody) to immobilize the target. 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.
For an indirect ELISA, the target is immobilized directly to the plate, then bound by a primary antibody specific for the target. This is followed by binding of a secondary antibody coupled to a reporter enzyme. Addition of a substrate results in a colorimetric read-out. The concentration of target protein can be determined by comparing the absorbance to a standard curve with known concentrations of control protein carried out on the same assay plate.
Example dilution series generated for A-beta fibrils using an indirect ELISA. Limit of detection (LOD) = 0.53 nM. Limit of quantitation (LOQ) = 1.78 nM. LOD = 3σ/s and LOQ = 10σ/s, where σ is the standard deviation of the blanks and s is the slope.
We can use ELISAs to confirm whether a target protein is present compared to a blank or control antigen (qualitative ELISA). We can use ELISAs to obtain semi-quantitative data as an indication of relative levels of antigen in test samples, where the absorbance signal is dependent upon the level of target in the sample. We are also able to run quantitative ELISAs, where precise concentrations of antigen can be determined by comparing absorbance signals to that of a standard curve run with protein of a known concentration.