ELISA (ENZYME-LINKED IMMUNOSORBENT ASSAY) is a widely used biochemical technique for the detection of an antigen in a sample. The sandwich ELISA utilizes two antigen specific antibodies; one capture antibody bound to a solid phase and one enzyme-linked detection antibody. Direct enzyme conjugation of the detection antibody ensures an easy-to-use and sensitive assay with minimal background signal.
In a sequence assay, sample is applied to an antibody-coated microtiter plate well. The capture antibody binds to the antigen in the sample. Unbound compounds are removed by a washing procedure before the enzyme-linked detection antibody is added to the well. The detection antibody binds to a second epitope (immunogenic part) on the antigen. Unbound antibodies are removed by washing before theaddition of a substrate, which is converted by the enzyme to a chromogenic signal. The enzyme reaction is stopped and the result is monitored spectrophotometrically. The more antigen in the sample, the stronger the signal.
The sandwich ELISA may also be constructed as a simultaneous assay where the sample and detection antibody is added in the same step.