ABSTRACT
Enzyme-linked immunosorbent assay (ELISA) is based on the specific reaction between an antibody and an antigen. One of the reagents in the reaction is labeled with an enzyme that generates a colorimetric product that can be measured with a spectrophotometric device. The color intensity correlates with the concentration of specific antibody and the respective antigen. The reaction can be formatted in various ways in a multiwell plate (microtiter plate) with the common formats being the sandwich assay, the competitive assay, and the direct assay. (See Figure 11.1.)
The sandwich assay is the format used most often to quantitate a target antigen or analyte. In the sandwich assay, two antibodies are used that bind to different parts of the antigen. One of the antibodies is bound to, or coated on, the solid surface (mictotiter plate wells), whereas the other has a label attached to it (Figure 11.1a). Alternatively, a secondary conjugated antibody can be used to detect the bound primary antibody (Figure 11.1b). If the antigen is present in the sample solution, it links the two antibodies. Therefore, the label is retained on the plate where it can be detected by use of a colorimetric substrate.
