The direct enzyme-linked immunosorbent assay (ELISA) is referred to as a solid-phase enzyme immunoassay used to identify and quantify the antibodies present in a sample. In most cases, it has been used to detect the anti-HIV antibodies present in a patient’s serum. In direct ELISA, the antigen is immobilized through charge interactions in the well of a microtiter plate. This is followed by the addition of a non-reacting protein, such as casein, to cover the plastic surfaces of the well. The well is then incubated with an enzyme-labeled antibody that is specific to that antigen. The unbound antibodies are then removed by washing, and a substrate for that enzyme is added. The substrate usually changes color after reacting with the enzyme. Therefore, the higher the primary antibody concentration in the serum, the bigger the color change. A spectrometer is used to quantify the color changes.
Unlike the direct ELISA, the indirect ELISA is a two-step ELISA since a primary and secondary antibody is used. However, in this scenario, the primary antibody is not enzyme-labeled but the secondary antibody. An antigen is immobilized in the well of a microtiter plate, and it is incubated with the primary antibody. After that, the secondary antibody is conjugated with the primary antibody. The substrate reacts with the enzyme bound to the secondary antibody to generate color changes, otherwise known as chromogenic signals. Nevertheless, the incubation of the two antibodies may result in the generation of non-specific signals because of the cross-reaction that the secondary antibody might trigger. In comparison to direct ELISA, indirect ELISA is much faster as it involves relatively more steps, hence is more prone to error.