Enzyme-linked immunosorbent assay (ELISA) is a very common biochemistry assay. It is often used as a diagnostic method or quality control.
In the main technique of this family, an antigen is coated or bound on a surface. The antibodies that target the antigen are applied and eventually bind to it. After washing, the antibody is revealed via a signal emitted thanks to an enzyme such as Horse Radish Peroxydase (HRP). Colorful dyes are often used as revelators and the signal is measured with spectrophotometer. This immunoassay, aiming at detecting specific antibodies, might be qualitative or quantitative given the intensity of the signal.
ELISA assays types
Many types of ELISA assays can be set up depending on the samples and target studied. Assays using no antibody are sometimes also named ELISA-like as they aim at assessing the binding of two molecules with the same principle. Four main types of ELISA have been identified.
In Direct ELISA the antigen that is studied is coated on the plate. The primary antibody that targets this antigen, conjugated to an enzyme is then added. The substrate of the enzyme is subsequently added to the reagents for the enzyme to reveal the presence of the antigen.
In Sandwich ELISA, a primary antibody, called capture antibody, is coated in the wells of the plate. The sample containing the antigen is then applied and washed. The antigen is now captured on the plate. A detection antibody, also specific to the antigen is applied on it to "form a sandwich". A secondary antibody conjugated to the detection enzyme is added to bind to the constant Fc region of the detection antibody. Subsequently, the enzyme is activated with its substrate.
In Competitive ELISA, antigens and antibodies are first incubated together. They are then added to the antigen coated plate and washed. The remaining antibodies from the incubated sample can bind to the plate. Their quantity is then assessed with a secondary antibody linked to an enzyme. The competition therefore takes place between the incubation media and the coated product.
In Reverse ELISA the antigen that is measured and its unlabeled antibody are incubated together. They are then passed through a flow through channel that contains scavenger antigens. They will attach the unbound antibodies. The sample containing the first antigen-antibodies complexes can be further washed away and analyzed to measure the antibodies and thus complexes quantity.