Systematic Veterinary Virology

INDIRECT ELISA

The steps of the general, "indirect," ELISA for determining serum antibody concentrations are:

• Apply a sample of known antigen of known concentration to a surface, often the well of a microtiter plate . The antigen is fixed to the surface to render it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient. These samples of known antigen concentrations will constitute a standard curve used to calculate antigen concentrations of unknown samples. Note that the antigen itself may be an antibody.
• The plate wells or other surface are then coated with serum samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards. Since antigen immobilization in this step is due to non-specific adsorption, it is important for the total protein concentration to be similar to that of the antigen standards.
• A concentrated solution of non-interacting protein, such as Bovine Serum Albumin (BSA) or casein , is added to all plate wells. This step is known as blocking , because the serum proteins block non-specific adsorption of other proteins to the plate.
• The plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins.
• The plate is washed to remove any unbound detection antibody. After this wash, only the antibody-antigen complexes remain attached to the well.
• Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. These secondary antibodies are conjugated to the substrate-specific enzyme. This step may be skipped if the detection antibody is conjugated to an enzyme.
• Wash the plate, so that excess unbound enzyme-antibody conjugates are removed.
• Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal.
• View/quantify the result using a spectrophotometer, spectrofluorometer, or other optical/electrochemical device.