Fig. 1. Solid phase membranes (e.g., a 96 well plate) are coated with antigen using a coating buffer that enhances binding. Sites unoccupied by the antigen are blocked with a blocking buffer to prevent non-specific binding of protein in the sample.

Positive sample       Negative sample

Fig. 2. Serum samples are added and incubated to allow specific antibodies to bind to specific antigen. Antibodies that do not recognize and bind specifically to the antigen remain unbound.

Positive sample       Negative sample

Fig. 3. Non-specific antibodies that do not bind to the antigen are washed away.

Positive sample       Negative sample

Fig. 4. An enzyme labeled (horseradish peroxidase) secondary antibody is incubated with the sample and binds tightly to the sample antibody.

Positive sample        Negative sample

Fig. 5. Unbound labeled antibody is washed away, and a colorimetric substrate (o-phenylenediamine dihydrochloride) is added.

Positive sample         Negative sample

Fig. 6. The enzyme cleaves the substrate causing a color change of the substrate solution. The intensity of the color is quantitated using a spectrophotometer and is proportional to the amount of antibody present in the sample.