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. |
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. |
Fig. 3. Non-specific antibodies that do not bind to the antigen are washed away. |
Fig. 4. An enzyme labeled (horseradish peroxidase) secondary antibody is incubated with the sample and binds tightly to the sample antibody. |
Fig. 5. Unbound labeled antibody is washed away, and a colorimetric substrate (o-phenylenediamine dihydrochloride) is added. |
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. |