Food Biochemistry and Food Processing

Figure 31.5. A.A schematic of direct ELISA. The antigen is used to coat a well of a microtiter plate. Excess antigen
is washed away and the wells blocked to prevent nonspecific binding by other proteins and antibodies. Primary anti-
body is added to bind the antigen in the well. Excess unbound antibody is washed away, and the secondary anti-
body, conjugated to a colorimetric enzyme is added. After a final wash, the enzyme substrate is added, and the inten-
sity of color produced is proportional to the quantity of antigen present. B.A schematic of competitive ELISA. This
format is similar to the direct ELISA except that the well is coated with a known amount of standard antigen and the
primary antibody is preincubated with the sample. The more antigen is present in the sample, the less primary anti-
body will be available to bind to the standard antigen. After adding the secondary antibody and substrate, the final
absorbance can be compared to a standard curve to determine the amount of antigen present in the sample.