In a plate trapped antigen ELISA the sample of interest (A) is
coated on to the microtitre plate. After a period of incubation the
unbound sample is removed by washing, any free sites on the plate
are blocked by the addition of a blocking buffer containing a high
proportion of protein. The blocking buffer is removed by washing
and an antibody (IgG) which has been raised against the antigen is
added to the plate. After washing, a second antibody which is
conjugated to enzyme (E) - in our case alkaline phosphatase - is
added to the plate and incubated. The secondary antibody is
specific for the species that the primary antibody is raised in. The
unbound antibody-enzyme conjugate is washed from the plate and
the substrate (S) for the enzyme is added. If there is any bound
antibody-enzyme conjugate the substrate will change colour.