FIG. 1.

Diagram of the assay. A test sample is preincubated with a biotinylated detection antibody for 10 min, resulting in formation of an antibody-antigen complex. The antibody-coated filter captures the antigen-antibody complex from solution, while unbound antibody flows through. Then a second solution, which contains HRP-labeled streptavidin that binds to the biotin, is added. Finally, an insoluble substrate for the HRP enzyme is added, which produces a visible band following precipitation of TMB where the HRP, and thus the antigen, is present. For the E. coli O157 assay, unbound cells were run through the column to bind to the capture antibody before addition of the detection antibody.