A sandwich fluorescent immunoassay in a microarray format was used to capture and detect E. coli O157:H7. Here, we explored quantitative aspects, limitations, and capture efficiency of the assay. When biotinylated capture antibodies were used, the signal generated was higher (over 5-fold higher with some cell concentrations) compared to biotinylated protein G-bound capture antibodies. By adjusting the concentration of reporter antibody, a linear fluorescent response was observed from approximately 3.0 x 10(6) to approximately 9.0 x 10(7) cells/mL, and this was in agreement with the number of captured bacteria as determined by fluorescence microscopy. Capture efficiency calculations revealed that, as the number of bacteria presented for capture decreased, capture efficiency increased to near 35%. Optimization experiments, with several combinations of capture and reporter antibodies, demonstrated that the amount of bacteria available for capture (10(6) versus 10(8) cells/mL) affected the optimal combination. The findings presented here indicate that antibody microarrays, when used in sandwich assay format, may be effectively used to capture and detect E. coli O157:H7.