Antibody Microarray Detection ofEscherichiacoliO157:H7:  Quantification, Assay Limitations, and Capture Efficiency

Abstract

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.

Antibody immobilization using pneumatic spray: Comparison with the avidin–biotin bridge immobilization method

“…This suggests that the sprayed films have a higher antibody density than the avidin-biotin bridge attached films. The corresponding mass of the deposited antibody was calculated to be around 31.69 ng/mm 2 for the pneumatic spray films, while the deposited mass for the avidin-biotin bridge has been reported to be between 2.2 ng/mm 2 (Moulin et al, 1999;Batalla et al, 2008) and 4.74 ng/mm 2 (Anderson et al, 1997;Gehring et al 2006). This is not surprising since the spray process is a non-equilibrium technique, whereas the covalent attachment in solution is equilibrium controlled.…”

“…The sprayed prepared slides have a higher antibody density with randomly immobilized antibody on the surfaces whereas the avidin-biotin bridge prepared slides have less antibody but clearly have a higher degree of orientation (Gehring et al, 2006). Studies suggest that random orientation reduces the ability of the antibody to react with the antigen due to the impact of steric hindrance generated by the arbitrary alignment of the antibodies (Lu et al, 1996;Xu and Zhao et al, 2006b).…”

“…Optimization studies to the capture surface of various biosensors have been performed with the goal of improving sensitivity and reproducibility of the antibody capture surface but not all have been successful (Anderson et al, 1997;Plowman et al, 1999;Sapsford et al, 2001;Vijayendran and Leckband., 2001;Vermette et al, 2003;Cui et al, 2003;Gehring et al, 2006;Ngundi and Anderson, 2007;Batalla et al, 2008;Ikeda et al, 2009;Feng et al, 2011). In addition, requirement of trained personnel, extra protocol steps and reagents increase preparation time and material usage.…”

Antibody-based biosensors for environmental monitoring

“…Gehring and coworkers used a sandwich fluorescent immunoassay in a microarray format using a biotinylated antibody for E. coli O157 : H7 on a streptavidincoated microarray slide 27 . They monitored linear fluorescence response from 3.0 10 6 to 9.0 10 7 cells/ mL.…”

Heightened sense for sensing: recent advances in pathogen immunoassay sensing platforms

“…Gehring and co-workers have recently optimized the fabrication of an antibody microarray for the detection of O157:H7 E. coli pathogenic bacteria. 28 For example, it was observed that direct contact printing of biotinylated anti-O157:H7 E. coli antibody on streptavidin coated microarray slides, as opposed to an indirect attachment via biotinylated protein G-bound captured antibodies, produced a readout signal which is ~5-times higher.…”