Antibody Microarray Detection of Escherichia coli O157:H7

Chem. , 2006, 78 (18), pp 6601–6607 ... with some cell concentrations) compared to biotinylated protein G-bound capture ... Analytical Chemistry 0 (...
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Anal. Chem. 2006, 78, 6601-6607

Antibody Microarray Detection of Escherichia coli O157:H7: Quantification, Assay Limitations, and Capture Efficiency Andrew G. Gehring,† David M. Albin,*,† Arun K. Bhunia,‡ Sue A. Reed,† Shu-I Tu,† and Joseph Uknalis†

Agricultural Research Service, United States Department of Agriculture, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038, and Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University, 745 Agriculture Mall Drive, West Lafayette, Indiana 47907

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 ∼3.0 × 106 to ∼9.0 × 107 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 (106 versus 108 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. Pathogenic bacteria may be naturally present in foods and are responsible for millions of illnesses and thousands of deaths annually in the United States.1 Recently, the perceived threat of intentional food contamination with multiple combinations of pathogens has enhanced interest in food safety and security. Therefore, efforts to develop biosensors for multiplexed screening and detection of pathogens have been undertaken. The biosensors need to be efficient, rapid, specific (to only detect the targeted organisms, while excluding others), sensitive, and preferably, quantitative. In addition, they must be capable of quick adaptations to allow for testing of new analytes, and they need to allow for multianalyte assays (i.e., the ability to perform numerous assays with a single sample). Especially for the latter purpose, microarrays, which contain hundreds or thousands of micrometerdiameter spots, pose enormous potential. In the case of biosensor * To whom correspondence should be addressed. E-mail: dalbin@ errc.ars.usda.gov. † United States Department of Agriculture. ‡ Purdue University. (1) Mead, P. S.; Slutsker, L.; Dietz, V.; McCaig, L. F.; Bresee, J. S., Shapiro, C.; Griffin, P. M., Tauxe, R. V. Emerg. Infect. Dis. 1999, 5, 607-625. 10.1021/ac0608467 CCC: $33.50 Published on Web 08/12/2006

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microarrays, each spot contains biorecognition molecules that capture the analyte of interest. Typically, for detection, a second, labeled biorecognition molecule is then used. Such assays, called “sandwich assays”, have been used extensively in the past, with antibodies serving as the biorecognition molecules.2-4 Current efforts to combine sandwich assays with the microarray format are being explored.5 Analytical methods that have been applied to the detection of bacteria include plate culture, ELISA, and PCR, among others.6-7 However, none of the reported methods have multiplex capabilities that approach microarrays, which allow for screening of samples for thousands of bacterial analytes in a single pass, combined with high-throughput analysis. We developed a microarray sandwich immunoassay biosensor for detection of Escherichia coli O157: H7. The procedure involved contact printing of biotinylated capture antibodies onto streptavidin-coated microarray slides, which were used to capture E. coli O157:H7 cells. Fluoresceinlabeled reporter antibodies were then used to detect the presence of captured bacteria. We examined how antibody microarrays may be used as biosensors and how well they meet some of the criteria listed above. The findings reported here examine the ranges in which microarray biosensors can be used for detection of E. coli O157:H7 and explore capture efficiency of the antibody-mediated binding of bacteria to the microarray substrate. EXPERIMENTAL SECTION Materials. Materials used in this research included the following (when listed, biological reagent concentrations were for initial working solutions that were used full strength unless reported otherwise): unlabeled goat anti-E. coli O157:H7 antibody (used as capture antibody; initial working solution concentration of 2 µg/µL), biotinylated goat anti-E. coli O157:H7 antibody (used (2) Gehring, A. G.; Albin; D. M., Irwin, P. L.; Reed, S. A.; and Tu, S. J. Microbiol. Methods. In press. (3) Saviranta, P.; Okon, R.; Brinker, A.; Warashina, M.; Eppinger, J.; Geierstanger, B. H. Clin. Chem. 2004, 50, 1907-1920. (4) Tu, S.; Uknalis, J.; Yamashoji, S.; Gehring, A.; Irwin, P. J. Rapid Methods Autom. Microbiol. 2005, 13, 57-70. (5) Nielsen, U. B.; Geierstanger, B. H. J. Immunol. Methods 2004, 290, 107120. (6) Ivnitski, D.; Abdel-Hamid, I.; Atanasov, P.; Wilkins, E. Biosens. Bioelectron. 1999, 14, 599-624. (7) Fratamico, P. M.; Gehring, A. G.; Karns, J.; van Kessel, J. In Improving the Safety of Fresh Meat; Sofos, J. N., Ed.; Woodhead Publishing: Ltd.: Cambridge, 2005; pp 24-55.

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Figure 1. Antibody microarray detection of E. coli O157:H7 (five 135-µm-diameter spots each of biotinylated and biotinylated protein G-bound capture antibody, each on 2 slides, at each data point) at different concentrations (3.8 × 104-3.8 × 109 cells/mL) using sandwich assay format. In (A), the capture antibodies exhibited markedly different detection of bacteria (nearly 5-fold greater with biotinylated capture antibody; data shown in AFU, background corrected). Representative spots, from the array scanner, for each biotinylated antibody data point are shown above. In (B), bacterial capture at 3.8 × 104 cells/mL, showing that biotinylated capture antibody spots produced a mean AFU signal greater than background (determined with samples that did not contain bacteria), and a SNR, approaching 10. Biotinylated protein G spots were similar to the background signal (when considering the error bars).

as capture antibody; 1 µg/µL), and fluorescein-labeled goat antiE. coli O157:H7 antibody (used as reporter antibody; 0.5 µg/µL) (note: the first 2 antibodies mentioned were from the same batch), from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD); biotinylated protein G (protein G; 0.1 µg/µL) from CalbiochemNovabiochem Corp. (San Diego, CA); biotin-labeled bovine albumin (1 µg/µL), streptavidin (0.2 µg/µL), phosphate-buffered saline (PBS) tablets, Trizma base, and bovine serum albumin (BSA; fraction V) from Sigma-Aldrich (St. Louis, MO); Superfrost Gold electrostatically coated microscope slides and LifterSlip m series microarray coverslips (22 mm × 50 mm) from Erie Scientific Co. (Portsmouth, NH); PAP hydrophobic barrier pens from BioGenex Laboratories (San Ramon, CA); E. coli O157:H7 B1409 from Centers for Disease Control (Atlanta, GA); and EC medium from Difco Laboratories (Detroit, MI). Other chemicals used were of reagent grade. Apparatus. Solutions were printed onto microarray slides using a SpotBot Personal Microarray Robot (protein version; TeleChem International, Inc., Sunnyvale, CA). Fluorescent images of the microarray slides were produced with a Tecan LS400 slide scanner (Research Triangle Park, NC). Incubations with shaking were conducted with an innOva 4000 from New Brunswick Scientific (Edison, NJ). A Petroff-Hausser counting chamber from 6602 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006

Thomas Scientific (Swedesboro, NJ) was used to enumerate bacterial cells. Washing of microarray slides was conducted with a Stain Train System (slide holders and washing jars) from MarketLab (Kentwood, MI). Microarray slides were coated with streptavidin by submersion in solutions contained in polypropylene Coplin jars and polystyrene slide washing jars from VWR (West Chester, PA). Streptavidin-Coating Microarray Slides. Streptavidincoated slides were prepared as described on Dr. Andrew Flavell’s website (The University of Dundee, Dundee, U.K.: http:// www.personal.dundee.ac.uk/∼ajflavel/TAM_protocol.htm; see Appendix 3). Briefly, five Superfrost Gold slides were placed in a siliconized polypropylene Coplin jar and incubated in 20 mL of biotin-labeled bovine albumin solution (prepared in T50 buffer (10 mM Trizma, 50 mM NaCl, pH 8.0) for 45 min at room temperature. Twenty milliliters covered approximately two-thirds of each slide, and the slides were periodically mixed. After two washes (10 min each) in T50 buffer in a polystyrene slide washing jar, the slides were incubated in another siliconized polypropylene Coplin jar in streptavidin solution (in T50 buffer) for 10 min at room temperature. After two washes (10 min each) in T50 buffer, the slides were briefly rinsed in distilled, deionized H2O and allowed to dry in a fume hood for ∼1 h. They were then stored at

Figure 2. Correlation between enumerated bacterial objects (shown as open circles; enumeration of individual, or clusters of, bacteria, called bacterial objects, was determined manually with fluorescence microscopy) and AFU measurements (shown above as closed circles) from microarray slide scanner. Bacterial concentrations (from 3.0 × 106 to 9.0 × 107 cells/mL) were exposed to microarray slides (duplicate 135µm-diameter spots with biotinylated capture antibody, each on 2 slides). In (A), the number of enumerated bacterial objects and AFU from array scanner were linear (r2 > 0.97 for each) and similar to each other. Linear trend lines for each were as follows: enumerated bacterial objects, open circles (y ) 0.92x + 14.7); AFU signal, closed circles (y ) 306.7x + 33.7). In (B), representative fluorescent micrographs, each of a microarray spot, of the 4 bacterial concentrations (upper left 9.0 × 107, upper right 3.0 × 107 lower left 9.0 × 106, and lower right 3.0 × 106 cells/mL) are shown.

7 °C in a slide box. The streptavidin-coated slides were used within 2 months. Antibody and Microarray Slide Preparation for Bacterial Capture. Lyophilized pellets of unlabeled and biotinylated antiE. coli antibodies were reconstituted in 60% PBS/40% glycerol (v/ v) in order to prevent evaporation of the droplets and keep the capture antibodies hydrated.8 Biotinylated anti-E. coli antibodies were then able to be printed onto the microarray slides. For (8) Macbeath, G.; Schreiber, S. L. Science 2000, 289, 1760-1763.

optimization studies, biotinylated capture antibodies were diluted with 60% PBS/40% glycerol (v/v) solution before printing. However, to investigate the potential beneficial effects of Fc-directed antibody orientation,9 the unlabeled antibodies were reacted with biotinylated protein G prior to printing. Unlabeled antibody solutions were diluted 1:2 with protein G (in PBS) and incubated for 4 h at 7 °C with periodic mixing.9 Longer incubations (∼20 h) did not improve protein G-antibody binding (data not shown). (9) Akerstrom, B.; Bjorck, L. J. Biol. Chem. 261 1986, 261, 10240-10247.

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Figure 3. Efficiency of bacterial cell capture on antibody microarray (calculated using data in Figure 2). As the number of cells delivered per spot decreased, the efficiency of capture (cells captured as enumerated by fluorescence microscopy) increased. At the 9.0 × 106 cells/mL data point (second from left; corresponded to 143 cells delivered per spot), capture efficiency increased rapidly with decreasing bacterial concentration, and standard deviation (error bars shown above) was markedly reduced.

The protein G-bound capture antibodies were then suitable to be printed onto microarray slides. Ten microliters of thoroughly mixed capture antibody solution was pipetted into a well of the 384-well source plate on the microarray printer. The array printing robot was controlled with SPOCLE software v. 1.1.0 (TeleChem). Contact printing, using default wash and contact settings, was conducted with SMP4 pins (135-µm spot diameter). The pin delivered 1.1-nL volumes per contact stroke. The pins were sonicated for 5 min in distilled H2O before each use. Spots were spaced 750 µm apart on the slide. Each slide was visually examined after printing to ensure that a spot was printed with each pin stroke. For slides that were going to be used without a coverslip, a hydrophobic barrier (9 mm × 10 mm) was created around the spots with a PAP pen. Within 30 min of printing completion, the slides were stored at 7 °C overnight before being used the next day (∼18 h). Growth and Enumeration of E. coli O157:H7. One milliliter of frozen stationary-phase E. coli O157:H7 was thawed and added to 10 mL of EC broth. This was incubated at 37 °C for 18 h with shaking at 160 rpm. Cultures were enumerated with a Petroff-Hausser counting chamber as described by Gehring et al.10 A 1-mL aliquot of cells was pelleted by centrifugation at 5000 rpm for 5 min, was resuspended in PBS, and serially diluted to the desired concentrations. Antibody Microarray Detection of E. coli O157:H7. Microarray slides and blocking solution were removed from refrigeration and allowed to warm briefly (to about room temperature). To prevent nonspecific binding, the slides were blocked by static incubation with 100 µL of PBS plus 1% BSA (w/v), pipetted onto the slide, for 1 h at room temperature. The slides were then washed with continuous mixing in PBS for 3 min using a slide holder and washing jars and were then dried with centrifugation at 2000 rpm for 2 min. One hundred microliters of bacterial solution was then added, and each array was statically incubated at room temperature for 1 h to allow bacterial capture. During this time, the reporter antibody solutions were prepared. Frozen aliquots of reporter antibody were thawed and diluted (10) Gehring, A. G.; Patterson, D. L.; Tu, S. Anal. Biochem. 1998, 258, 293298.

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(typically 1:10, unless specified otherwise) with PBS plus 0.5% BSA (w/v). The reporter antibody was carefully light-protected during all experiments. The slides were washed 3 times (3 min each) and dried as above. Next, 100 µL of reporter antibody solution was added to each slide, which was incubated for 1 h at room temperature. Slides were washed twice (3 min each), dried as above, and then scanned at the appropriate fluorescence setting (excitation 488 nm; emission filter 535 nm) on the LS400 array scanner using default settings (scan mode, single; pinhole, large; scan resolution, 20 µm; oversampling factor, 1; PMT gain, 125 (or 175 for fluorescence microscopy studies); slide thickness, 1000 µm). Fluorescence Microscopy of Microarray Slides. Select microarray spots were visualized with fluorescence microscopy using procedures similar to those reported previously.11 To enumerate captured bacterial objects, reporter antibody solutions were diluted 1:100 in PBS plus 0.5% BSA (w/v). Fluorescent microspheres (Fluorospheres, 9.7-µm diameter; Invitrogen, Carlsbad, CA) were used to estimate the size of bacterial objects that were microscopically enumerated. Briefly, the slides were placed on an inverted microscope (Nikon Diaphot, Garden City, NY). The fluorescent images that were produced with a 100-W mercury lamp, with light traveling through a cube filter (Nikon; excitation, 470 ( 20 nm; emission, >520 nm), were captured digitally using software (IPLab v. 3.55, Scanalytics, Inc., Rockville, MD). Data Analysis. Each microarray slide, which contained at least duplicate printed spots, was considered an experimental unit. Background measurements were taken from preselected locations on the microarray substrate that were proximal to, yet outside of, the printed capture antibody spots using setting number 15 × 15 with ScanAlyze software v. 2.4.0 (Dr. Michael Eisen Laboratory, University of California at Berkeley). The data shown throughout indicate means ( standard deviations. The signal-to-noise ratio (SNR) was calculated using the following equation:

SNR ) [(raw fluorescence signal) (background fluorescence signal)]/ (standard deviation of background fluorescence signal).

Figure 4. Antibody microarray detection of E. coli O157:H7, with varying concentrations of capture and reporter antibodies. Duplicate 135µm-diameter spots of biotinylated capture antibody (each on 2 slides) were exposed to bacteria and, following capture and washing, were then labeled with fluorescein-labeled reporter antibody. (A) When 108 cells/mL was applied to the slides, the concentration of reporter antibody, and not the concentration of capture antibody, increased the AFU signal to plateau levels. The mean coefficient of variation was 25%. (B) Conversely, at the same ranges of capture and reporter antibodies, yet with fewer bacteria (106 cells/mL) available for capture, AFU signal was elevated with increasing concentrations of capture but not reporter antibody. The mean coefficient of variation was 29%.

RESULTS AND DISCUSSION When samples were analyzed without bacteria in the presence of reporter antibody, as well as when samples of bacteria were captured without reporter antibody, the signal generated was less than, or equal to, the localized background arbitrary fluorescence unit (AFU; data not shown) measurements. Range of E. coli O157:H7 Detection. To establish the limits of E. coli O157:H7 detection with an antibody microarray, slides were exposed to a range of cell concentrations (3.8 × 104-3.8 × 109 cells/mL; see Figure 1A). Two slides, each with five spots of both biotinylated and protein G capture antibodies, were used at each cell concentration. Interestingly, the biotinylated capture antibody produced a much larger signal (in AFU), especially at 107-109 cells/mL, where the signal was over 5-fold greater and appeared to saturate. (Note, the instrument employed to measure sample fluorescence was not overwhelmed at this point; therefore, saturation of response was presumed to be from the capture antibody being at a limiting concentration.) At 106 cells/mL and (11) Tu, S.; Uknalis, J.; Patterson, D. L.; Gehring, A. G. J. Rapid Methods Autom. Microbiol. 1998, 6, 259-276.

lower, protein G-bound antibody was unable to produce a signal above background, while the biotinylated capture antibody was able to give a detectable signal. Even at 104 cells/mL (Figure 1B), biotinylated capture antibody was able to produce a mean AFU signal that was greater than background and with a SNR approaching 10. It should be noted that these two approaches of antibody surface attachment produced very similar amounts of immobilized capture antibody, as indicated by experiments with fluorescein-labeled anti-IgG (data to be published elsewhere). Therefore, protein G-bound capture antibody may have been oriented improperly compared to biotinylated antibody. Protein G binds antibodies at the Fc region,9,12 which in the current study theoretically produced a higher proportion of antibodies with their functional, antigen-binding portions (i.e., Fab) oriented away from the glass slide. However, protein G also contains membrane, Fab, and albumin binding sites13 (Sigma Chemical Co., St. Louis, MO). In addition, protein G selectively binds subclasses of IgG.14 (12) Akerstrom, B.; Brodin, T.; Reis, K.; Bjorck, L. J. Immunol. 1985, 135, 25892592. (13) Oda, M.; Kozono, H.; Morii, H.; Azuma, T. Int. Immunol. 2003, 15, 417426.

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Figure 5. Background AFU (from data in Figure 4) measurements, as affected by reporter (x axis) and capture (separate curves, see legend) antibody concentration, at 108 (A) and 106 (B) cells/mL. Background measurements were taken from preselected locations on the microarray substrate that were proximal to, yet outside of, the printed capture antibody spots. Background measurements did not appear to be affected by reporter and capture antibody concentrations. Overall, 106 cells/mL exposure (B) produced higher background signal compared to 108 cells/mL exposure (A).

Therefore, the differences between the capture antibodies reported here could easily be due to one or both of these factors. It should be noted, however, that the selective binding of protein G to subclasses of IgG could be beneficial if one or more subclasses of IgG are highly specific for the analyte in question. Another potential benefit of protein G-bound capture antibodies involves affinity. Capture antibodies with low affinity may reduce or eliminate cross-reactivity (and produce highly specific biosensor assays), as nonspecific binding affinity with antibodies is usually lower than specific binding, and thus, nonspecific binding energy may be less than the physically possible threshold necessary for antigen-antibody bond formation.15 Array Scanning Response versus Enumerated Bacterial Objects, and Capture Efficiency. For quantification of E. coli O157:H7, it was necessary to determine the bacterial concentration range that produced a linear response and to determine whether this correlated with the amount of captured bacteria (Figure 2A). To count bacterial objects (presumed to be a mixture composed of at least live or dead bacterial cells, membrane particles, blebs, LPS, flagella, and toxins), it was necessary to dilute the amount of reporter antibody in PBS plus 0.5% BSA (w/v) by 1:100. Using fluorescent microspheres of known diameter (see above), it was (14) Shearer, M. H.; Dark, R. D.; Chodosh, J.; Kennedy, R. C. Clin. Diag. Lab. Immunol. 1999, 6, 953-958. (15) Karush, F. Immunoglobulins; Plenum: New York, 1979.

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estimated that many enumerated objects were solely composed of bacteria (Figure 2B). Bacterial cell concentrations, captured with biotinylated capture antibodies (duplicate spots, each on 2 slides) from 3.0 × 106 to 9.0 × 107 cells/mL, produced a linear response (r2 > 0.97) with the fluorescence microarray scanner (left y axis, solid dots; Figure 2A). This is in general agreement with data shown in Figure 1A. Accordingly, the number of enumerated bacterial objects (representative images, each of a microarray spot, shown in Figure 2B; see figure caption) produced a similar linear response (r2 > 0.97) over the same range of concentrations (right y axis, open dots; Figure 2A). Therefore, the AFU signal produced with the array scanner was in excellent agreement with the number of captured bacterial objects and produced a linear response, which is important for quantification. Other papers using antibody microarrays have reported similar linear ranges. For example, Bacillus globigii spores exhibited linearity from ∼1.0 × 106 to ∼1.0 × 108 cfu/mL, while MS2 bacteriophage particles were detected linearly from ∼1.0 × 107 to ∼1.0 × 109 pfu/mL.16 Also, whole leukocytes, bound to array spots, increased linearly with increasing concentration of leukocyte solution (from 1.25 to 10 × 106 cells/mL).17 (16) Rao, R. S.; Visuri, S. R.; McBride, M. T.; Albala, J. S.; Matthews, D. L.; Coleman, M. A. J. Proteome Res. 2004, 3, 736-742. (17) Belov, L.; de la Vega, O.; dos Rmedios, C. G.; Mulligan, S. P.; Christopherson, R. I. Cancer Res. 61 2001, 61, 4483-4489.

In terms of capture efficiency, the number of enumerated bacterial objects (Figure 2A) was compared with the calculated amount of cells delivered per spot. (The number of cells delivered per spot was estimated to be the fraction of the sample concentration that corresponded to the ratio of the area of the spot of printed capture antibody in question to the total substrate area to which the sample was exposed/applied.) For example, with 9.0 × 107 bacterial cells/mL, 100 µL of this solution spread over the area formed with a hydrophobic barrier (9 mm × 10 mm) resulted in slightly over 1400 cells exposed per microarray spot (the rightmost data point in Figure 3). As the number of cells delivered per spot decreased, the capture efficiency increased, approaching 35% (Figure 3). However, assuming that the amount of clusters formed is somewhat constant over the range of bacterial concentrations used here, it is very likely that capture efficiency decreased with increasing concentration. Considering this from an immunoassay perspective, at the highest bacterial concentration used here, 1430 bacterial cells (each 1-2 µm in diameter) were competing for antibody binding sites on a 135-µm-diameter spot and saturating the antigen-antibody interactions. When the cell concentration was reduced 10-fold, 143 bacterial cells were delivered per spot. Interestingly, at this point the standard deviation was markedly reduced and the capture efficiency began to rapidly increase with reduced bacterial concentration (Figure 3). Another consideration raised from these data (Figure 3) concerns the amount of bacteria exposed to each spot. With 3.0 × 106 cells/mL (left-most data point), the amount of cells exposed per spot was less than 100. Thus, reducing the amount of bacteria for capture will reach a point where less than 1 cell, on average, is exposed to each spot. So, data in Figure 1B, which shows detection using 3.8 × 104 cells/mL, likely only represent the capture of a few cells per spot. Therefore, spot size, number of spots, and slide area appear to be important considerations when capturing whole bacteria, in terms of efficiency, variation, and possibility of false negatives. Optimization of Capture and Reporter Antibodies, and Background Effects. To determine optimal concentrations of capture and reporter antibodies for detection of E. coli O157:H7, a 3 × 3 factorial design, with three concentration levels each of capture and reporter antibody, was used. Nine combinations of capture and reporter antibody concentrations were examined (duplicate spots with biotinylated capture antibody, each on 2 slides at each capture/reporter antibody combination), with either 3.7 × 106 or 3.7 × 108 cells/mL (Figure 4A,B). At the higher cell concentration, although AFU signal increased with reporter antibody concentration, the signal converged as capture antibody concentration was increased (Figure 4A). According to data presented here (Figures 1A, 2A, and 3), 3.7 × 108 cells/mL represented a saturation level with this antibody microarray procedure. Therefore, at this cell concentration, the reporter antibody would be expected to affect the AFU signal much more than the concentration of capture antibody. The mean coefficient of variation among all data points was 25%. At the lower cell concentration, the AFU signal was affected by capture antibody concentrations rather than reporter antibody

(Figure 4B). In fact, with constant reporter antibody concentrations of either 0.01 or 0.005 µg/µL, AFU measurements increased linearly (r2 ) 0.99 for both) with increasing capture antibody concentration. With 3.7 × 106 cells/mL, the number of cells was likely limiting the AFU signal (Figures 1A, 2A, and 3). Therefore, when the amount of capture antibody was increased, the AFU signal was linearly increased. This occurred with little influence from reporter antibody concentration, which was likely in excess for the number of captured cells. The mean coefficient of variation among all data points was 29%. To determine the effect of capture and reporter antibody concentrations on localized background fluorescence (at 106 and 108 cells/mL), background measurements, taken from data shown in Figure 4A,B, were examined (Figure 5A,B). Interestingly, with 106 cells/mL (Figure 5B), AFU signal, regardless of capture and reporter antibody concentrations, was higher than with 108 cells/ mL (Figure 5A). Localized background measurements were taken near the array spots using preselected locations on the microarray substrate. With fewer captured cells (106 cells/mL; Figure 5B) at the same reporter antibody concentration as above (i.e., reporter concentration in excess), surplus fluorescence from unbound or unreacted reporter antibody may have contributed to the localized background signal. Thus, in situations with low levels of captured cells, increased localized background measurements from excess reporter antibody could produce false negatives. Therefore, optimization of capture and reporter antibody levels may be critical for the reduction of background for select applications. CONCLUSIONS Antibody microarrays, used in sandwich assay format to detect E. coli O157:H7, met many important criteria for use as a biosensor. With the streptavidin-coated surface functionality, they are readily adaptable to multianalyte analyses with appropriate biotinylated capture antibodies as additional capture antibodies could be easily and rapidly incorporated to meet changing needs. This rapid method using antibody microarray biosensors was also capable of quantitative testing as indicated by the linear range (3.0 × 106-9.0 × 107 cells/mL) in Figure 2A. This and the data in Figure 1A infer an apparent limit of detection of ∼3.0 × 106 cells/mL. Future work will seek to adapt and integrate microarray analysis into existing regulatory protocols by initially testing these developed methods with culture-enriched food samples (e.g., ground beef) artificially inoculated with pathogenic bacteria. ACKNOWLEDGMENT Reference of brand or firm names does not constitute an endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned. We thank Drs. Jeffrey Brewster and Yanhong Liu,, from the Eastern Regional Research Center, Agricultural Research Service, U. S. Department of Agriculture, for advice and discussions on microarrays. Received for review May 8, 2006. Accepted July 17, 2006. AC0608467

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