Bacillus globigii Bugbeads: A Model Simulant of a Bacterial Spore

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Anal. Chem. 2005, 77, 549-555

Bacillus globigii Bugbeads: A Model Simulant of a Bacterial Spore Svetlana Farrell, H. Brian Halsall,* and William R. Heineman*

Department of Chemistry, University of Cincinnati, P.O. Box 210172, Cincinnati, Ohio 45221-0172

Nonpathogenic microorganisms are often used as simulants of biological pathogens during the initial phase of detection method development. While these simulants approximate the size, shape, and cellular organization of the microorganism of interest, they do not resemble its surface protein content, a factor particularly important in methods based on immunorecognition. Here, we develop and detect an artificial bacterial sporesB. globigii (BG) Bugbeadsa particle mimicking the antigenic surface of BG spores. Two methods of spore protein extraction were compared both quantitatively (by protein concentration assay) and qualitatively (by SDS-PAGE and Western blot): extraction by mechanical disruption and extraction by chemical decoating. The former method was more efficient in producing more protein and a greater number of antigens. BG Bugbeads were made by conjugating the extracted proteins to 0.8-µm carboxyl-coated polystyrene particles via carbodiimide coupling. BG Bugbeads were successfully detected by a bead-based enzyme-labeled immunoassay with fluorescence detection with a detection limit of 6.9 × 103 particles/mL. Formation of the Bugbead-capture bead complex was confirmed by ESEM. The concept of a harmless artificial spore can be applied to developing improved simulants for pathogenic sporeforming microorganisms such as B. anthracis, C. botulinum, and B. cereus, which can to be used for method validation, instrument calibration, and troubleshooting. Different methods exist for detecting bacterial agents and their toxins in various media (i.e., air, water, food, and bodily fluids), and many still remain to be developed.3 The most common detection methods are based on cell culture,1,2 immunoassay technology,4-6 PCR.7,8 and flow cytometry.9,10 Each detection technique has advantages and disadvantages, and the current * To whom correspondence should be addressed. E-mail: [email protected]. (1) Ball, A. S. Bacterial Cell Culture: Essential Data; Wiley: New York, 1997. (2) James, G. S.; Sintchenko, V. G.; Dickeson, D. J.; Gilbert, G. L. J. Clin. Microbiol. 1996, 34, 1572-1575. (3) Ivnitski, D.; Abdel-Hamid, I.; Atanasov, P.; Wilkins, E. Biosens. Bioelectron. 1999, 14, 599-624. (4) King, K. D.; Anderson, G. P.; Bullock, K. E.; Regina, M. J.; Saaski, E. W.; Ligler, F. S. Biosens. Bioelectron. 1999, 14, 163-170. (5) Brewster, J. D.; Gehring, A. G.; Mazenko, R. S.; Houten, Van, L. J.; Crawford, C. J. Anal. Chem. 1996, 68, 4153-4159. (6) Abdel-Hamid, I.; Ivnitski, D.; Atanasov, P.; Wilkins, E. Biosens. Bioelectron. 1999, 14, 309-316. (7) Meng, J.; Shaohua, Z.; Doyle, M. P.; Mitchell, S. E.; Kresovich, S. Int. J. Food Microbiol. 1996, 32, 101-113. 10.1021/ac049156y CCC: $30.25 Published on Web 12/07/2004

© 2005 American Chemical Society

tendency is to use a combination of different methods of pathogen detection to overcome such limitations. The exposure of laboratory personnel to biological pathogens during method development presents a potential health hazard to a varying degree. Working with highly infective and toxinproducing bacteria, such as Bacillus anthracis, requires special training and BL3/BL4 research facilities.11 The health risk and the overall project cost can be minimized by replacing pathogens with nonpathogenic simulants, thus avoiding unnecessary exposure to pathogens. Over the years, harmless bacteria, viruses, and spores have been used to mimic microorganisms of interest. For example, MS2 bacteriophage is used as a model virus,12 Bacillus globigii spores are a spore model,12 and Erwinia herbicola is a bacterial simulant.13 Although these simulants mimic the size, shape, and general cellular organization of the pathogens, they do not represent accurately their antigenic composition. For some detection methods, especially those based on immunoreactions, the antigenic content of the cell surface is the sole basis of molecular recognition. Consequently, the ideal simulant should have a surface that morphologically and antigenically resembles the surface of a pathogen and be nonhazardous at the same time. The concept of an “artificial microorganism”sa Bugbeadswas developed in our laboratories.14 A Bugbead consists of a particle coated with the antigens of interest, which can originate from the bacterial, spore, or viral outer surface and thus represents the antigenic surface of the microorganism. In immunological methods of detection, such particles can be recognized by the antibodies raised against the pathogen’s exposed outer surface, but be completely harmless, due to their lack of genetic material and inability to produce and release toxin. The size of the core particles varies from 0.05 to 18 µm, which allows the size of a Bugbead to be matched to the size of the bioagent of interest. For concept demonstration, a Bugbead coated with a model proteinsIgGswas created and successfully detected by a beadbased immunoassay.14 An IgG-coated particle, however, did not (8) Way, J. C.; Josephson, K. L.; Pillai, S. D.; Abbaszadegan, M.; Gerba, C. P.; Pepper, I. L. Appl. Environ. Microbiol. 1993, 59, 1473-1479. (9) Clarke, R. G.; Pinder, A. C. J. Appl. Microbiol. 1998, 84, 577-584. (10) Nebe-von Caron, G.; Stephens, B. J. Appl. Microbiol. 1998, 84, 988-998. (11) Food, U. S.; Drug Administration. Federal Register. Rules and Regulations, 2002; Vol. 67, p 105. (12) Rowe. C. E.; Tender, L. M.; Feldstein, M. J.; Golden, J. P.; Scruggs, S. B.; MacCraith, B. D.; Cras, J. J.; Ligler, F. S. Anal. Chem. 1999, 71, 38463852. (13) Walter, K. Science and Technology Review, Lawrence Livermore National Laboratory, June 1998; pp 4-10. (14) Kradtap, S.; Wijayawardhana, C. A.; Schlueter, K. T.; Halsall, H. B.; Heineman, W. R. Anal. Chim. Acta 2001, 444, 13-26.

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represent the complex surface of a real microorganism where macromolecules of different classes vary by structure and function, and so an artificial bacterium was also previously created and detected by an immunoassay.14 Here, we develop an artificial spore model for B. globigii (BG) sporessa BG Bugbeadsand detect it by a bead-based immunoassay with fluorescence detection. Two challenges have to be overcome in preparing the BG Bugbead: extraction of the predominantly protein antigens from the spores and attaching the proteins to the surface of the core particles. Spores are known to be the most resistant life forms. They can withstand boiling, UV radiation, free-radical damage, and disinfection treatment.15,16 Some spores are known to survive for hundreds of years under harsh environmental conditions.17 These properties of the spores are due to their rugged, multilayered coat. To prepare a soluble BG spore coat extract, it was necessary to disrupt the spore coat and extract a sufficient amount of proteins, but also to ensure that these proteins are recognized by the available anti-BG antibodies. When attaching the proteins to the surface of the beads, an efficient coupling procedure that maximizes the protein conjugation had to be used to result in a detectable immunoassay signal. If an artificial spore is created and detected, the same principle can be applied to make an artificial virus. Because the viruses are more susceptible to disruption by physical force or chemical agents compared to the spores, no major problems during protein extraction would be expected once the procedure is established for the spores. If a BG Bugbead is successfully developed, the same methods can be used to prepare artificial spores for other spore-forming microorganisms such as B. anthracis, Clostridium botulinum, and Bacillus cereus. Bugbead pathogen simulants can be used not only for method developing and validation but also for instrument calibration and troubleshooting. EXPERIMENTAL SECTION Apparatus. All fluorescence measurements were done in an LS 45 luminescence spectrometer (Perkin-Elmer Life and Analytical Science, Boston, MA). A Lambda 45 UV/visible system (Perkin-Elmer Life and Analytical Science) was used for analyzing the extract of BG spore proteins. Two types of fluorescence cuvettes were used in the spectroscopic measurements: a 1-cmpath length cuvette and a 0.4-cm-path length cuvette equipped with an adaptor (McCarthy Scientific Co., Fullerton, CA). A Zenith Dental Amalgamator (Speedco Dental, Los Angeles, CA) was used to mechanically disrupt the BG spores. A Sorvall RC5 floor model centrifuge with the GSM rotor from Kendro Laboratory Products (Newtown, CT) was used to preconcentrate the BG spore protein extracts. A Micro 14 tabletop microcentrifuge (Fisher Scientific, Pittsburgh, PA) was used to separate the Bugbeads from the buffer during the washing step. Polypropylene microcentrifuge tubes with locking lids (1.5 mL, Fisher) were centrifuged at 10 000 rpm for various running times. A computer-controlled electrophoresis power supply model 3000 Xi, Mini-Protean II Electrophoretic Cell, and Mini-Trans-Blot Electrophoretic Transfer Cell (BioRad, Hercules, CA) were used (15) Riesenman, P. J.; Nicholson, W. L. Appl. Environ. Microbiol. 2000, 66, 620626. (16) Driks, A. Microbiol. Mol. Biol. Rev. 1999, 63, 1-20. (17) Gould, G. W.; Russell, A. D.; Tull, D. S. Fundamental and Applied Aspects of Bacterial Spores; Blackwell Publishing: Malden, MA, 1994.

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in SDS-PAGE and Western blot (WB) analysis. AlphaImager high-performance gel documentation/image analysis system (Biotechnology and Life Sciences, Maarssen, The Netherlands) was used for SDS gels and WB membrane imaging. The interactions between the BG Bugbeads and the capture beads were imaged using an XL 30 environmental scanning electron microscope, which was from Philips Electron Optics (Hillsboro, OR). Buffers and Reagents. Streptavidin-coated M-280 Dynabeads were from Dynal (Lake Success, NY). Carboxyl-coated 0.8-µm polystyrene beads were from Spherotech (Libertyville, IL). B. globigii spores were from Aberdeen Proving Ground (Aberdeen, MD). Rabbit anti-B. globigii IgG raised against the intact spores was from Tetracore Inc. (Gaithersburg, MD). Custom alkaline phosphatase (AP) conjugation to rabbit anti-B. globigii IgG was done by American Qualex (San Clemente, CA). Biotin-SPconjugated donkey anti-mouse IgG (H+L) and goat anti-rabbit IgG conjugated to β-galactosidase were from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). Centriprep-3 centrifugal filter units and Immobilon PVDF membranes were from Millipore (Billerica, MA), and 800-µm silica/glass homogenizing beads were from OPS Diagnostics, LLC (Bridgewater, NJ). Polyacrylamide gels (4-15% Tris-HCl, linear gradient) were from BioRad. Fluorescein diphosphate (FDP) was from Molecular Probes (Eugene, OR). SigmaMarker Low Range for proteins (6.5-66-kDa range), Folin-Ciocalteu’s phenol reagent, Tris-HCl, 2-mercaptoethanol, dithiothreitol (DTT), 4-morpholinoethanesulfonic acid monohydrate (MES), sodium azide, and 5-bromo-4-chloro-3-indolyl β-Dgalactopyranoside (X-Gal) were from Sigma (St. Louis, MO), and Tween-20 was from Aldrich (Milwaukee, WI). Glycine, potassium phosphate monobasic, and potassium phosphate dibasic were from Matheson Coleman & Bell Manufacturing Chemists (Norwood, OH). Tris(hydroxymethyl)aminomethane (THAM), magnesium chloride, bovine serum albumin (BSA), urea, sodium dodecyl sulfate (SDS), bromophenol blue, sodium chloride, sodium carbonate, sodium hydroxide, sodium tartrate, copper sulfate, methanol, N,N-dimethylformamide (DMF), and 12 × 75 mm borosilicate culture tubes were from Fisher Scientific (Fair Lawn, NJ). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and GelCode Blue Staining Reagent were from Pierce (Rockford, IL). The neodymium-iron-boron rare earth magnets were from Radio Shack (Fort Worth, TX). All chemicals were used without further purification. Two buffers were prepared for SDS-PAGE: stock running buffer (0.12 M THAM, 0.96 M glycine, 0.5% w/v SDS) and sample buffer (1 mL of 0.5 mM THAM + 1.6 mL of 10% w/v SDS + 0.8 mL of glycerol + 0.4 mL of mercaptoethanol + 0.4 mL of 1% w/v bromophenol blue + 3.8 mL H2O). WB transfer buffer (48 mM THAM, 39 mM glycine, 0.1% w/v SDS, pH 9.2) and WB working buffer (44 mM KH2PO4, 56 mM K2HPO4, 0.1 M NaCl, 0.5% (w/v) BSA, 0.02% (w/v) NaN3, pH 7.0) were used in the WB analysis of BG spores protein extract. Phosphate buffer (PBS) (44 mM KH2PO4, 56 mM K2HPO4, 0.1 M NaCl, 1% (w/v) BSA, 0.02% (w/v) NaN3, 0.02% (v/v) Tween20, pH 7.0) was used in the assay preparation and as a blocking buffer for the WB experiment. The presence of BSA and Tween20 was to reduce nonspecific adsorption. MES/saline buffer (0.1 M MES, 0.5 M NaCl, pH 6.0) was used in the Bugbead

preparation. Tris buffer (0.1 M THAM, 10 mM MgCl2, 0.02% (w/ v) NaN3, 10 mM glycine, pH 9.0) was used in the immunoassay detection procedure. BG Spore Coat Protein Extraction. Extraction by Mechanical Grinding. A dental amalgam capsule was thoroughly washed first with methanol and then with diH2O, and then 25 mg of dry BG spores, 25 mg of 800-µm glass homogenizing beads, and 250 µL of PBS, pH 7.0, were added to the capsule. The capsule was placed in a dental amalgamator and shaken for 2 min. After that, the mixture was transferred to a microcentrifuge tube and centrifuged at 10 000 rpm for 10 min. The amber supernatant was transferred to a test tube. The remaining pellet was washed 3 times with 250 µL of PBS by centrifuging at 10 000 rpm, and the supernatant was collected each time and transferred to a test tube. All supernatants were combined, and the resulting solution was filtered in a Centricon-3 filter unit at 4500 rpm. The protein retentate was washed 3 times with 250 µL of PBS at 4500 rpm and resuspended in 1 mL of PBS. The stock protein extract was stored at -70 °C. Extraction by Chemical Decoating. A procedure described by Hayes18 was followed for chemical decoating. A decoating solution containing 50 mM Tris-HCl, pH 8.0, 8 M urea, 50 mM DTT, 10 mM EDTA, and 1% w/v SDS was made in diH2O. One milliliter of the decoating solution was added to 25 mg of BG spores, and the suspension was incubated for 1.5 h at 37 °C. The suspension was then centrifuged at 10 000 rpm, and the supernatant was removed. The spores were washed 3 times with 250 µL of PBS by centrifuging at 10 000 rpm, and the supernatants were collected. All supernatants were combined together, and the resulting solution was filtered in a Centricon-3 filter unit at 4500 rpm. The protein retentate was washed 3 times with 250 µL of PBS at 4500 rpm and resuspended in 1 mL of PBS. The stock protein extract was stored at -70 °C. Analysis of BG Spore Protein Extracts. Protein Concentration Assay. Proteins of the two BG spore extracts (one obtained by chemical decoating, the other by mechanical grinding) were quantitated by a Folin-Ciocalteu assay.19 A set of BSA protein standards between 17 and 250 µg/mL was prepared in diH2O (final volume 1.2 mL). BG protein extracts obtained by mechanical grinding and chemical decoating (25 µL each) were also diluted in diH2O to a final volume of 1.2 mL. All BSA standards and diluted extracts were prepared in triplicate. The assay was continued as described.19 A protein standard curve was generated by plotting the average absorbance versus BSA concentration. Concentrations of protein extracts were calculated from the calibration curve. Analysis by SDS-PAGE. Running buffer was prepared by diluting the stock running buffer 1:4 with diH2O, and adjusting the pH to 8.3. The buffer was deoxygenated by nitrogen gas for 30 min and kept at 4 °C. The concentrations of the two protein extracts were adjusted to 1.5 mg/mL with sample buffer. A set of molecular weight marker proteins (6500-66 000 MW range) was used as a protein standard. Two protein samples and the MW markers were placed in a water bath and incubated for 5 min at 70 °C. Two BioRad polyacrylamide gels (4-15% Tris-HCl, linear gradient) were then loaded with 5 µL of the MW markers (well 1 of each gel), and 10 µL of the spore protein extracts obtained by (18) Hayes, C. S.; Setlow, P. J. Bacteriol. 1997, 179, 6020-6027. (19) Switzer, R. L.; Garrity, L. F. Experimental Biochemistry. Theory and Exercises in Fundamental Methods, 3rd ed.; W. H. Freeman Publishing: New York, 1999.

chemical decoating and by mechanical grinding (wells 2 and 3, respectively). Both gels were run in parallel in deoxygenated running buffer at E ) 180 V, Iinitial ) 46 mA, and Ifinal ) 25 mA for 40 min. The gels were then removed from the apparatus and washed 3 times each with 50 mL of diH2O. One gel was stored in diH2O at 4 °C for the WB analysis. The other gel was stained with the GelCode Blue Staining Reagent for 1 h at room temperature on a plate shaker. The stained gel was then destained by washing in diH2O for 24 h. The water was changed three times. Destained gel was imaged by the AlfaImager gel scanner. The rf values of the MW standards were calculated using the AlfaImager software, and the MWs of the resolved sample proteins were determined for BG spore extracts obtained by mechanical grinding and by chemical decoating. Analysis by Western Blot. The SDS-PAGE gel prepared and set aside for the WB analysis was thoroughly washed with diH2O and kept at 4 °C. A PVDF membrane was incubated in methanol for 3 min. After that, the membrane, SDS gel, filter papers, and WB fiber pads were equilibrated in cold transfer buffer, pH 9.2, for 30 min at 4 °C. A gel holder cassette was then loaded according to the manufacturer’s recommendations. The cassette was placed into a buffer tank of a Mini-Trans-Blot Electrophoretic Transfer Cell, and a Bio-Ice cooling unit was put next to the cassette to maintain the buffer temperature. The gel was run in cold transfer buffer for 15 h at a constant E ) 30 V, with Iinitial of 90 mA and P ) 4 W. After the power supply was disconnected, the gel was washed 3 times with 50 mL of diH2O and stained and destained according to the procedures described in the previous section. The PVDF membrane was rinsed thoroughly with diH2O and incubated in 10 mL of the WB blocking buffer for 1 h at room temperature on a plate shaker. The membrane was washed in 50 mL of PBS for 15 min with a buffer change every 5 min. Rabbit anti-BG IgG and goat anti-rabbit IgG-β-gal conjugate were diluted to 50 µg/mL each in PBS. The PVDF membrane was first incubated in a solution of rabbit anti-BG IgG for 24 h at room temperature on a plate shaker and then washed in 50 mL of PBS for 15 min with the buffer being changed every 5 min. After that, the membrane was immersed in goat anti-rabbit IgGβ-gal conjugate and incubated for 24 h at room temperature on a plate shaker. The membrane was washed in 50 mL of PBS for 15 min, with a buffer change every 5 min. A β-gal substrate, X-Gal, was dissolved in 80 µL of a 1:1 DMF/methanol mixture. PBS was then added to make a 2 mM X-Gal solution in the final volume of 10 mL. A PVDF membrane was incubated with 2 mM X-Gal at room temperature on a plate shaker until the blue precipitation spots developed. The membrane was then washed thoroughly with diH2O and air-dried. Images of the PVDF membrane and SDS gel were obtained with the AlfaImager gel scanner, and the rf values of the proteins transferred to the membrane were calculated using the AlfaImager software and compared to those obtained in the previous section. Preparation of BG Bugbeads. The extracted BG spore proteins were attached to the carboxyl-coated polystyrene beads by carbodiimide coupling as described.20 Briefly, 200 µL of 1.8 × 1011 beads/mL 0.8-µm carboxyl-coated polystyrene beads were activated by EDC, and then incubated for 2 h at room temperature on a vortex with 36 µL of 5.3 mg/mL BG spores protein extract (20) Spherotech Technical Note.

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obtained by mechanical grinding. The beads were then washed 3 times with 200 µL of MES by centrifuging at 14 000 rpm, and resuspended in PBS to a final concentration of 1.8 × 1011 beads/ mL. Bugbeads prepared in this way and stored in PBS at 4 °C were stable for the 3-week testing period. Sandwich immunoassay for BG Bugbeads. A 20-µL aliquot of 6.7 × 108 streptavidin-coated Dynabeads/mL was added to 100 µL of 12 µg/mL biotinylated rabbit anti-BG IgG in PBS. The beads were incubated for 10 min at room temperature on a vortex, washed 5 times with 120 µL of PBS, and resuspended in 20 µL of PBS. From that, 2.5-µL aliquots of beads were transferred into six fresh test tubes. One tube was set aside as a control for NSA. BG Bugbeads were added to the rest of the tubes to between 1 × 104 and 1 × 105 beads/mL in a final volume of 30 µL adjusted with PBS. The beads were incubated for 10 min at room temperature on a vortex and washed 5 times with 50 µL of PBS. Rabbit anti-BG IgG-AP conjugate was added to each tube and the control to 30 µg/mL in a final volume of 30 µL adjusted with PBS. The beads were incubated for 5 min at room temperature on a vortex, washed 5 times with 30 µL of Tris, and resuspended in 30 µL of Tris. Fluorescence detection was done in real time with λex ) 490 nm and λem ) 520 nm. The detection cell contained 298 µL of 16 µM FDP in Tris and 2 µL of beads. After the beads were added to the cuvette, the latter was inverted a few times to ensure proper mixing and placed in the instrument. All Bugbead concentrations were run in triplicate. Reaction velocity was calculated from the most linear portion of each reaction progress curve and plotted versus the BG Bugbeads concentration. The limit of detection was calculated as 3 times the standard deviation of the lowest concentration detected/slope of the calibration plot.21 Imaging Interactions between the BG Bugbeads and the Capture Beads by ESEM. Three samples were imaged: (1) BG Bugbeads (control), (2) specific interactions between the BG Bugbeads and the capture beads, and (3) nonspecific interactions between the BG Bugbeads and the capture beads. Samples 1 and 2 were prepared by transferring 5 µL of BG Bugbeads and 5 µL of the BG Bugbeads-capture beads complex, respectively, to clean microcentrifuge tubes, washing 5 times with 30 µL of diH2O and resuspending in 30 µL of diH2O. Nonspecific interactions between the Bugbeads and the capture beads (3) were analyzed using anti-mouse IgG capture beads prepared by adding 25 µL of 12 µg/mL biotinylated donkey anti-mouse IgG to 5 µL of 6.7 × 108 streptavidin-coated Dynabeads/mL. The beads were incubated for 10 min at room temperature on a vortex, washed 5 times with 50 µL of PBS, and resuspended in 5 µL of PBS. BG Bugbeads were then added to the capture beads to make a final Bugbead concentration of 1 × 105 beads/mL. The beads were incubated for 30 min at room temperature on a vortex, washed 5 times with 50 µL of diH2O, and resuspended in 30 µL of diH2O. A 5-µL aliquot of that bead suspension was transferred to a fresh test tube, and 25 µL of diH2O was added to the beads. This tube was used to analyze for nonspecific interactions. As a control check for analysis of nonspecific interactions, anti-BG IgG-AP conjugate was added to sample 3 following the imaging, and the fluorescence signal from enzymatic conversion of FDP was recorded. (21) Thompson, R. Q.; Barone, G. S., III; Halsall, H. B.; Heineman, W. R. Anal. Biochem. 1991, 192, 90-95.

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Figure 1. Protein concentration assay for BG spore protein extracts. Standard protein concentration range 17-250 µg/mL. Sample A is a BG extract prepared by chemical decoating, Sample B is a BG extract obtained by mechanical grinding. Absorbance was recorded at 500 nm.

RESULTS AND DISCUSSION Preparation of the BG Spores Protein Extract. To prepare the Bugbeads that mimic the surface of the BG spores, it was necessary to prepare a soluble extract of the spore proteins prior to attaching them to the surface of the core particles. It was desired that the protein extract contained antigens for the available anti-BG antibodies and also be of a sufficient concentration to produce a detectable signal in the immunoassay. Three methods have been used for microbial protein extraction: physical disruption22 by mechanical grinding or sonication, chemical decoating,23 and enzymatic treatment.24 Enzymatic treatment of bacterial spores for the purpose of protein extraction proved to be ineffective16 and was not tried here. Instead, we disrupted BG spores by mechanical grinding with silica/glass homogenizing beads in a dental amalgamator and by chemical decoating as described by Hayes,18 and followed the disruption by protein extraction and purification. Both extracts were characterized qualitatively and quantitatively. Analysis of BG Spores Protein Extracts. Protein Concentration Assay. The efficiency of a protein extraction procedure can be quantitated by a protein concentration assay. More efficient extraction gives higher amounts of soluble proteins. The Folin (Lowry) method25 that relies on the presence of the aromatic amino acids, such as tryptophan and tyrosine, in a protein19 was used to determine the protein concentrations of the two BG spores extracts. Figure 1 shows a calibration plot obtained for the Folin assay with BSA as a standard. The plot is linear from 17 to 250 µg/mL BSA, and the absorbance values of the unknown protein samples fall within this linear range, making a more sensitive assay unnecessary. The protein concentrations of the two diluted extracts were determined from the calibration plot and then used to calculate the protein concentration of the stock BG extracts obtained by mechanical grinding and chemical decoating to be 5.3 and 3.5 mg/mL, respectively. Protein extraction by mechanical (22) Mett, H.; Schacher, B.; Wegmannet, L. J. Antimicrob. Chemother. 1988, 22, 293-298. (23) Quinlan, J. J.; Foegeding, P. M. Appl. Environ. Microbiol. 1997, 63, 482487. (24) Masschalck, B.; Van Houdt, R.; Van Haver, E. G. R.; Michiels, C. W. Appl. Environ. Microbiol. 2001, 67, 339-344. (25) Lowry, O. H.; Rosebrough, N. J.; Farr, R. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265-275.

Figure 3. Schematic of a sandwich immunoassay for BG Bugbeads with fluorescence detection. Dynal capture bead is a solid support, biotinylated (B-) rabbit anti-BG IgG is the primary antibody, BG Bugbead is the antigen; rabbit anti-BG IgG-alkaline phosphatase (AP) conjugate is a secondary antibody. Fluorescein diphosphate (FDP) is a fluorogenic AP substrate.

Figure 2. Analysis of BG spore protein extracts by SDS-PAGE (A) and Western blot (B). Lane 1, molecular weight marker (MWs shown); lane 2, proteins extracted by chemical decoating; lane 3, proteins extracted by mechanical grinding. SDS-PAGE running conditions: E ) 180 V, Iinitial ) 46 mA, Ifinal ) 25 mA, and t ) 40 min. WB running conditions: E ) 30 V, with Iinitial of 90 mA and P ) 4 W, t ) 15 h. Proteins identified as antigens are shown in boxes with the corresponding MWs.

grinding proved to be more efficient in producing more protein than chemical decoating. Analysis by SDS-PAGE and Western Blot. In comparing the two methods of protein extraction, it was important to determine which extraction method produces larger amounts of proteins and to characterize the resulting proteins according to their antigenic properties. Immunorecognition of an artificial spore relies primarily on the presence of antigenic proteins on the surface of a particle and on the ability of antibodies to recognize these proteins. To ensure the presence of antigenic proteins in the extract samples and their immunorecognition both extracts were analyzed by SDS-PAGE and Western blot. SDS-PAGE and WB are two common methods for characterizing protein mixtures. SDS-PAGE separates proteins on the basis of their MW, and WB detects proteins in the mixture based on the immunological reaction of these proteins with their antibodies. A 15% polyacrylamide gel was used for SDS-PAGE analysis, which gives an effective separation in the range of 5000 and 70 000 Da, and is commonly used to screen protein mixtures of unknown composition.19 Figure 2A is a gel image obtained in the SDS-PAGE separation of the BG spore proteins with different methods of extraction. Lane 1 contains MW standards, lane 2 contains the proteins resolved from the mixture obtained by chemical decoating, and lane 3 contains the proteins resolved from the mixture obtained by mechanical grinding. It can be seen that chemical decoating produced mostly low molecular weight proteins. Mechanical grinding, however, resulted in a mixture of proteins with a wide MW distribution (23 protein bands were seen on the gel). The MWs of all sample proteins were calculated with respect to the standard, and the MW distributions of the mixtures obtained by chemical decoating and mechanical grinding were 6500-47 000 and 6500-130 000, respectively.

A WB experiment was done to determine whether the proteins separated by the SDS-PAGE were antigenic to the polyclonal antiBG antibody available. A PVDF membrane was used to transfer proteins with MW > 10 000,26 and β-galactosidase-X-Gal was the enzyme-substrate couple. X-Gal is a soluble, colorless indole derivative that reacts with β-Gal to form an insoluble, precipitating, blue product that remains within the membrane, helps visualization, and improves resolution. Figure 2B shows the PVDF membrane image with that of the corresponding gel (Figure 2A). After the transfer, no protein bands could be seen on the gel, indicating that most proteins were successfully transferred to the membrane. Three precipitate spots that can be seen on the transfer membrane (one for the chemically decoated spores and the others for the mechanically ground spores) indicate the proteins that complexed with the anti-BG IgGs and were antigenic to the available antibodies. The rf values for the visualized proteins had relative MWs of approximately 34 000 and 48 000 (mechanically ground) and 12 000 (chemical decoating). The absence of high-MW bands from the extract prepared by chemical decoating and the absence of a 12 000 band from the sample prepared by mechanical grinding suggest that the lowMW band represents antigenic polypeptide fragments of larger proteins or, more likely, reflects the relative efficiencies of the two extraction procedures for the constituent antigens. Since protein extraction by mechanical grinding was more efficient and yielded reactive spore surface antigens, it was used in the BG Bugbead preparation. The weight distribution of the antigen also suggests that when pathogen-Bugbeads are built, consideration should be made to generate an accurate statistical representation of the serotype antigens on their surface. Sandwich Immunoassay for BG Bugbeads. Our previous Bugbead work was directed toward demonstrating the concept of an artificial microorganism.14 For this purpose, mouse IgG, a protein that has been extensively studied and well characterized, was attached to the surface of a polystyrene particle, and the particle was detected by an immunoassay. While the concept was successfully demonstrated, a practical application of a mouse IgG particle was questionable, and Bugbeads that mimic Escherichia coli bacteria were developed.14 Having a particle that mimics the surface of a real microorganism can be of a particular interest to those who design methods and instrumentation for detection and (26) Immobilon-P Transfer Membrane User Guide; Millipore, http://www. millipore.com/userguides.nsf/docs/P15372.

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Figure 4. Sandwich immunoassay for BG Bugbeads with fluorescence detection. BG Bugbeads concentration range 1 × 104-1 × 105 beads/mL. Detection is done in real time with λex ) 490 nm and λem ) 520 nm. Each data point is the average of three measurements. Nonspecific adsorption (NSA) signal results from the adsorption of the secondary antibody to the beads in the absence of BG Bugbeads.

quantitation of bacterial agents. Being completely harmless, such simulants can be used in the initial development steps to avoid health risk, and they can also be used later for instrument calibration. Although it is possible that toxins from toxin-producing pathogens may be present in coat or membrane preparations and become covalently attached to the beads, the concentration should be extremely small and nonreleasable from the Bugbeads. Nevertheless, validation of these statements would seem prudent. For this study, we chose B. globigii spores because they are nonpathogenic and are commonly used as a spore model. If a particle that simulates a BG spore is created and successfully recognized in the immunoassay, the same approach can be taken to mimic other bacterial spores. It is necessary to note, however, that a Bugbead simulant resembles a bacterial surface only in antigenic content and does not reflect a shape or surface topology of a microorganism. The average size of a BG spore is 1.1 × 0.6 µm (Figure 5A), so spherical polystyrene particles with a diameter of 0.8 ( 0.1 µm were chosen as the Bugbead core. The resulting BG Bugbeads were detected by a sandwich immunoassay with

fluorescence detection. The schematic of the immunoassay is shown in Figure 3. Figure 4 is a calibration plot obtained for BG Bugbeads. The plot is linear in the range of the concentrations studied with a detection limit of 6.9 × 103 beads/mL. This limit of detection is comparable to those of 5 × 104 27 and 3 × 103 spores/mL28 for intact BG spores detected by similar methods. This indicates that the BG Bugbeads can be successfully detected by a bead-based immunoassay and that they can be used as a simulant for BG spores. Imaging Interactions between the BG Bugbeads and the Capture Beads by ESEM. In a bead-based sandwich immunoassay for a microorganism as an antigen, two different kinds of particles come into contact with each other. One is a capture bead coated with the primary antibody, and the other is a microorganism or its simulantsa Bugbead in this case. Once the two come into proximity, two general types of interactions between the particles are possible: specific and nonspecific. Specific interactions involve recognition of the antigens on the Bugbead by the primary antibody on the capture bead. The other nonspecific interactions do not involve the desired molecular recognition and are an undesirable side effect. Specific interactions are desired in designing a detection method based on immunorecognition, because they are a primary basis of the method’s selectivity. Excessive nonspecific interactions result in higher detection limits and false positive results and question the assay’s selectivity and validity. Two samples were prepared to determine what contributes to the formation of a BG Bugbead-capture bead complex. In the first sample, capture beads were coated with rabbit anti-BG IgG, an antibody raised against BG spores. In the second sample, antimouse IgG was used to coat the capture beads. If the interactions between the capture beads and BG Bugbeads were specific, the latter should form a complex with the complementary anti-BG but not with the anti-mouse capture beads. If the formation of the complex were due to nonspecific interactions, BG Bugbeads would complex with both complementary and noncomplementary cap-

Figure 5. Imaging interactions between the BG Bugbeads and the capture beads. (A) control BG Bugbeads, (B) specific interactions between the BG Bugbeads and the anti-BG IgG-coated capture beads, and (C) nonspecific interactions between the BG Bugbeads and anti-mouse IgG-coated capture beads. 554

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ture beads. The ESEM allows the formation of both types of complexes to be observed. BG Bugbeads were used as a control for shape and size distribution. Figure 5 shows the images of the BG Bugbeads and the capture beads obtained by the ESEM. The size and the shape of 0.8-µm Bugbead core particles (Figure 5A) are consistent with the manufacturer’s specifications. Specific interactions between the BG Bugbeads and the capture beads are imaged in Figure 5B, where the Bugbeads can be seen on the surface of the larger capture beads. When BG Bugbeads were introduced to anti-mouse capture beads (nonspecific interactions), no Bugbeads could be seen on the surface of the capture beads in the fields viewed (Figure 5C) compared to Figure 5B. Aggregation of capture beads is due to the effect of vacuum applied to the sample prior to imaging according to the manufacturer’s procedure. When the same sample was incubated with anti-BG IgG-AP conjugate followed by fluorescence detection as a control check, a fluorescence signal could not be distinguished from the noise (data not shown), indicating few or no Bugbeads captured by anti-mouse IgG-coated capture beads in a nonspecific manner. Complex formation between the Bugbeads and the complementary capture beads and the lack of such complex formation with the noncomplementary capture beads suggests that the interactions between the two particles are specific, due to the antigen-antibody recognition. CONCLUSIONS A simulant for a BG sporesa BG Bugbeadsthat resembles the antigenic surface of the spore was prepared and successfully (27) Anderson, G. P.; Rowe-Taitt, C. A.; Ligler, F. S. Proceedings of the First Conference on Point Detection for Chemical and Biological Defense, October 2002. (28) Farrell, S. Bead-based Immunoassay Technology in Protecting the Nation’s Water Supply from Bioterrorism Agents. Ph.D. Dissertation Thesis, University of Cincinnati, 2004.

detected by a bead-based immunoassay with fluorescence detection. In the BG Bugbead preparation, it was shown that spore protein extraction by mechanical grinding was more effective than chemical decoating, with resulting extracts of higher protein concentration and antigen content. A bead-based immunoassay for BG Bugbeads performed similarly to the assay of the same format for BG spores, and the detection limits were comparable.27,28 These results demonstrate that an “artificial spore” can be prepared and recognized by the antibodies raised against the original spore. Although the proposed approach was not tested with real pathogens due to the lack of appropriate facilities, we consider working with artificial spores to be much safer than with real spores. This is mainly due to the lack of a toxin production mechanism by Bugbeads. Although some bacteria do have toxin proteins on the surface, the toxin surface concentration is extremely low. Also, microorganisms have certain toxin release mechanisms such as excretion by lysis. Since all proteins are covalently attached to a polystyrene core particle of the Bugbead, Bugbeads lack such mechanisms and the toxins cannot be released into the outside environment. The proposed method could be used to prepare simulants of other spore-forming bacteria, such as B. anthracis, C. botulinum, and B. cereus. Nonpathogenic simulants of these spore species should find commercial applications in method development for detecting pathogenic microorganisms or for calibrating and troubleshooting existing devices, when health hazardous exposure to the pathogens needs to be avoided. ACKNOWLEDGMENT We thank Michael Goode (Aberdeen Proving Ground) for his generous gift of B. globigii spores. Received for review June 8, 2004. Accepted October 14, 2004. AC049156Y

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