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A novel antibody based capture matrix utilizing human serum albumin and streptococcal Protein G to increase capture effi...

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Title:
A novel antibody based capture matrix utilizing human serum albumin and streptococcal Protein G to increase capture efficiency of bacteria
Physical Description:
Book
Language:
English
Creator:
McCabe, Christie Renee
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
Publication Date:

Subjects

Subjects / Keywords:
ELISA
RAPTOR
Biosensor
Waveguide
Immobilization
Dissertations, Academic -- Cell Biology, Microbiology, and Molecular Biology -- Masters -- USF   ( lcsh )
Genre:
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: A novel capture matrix utilizing human serum albumin (HSA) and streptococcal Protein G (PG), which possesses an albumin binding domain (ABD), was used to immobilize antibodies for improved bacterial capture efficiency in immunoassays. Enzyme linked immunosorbent assays (ELISA) were used to characterize and optimize a specific protocol for the HSA-PG capture matrix; which revealed several critical factors that should be considered. The Fc binding domain, on PG, should have high affinity for the species of capture antibody used in the assay. Goat and rabbit species antibodies bound strongly to the Fc binding domain of PG. Displacement of the capture antibody, by the detector antibody should be avoided to reduce background signals. The Fc binding domain on PG should have equivalent or lower affinity for the detector antibody, when compared to the capture antibody. Goat species antibody, used as a detector antibody, did not displace the same-species capture antibody. ELISA analysis showed detection of Escherichia coli O157:H7 cells at 1.0 x 10⁴ CFU/ml using HSA-PG and goat antibody raised against Escherichia coli O157:H7; unlabeled antibody was used for capture while HRP labeled antibody was used for detection. Studies were performed on an automated fiber optic biosensor, RAPTOR, which was used for the rapid detection of pathogens. Biosensor assays showed detection of E. coli O157:H7 at 1.0 x 10³ CFU/ml in PBS and 1.0 x 10⁵ CFU/ml in homogenized ground beef supernatant. Capture efficiency of the HSA-PG capture matrix was studied using the biosensor and GFP-E. coli O157:H7. The amount of cells captured was less than one percent of the sample concentration. This limit of detection and capture efficiency was comparable to the streptavidin-biotin capture matrix.
Thesis:
Thesis (M.S.)--University of South Florida, 2009.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
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System requirements: World Wide Web browser and PDF reader.
Statement of Responsibility:
by Christie Renee McCabe.
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Title from PDF of title page.
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Document formatted into pages; contains 85 pages.

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aleph - 002028869
oclc - 436688512
usfldc doi - E14-SFE0002811
usfldc handle - e14.2811
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SFS0027128:00001


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A Novel Antibody Based Capture Matrix Utilizing Human Serum Albumin and Streptococcal Protein G to Increase Capture Efficiency of Bacteria by Christie Renee McCabe A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Cellular Microbio logy and Molecular Biology College of Arts and Sciences University of South Florida Major Professor: Daniel V. Lim, Ph.D. Lindsey N. Shaw, Ph.D. My Lien Dao, Ph.D. Date of Approval: April 7, 2009 Keywords: ELISA, RAPTOR, biosensor, waveguide, immobilization, detection, Escherichia coli O157:H7, orientation Copyright 2009, Christie Renee McCabe

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DEDICATION To my family, for their words of encouragement and their works of courage; without them you wouldnt be reading this. To the loves of my life Sam, Mom, Julie, John, and Joal; thank you for lifting me up when I fell, ma king me laugh when it wasnt funny, and protecting our country. With such inspiration all th ings are possible.

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ACKNOWLEDGEMENTS Thanks to Sonia Magana, Sarah Schlemme r, Dawn Hunter and Sonja Dickey at the Advanced Biosensors Laboratory, for incred ible technical assistan ce, strategic advice, and fun conversations. Thanks also to Dr Harvey George, and Joseph Peppe, at the Massachusetts Department of Public Health State Laboratory Institute, for their kind donation of the E. coli O157:H7 strain used in this study. Special thanks to Dr. Joyce Stroot for developing the capture efficiency procedure. Eternal grat itude belongs to Dr. Betty Kearns for her gentle guidance, and tireless support. Appreci ation is extended to my committee members: Dr. MyLien Dao for fostering confidence in me, and to Dr. Lindsey Shaw for sharing his authoring expertise with me. Ultimately, I would like to thank Dr. Daniel Lim for believing in my idea, providing opportunities for success, and for being an extraordinary mentor.

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i TABLE OF CONTENTS LIST OF TABLES iv LIST OF FIGURES vi ABSTRACT viii INTRODUCTION 1 Objective 1 Biosensor Assay 2 Streptavidin-Biotin 3 Random Antibody Orientation 3 Human Serum Albumin 4 Streptococcal Protein G 5 The PG-IgG Complex 6 Antibody Orientation by HSA-PG 7 Capture Efficiency 7 Hypothesis 8 MATERIALS AND METHODS 9 Bacterial Strains 9 Buffers 9 Media and Culture Conditions 10 Stock Cultures 10

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ii Sample Cultures 10 Capture Matrix Proteins 11 Albumins 11 Protein G and Streptavidin 11 Antibodies 12 Antibody Labeling 13 Cy5 Antibody Labeling Column 13 Cy5 Antibody Labeling Procedure 13 DyLight Antibody Labeling Procedure 14 ELISA s 16 PG Fc Binding Domain Specificity Assays 16 PG Albumin Binding Domain Functiona lity and Specificity Assays 17 Direct Assays for E. coli O157:H7 18 Statistical Analysis 19 RAPTOR ASSAYS 19 Coupon Preparation 19 Waveguide Preparation 20 RAPTOR Assay Procedure 22 Determination of Capture Efficiency 23 Detection of E. coli O157:H7 in Food Samples 23 Data Analysis 23 Statistical Analysis 27

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iii RESULTS AND ANALYSIS 28 Functional Albumin Binding Domain 28 Alternative Albumin Species 29 Optimal Ratio of HSA to PG 31 The Role of HSA in the Capture Matrix 32 The Role of HSA in Capturing Bacteria 33 Species Specific Fc Binding Domain 35 Optimal Capture Antibody Species 37 Optimal Concentration of Detector Antibody 38 Capture Antibody Displacement 40 The Limit of Detection for Two Captur e Matrices Using ELISA Analysis 41 The Limit of Detection for Two Captur e Matrices Using RAPTOR Analysis 43 The Limit of Detection for Ground Beef Homogenate Supernatant 45 The Limit of Detection for Spinach Leaf Homogenate Supernatant 46 The Capture Efficiency for Two Capt ure Matrices on Waveguide Surfaces 47 A Comparison of DyLight and Cy5 Labeled Detector Antibody 50 DISCUSSION 53 REFERENCES 66 APPENDICES 71 Appendix A: ELISA Analysis 71 Appendix B: RAPTOR Analysis 75

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iv LIST OF TABLES TABLE 1. Species Specificity of the Fc Binding Domain on PG 6 TABLE 2. Equations for Cy5 Labeling of Antibody 14 TABLE 3. Equations for DyLi ght Labeling of Antibody 15 TABLE 4. Capture Effici ency Calculations 23 TABLE 5. Baseline Values 24 TABLE 6. Normalization Coefficients 24 TABLE 7. Normalized Baseline Values 25 TABLE 8. Normalized Baseline Variability 25 TABLE 9. Limit of Detection 26 TABLE 10. Bacterial Sample Values 26 TABLE 11. Normalized Bacterial Sample Values 26 TABLE 12. Final Detection Values 27 TABLE 13. Precision of Capture Matrices 65 TABLE 14. Functional Albumin Binding Domain, HSA Role in Capture Matrix 72 TABLE 15. Alternativ e Albumin Species 72 TABLE 16. HSA Role in Capturing Bacteria 73 TABLE 17. Optimal Ratio of HSA to PG 73 TABLE 18. Species Specific Fc Binding Domain 74 TABLE 19. Optimal Capture Antibody Species 74

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v TABLE 20. Optimal Concentra tion of Detector Antibody 75 TABLE 21. The Limit of Detect ion Using ELISA, Capture Antibody Displacement 75 TABLE 22. The Limit of Detection of Two Capture Matrices Using RAPTOR 76 TABLE 23. The Limit of Detection in Ground Beef Homogenate Supernatant Fluid 79 TABLE 24. The Limit of Detection in Sp inach Homogenate Supernatant Fluid 80 TABLE 25. Cells Capture on Waveguide Surface, Capture Efficiency 81 TABLE 26. A Comparison of DyLight and Cy5 85

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vi LIST OF FIGURES FIGURE 1. Biosensor Phases 2 FIGURE 2. Orientation of Bi otinylated IgG on a Streptavidin Coated Waveguide 4 FIGURE 3. Orientation of IgG on PG -HSA Coated Waveguide Surface 7 FIGURE 4. Verification of a Functional Albumin Binding Domain in Native PG 28 FIGURE 5. Verification of th e Specificity of Different Albumin Species for PG Using ELISA Analysis 30 FIGURE 6. Verification of the Optimal Ratio of HSA to PG Using ELISA Analysis 31 FIGURE 7. The Role of HSA in the Alternative Capture Matrix Clarified Using ELISA Analysis 33 FIGURE 8. The Role of HSA in Captur ing Bacteria Clar ified Using ELISA Analysis 34 FIGURE 9. Verification of Species Specificity for the Fc Binding Domain of PG Using ELISA Analysis 36 FIGURE 10. Determination of the Optimal Capture Antibody Species Using ELISA Analysis 37 FIGURE 11. Determination of the Optimal Concentration of Detector Antibody Using ELISA Analysis 39 FIGURE 12. Capture Antibody Displacement Evaluated Using ELISA Analysis 40

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vii FIGURE 13. Determination of the Limit of Detection for Two Capture Matrices Using ELISA Analysis 41 FIGURE 14. Determination of the Limit of Detection for Two Capture Matrices Using RAPTOR Analysis 43 FIGURE 15. Determination of the Limit of Detection for Two Capture Matrices in Homogenized Ground Beef Supernatant Fluid 45 FIGURE 16. Determination of the Limit of Detection for the HSA-PG Capture Matrix in Homogenized Spinach Supernatant Fluid 46 FIGURE 17. Average Number of Cells Captured on the Waveguide Surface After RAPTOR Analysis 48 FIGURE 18. Average Capture Efficien cy on the Waveguide Surface After RAPTOR Analysis 49 FIGURE 19. A Comparison of DyLight649 and Cy5 Labeled Detector Antibody 51

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viii A NOVEL ANTIBODY BASED CAPTURE MATRIX UTILIZING HUMAN SERUM ALBUMIN AND STREPTOCOCCAL PROTEIN G TO INCREASE CAPTURE EFFI CIENCY OF BACTERIA CHRISTIE RENEE MCCABE ABSTRACT A novel capture matrix utilizing human serum albumin (HSA) and streptococcal Protein G (PG), which possesses an albumin binding domain (ABD), was used to immobilize antibodies for improved bacter ial capture efficiency in immunoassays. Enzyme linked immunosorbent assays (ELISA) were used to characterize and optimize a specific protocol for the HSA-PG capture matr ix; which revealed seve ral critical factors that should be considered. The Fc binding domain, on PG, should have high affinity for the species of capture antibody used in th e assay. Goat and rabbi t species antibodies bound strongly to the Fc binding domain of PG Displacement of the capture antibody, by the detector antibody should be avoided to reduce backgr ound signals. The Fc binding domain on PG should have equivalent or lowe r affinity for the detector antibody, when compared to the capture antibody. Goat species antibody, used as a detector antibody, did not displace the same-species capture antibody. ELISA analysis showed detection of Escherichia coli O157:H7 cells at 1.0 x 104 CFU/ml using HSA-PG and goat antibody raised against Escherichia coli O157:H7; unlabeled antibody was used for capture while HRP labeled antibody was used for detection. Studies were performed on an automated fiber optic biosensor, RAPTOR which was used for the rapid detection of pathogens.

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ix Biosensor assays showed detection of E. coli O157:H7 at 1.0 x 103 CFU/ml in PBS and 1.0 x 105 CFU/ml in homogenized ground beef supernatant. Capture efficiency of the HSA-PG capture matrix was studied using the biosensor and GFP-E. coli O157:H7. The amount of cells captured was less than one pe rcent of the sample concentration. This limit of detection and capture efficiency wa s comparable to the streptavidin-biotin capture matrix.

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1 INTRODUCTION Objective Fluorescent immunoassays have been growi ng in popularity for use in the field of pathogen detection. In 1984, Hirschfeld patent ed the use of evanescent wave and optical fiber in an immunoassay format to detect fluo rescent labeled analytes (15). Since this invention, optical fiber and fluorescent immunoassay have been developed for use in biosensor technology to rapidly detect pat hogens in environmental samples (6, 19, 23). Biosensors detect targets that have been captured by a matrix of capture molecules attached to a solid surface su ch as an optical fiber or waveguide. However, antibody based biosensor assays are plagued by poor cap ture efficiency and low sensitivity (36). The goal of this research was to orient the capture antibody to enhance capture efficiency of a target bacterium. This increase in captu re efficiency may improve assay sensitivity so that fewer bacterial cells are required fo r positive detection by the biosensor. This improved detection would benefit the public directly by promoting advances in food safety inspections and homeland security effo rts. In order to i nvestigate the hypothesis that orientation of antibodies would improve detection sensitivity, a novel capture matrix that presented antibodies in a uniform form ation on a solid surface was developed and then examined to assess improvements made to capture efficiency or assay sensitivity.

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Biosensor Assay A sandwich biosensor assay consists of three phases as shown in Figure 1. The purpose of the Capture Phase is to immobilize antibodies which are specifically able to capture the target antigen. The Sample Phas e is the introduction of liquid containing whole bacterial cells or small toxins. The samp le may come from a variety of liquids such as homogenized ground meat supernatant, e nvironmental water or phosphate buffered saline. The purpose of the Reporter Phase is to detect captured antigens by using a fluorescently labeled detector antibody specific for the target. The fluorescently labeled antibody is excited by a 635 nm laser focuse d through the core of the waveguide. The evanescent wave produced by the laser penetrates the surface of the waveguide to excite fluorophores within 100-1000 nm of the wavegui de surface (11, 23). Emissions from the fluorophore are recoupled into the optical fibe r and converted to picoamperes (pA) by a photodiode. Ultimately, the biosensor is a dedicate d fluorometer that is able to collect and quantitate emitted wavelengths above 650 nm. Any of these phases can be modified in order to produce a more efficient and sensitive biosensor assay. FIGURE 1. Biosensor Phases 2

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3 Streptavidin-Biotin Currently, biosensor capture surfaces ar e coated with a large homo-tetrameric protein isolated from the bacterium Streptomyces avidinii known as streptavidin (39). Streptavidin (60kDa) has an extrem ely high binding affinity (Ka = 2.5 x1013) for the much smaller vitamin H (244 Da), more common ly called biotin (13) Streptavidin-biotin conjugation is widely used in microbi ology and immunology due to this strong noncovalent interaction. Streptav idin has four subun its and each subunit can bind one biotin molecule. In solution, one stre ptavidin molecule can bind up to four biotin molecules simultaneously and with equivalent affinity (22). This strong binding ability has been utilized in a variety of assa ys, e.g., biotinylation of nuc leic acids, amino acids and antibodies. The biotinylation enab les the capture of targets by indirectly attaching them to a streptavidin coated surface. Biotinylated an tibodies, anchored via streptavidin to fiber optic waveguides, have been reported in a number of recent biosensor manuscripts (6, 19, 36). Random Antibody Orientation Biotin can be attached to the carbohydrate moiety found on the crystallizable fragment (Fc) region, or to the primary amines (-NH2) located on the numerous lysine residues found on the Fc, and antigen bi nding fragment (Fab) regions of an immunoglobulin G (IgG) molecule (16, 31). Hnatowichs method uses succinimidyl-6(biotinamido) hexanoate (NHS-LC-Biotin) to biotinylate lysine residues on an IgG molecule (6). This labeling method results in a random orientation of the antibody, tilted at various angles on the waveguide surface, with paratopes that are not aligned for antibody-antigen interaction (31). As depicted in Figure 2, the antibodi es are not oriented

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efficiently on the waveguide for antigen capture If the antibody is angled slightly, or laid on its side the antigen binding site is not lik ely to come into contact with the antigen, which results in missed capture opportunitie s. These missed opportunities may lead to poor bacterial capture efficien cy by the capture matrix. Capture IgG Biotin Streptavidin Waveguide FIGURE 2. Orientation of Bio tinylated Antibody on a Strept avidin Coated Waveguide Human Serum Albumin Working with human serum albumin (HSA) has many advantages beyond its common usage as a blocking agent in ELISA protocols (5). HSA (66 kDa) is an inexpensive transporter protei n found abundantly in human plasma (5 g/ 100 ml). The ability of HSA to transport molecules to target organs has been exploited to deliver therapeutic drugs in vivo (38). Like many species of albumin, the structure of HSA is a single asymmetrical polypept ide contained in three, almost identical, homologous domains resultant from gene multiplication (8, 38). This simple albumin structure allows for high affinity and rapid binding to liga nds, such as the albumin binding domain of streptococcal PG (29). The efficient coati ng of polystyrene surfaces by HSA removes the need for any additional blocking step, which allows the assay to be performed quickly (18). 4

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5 Streptococcal Protein G Lancefield group C and G streptococcal strains produce transmembrane bound Protein G (PG) to evade host immune responses. Membrane-bound PG binds to the Fc domain of an opsonizing antibody i n vivo which prevents C1q, a subcomponent of the complement system, from recognizing the an tibody and initiating th e classical pathway (27). PG is a highly versatile protein; however almost all commercially available PG is in a recombinant form. This recombinant form lacks the albumin binding domain located on the amino terminus of PG (17, 20, 29, 37). The albumin binding domain, if left intact, binds to serum albumin, making the protein inefficient at extr acting and purifying antibodies from blood samples. The albumin binding domain of PG has been used to anchor antibody fragments to HSA coated polystyrene ELISA wells (18). Native PG, containing the albumin binding domain, can be us ed as an anchor to polystyrene surfaces that have been non-covalently coated with HSA, such as a fiber optic waveguide or ELISA wells. PG has a high amount of sec ondary structure and contains a hydrophobic core, which makes it a very heat-stable protei n (7). The robust nature of PG contributes greatly to its appeal for use in biosensor assays. In addition to the albumin binding domai n, PG (65 kDa) has three identical IgG binding domains near the carboxyl terminus, which is structurally opposite from the albumin binding domain (14, 17, 20, 28, 30, 37). Only one of the three IgG binding domains, the most distal, has shown the ability to bind the carboxyl terminus of the heavy chain of intact IgG, or to the Fab region of fragmented IgG. This bindin g occurs without large conformational changes in the structure of either participant (7). PG has a high binding affinity (Ka = 5-10 x 1010) for the heavy chain of the Fc domain of IgG

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6 belonging to several different species, including goat, rabbit and human. The affinity of the PG Fc binding domain is minimal for mous e IgG (1, 4), and completely absent for chicken IgG (1, 14). This species specificity is due to the relatively conserved nature of the four gamma ( ) chains of the IgG molecule (14). Table 2 shows the relative affinities of several species of IgG. Species of Polyclonal Immunoglobulin Amount (ng) of Ig Required to Give a 50% Inhibition In Competitive ELISA Protein G Rabbit 151 Goat 217 Human 556 Mouse 1020 Chicken Indicates Species Not Reactive with Fc Binding Domain on PG TABLE 1. Species Specificity of the Fc Binding Domain on PG (14) The PG-IgG Complex The binding of the PG IgGbinding domain to the carboxyl terminus of the heavy chain of IgG involves a large amount of surface area on both mo lecules, creating a binding affinity comparable to antige n-antibody complexes (Ka = 5-10 x 1011) (7). In addition to multiple hydrogen bonds and va n der Waals attractions, the bound complex remains intact in so lution due to a hydrophobic area, creat ed by the intera ction of side chains of charged residues. This conformational binding was observed while the molecule was in crystal form, as well as in solution, indicating that liquid would not denature the PG-IgG complex (7).

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Antibody Orientation by HSA-PG The uniform orientation of an antibody on a solid surface increases its activity by promoting interactions between antigen, and antigen binding sites, as compared to random antibody orientation (40) Streptococcal PG binds sp ecifically to the carboxyl terminus of the Fc region of an IgG mol ecule, which causes the Fab region to face outward (21, 25). This uniform orientati on of immobilized IgG on HSA-PG can be attached to a solid surface for use in a bi osensor assay or ELISA, and may lead to increased capture efficiency and sensitivity. PG binding to the Fc domain of an IgG molecule dictates the orientation of the an tibody when attached to an albumin protein coated surface (7). Capture IgG Protein G HSA Waveguide FIGURE 3. Orientation of Antibody on a HSA-PG Coated Waveguide Surface Capture Efficiency Recently, the capture efficiency of evanescent waved based biosensor assays using a streptavidin-biotin-IgG capture matrix has been under review (36). Escherichia coli O157:H7 expressing green fluorescent protei n was used to quantitate target cell capture on planar and cylindrical waveguides using an automated and manual biosensor. Capture efficiencies were inversely related to the concentration of sample introduced into the matrix (36). One possible explanation for the poor capture efficiency was the random 7

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8 orientation of capture antibody on the wa veguide surface. Random antibody orientation reduces antibody activity when the antibody is attached to a sold surface (24, 40). Capture efficiency may be improved by replacing the streptavidin-biotin-IgG capture matrix with an HSA-PG-IgG capture matrix, which uniformly orients the capture antibody to increase interactions with the targeted antigen. Hypothesis A novel capture matrix consisting of human serum albumin, streptococcal Protein G and capture antibody (HSA-PG-IgG) would uni formly orient captu re antibodies on a solid surface, increasing capture efficiency of bacteria. More efficient bacterial capture would result in a higher signal-to-noise ra tio, enhancing sensitivity of the assay.

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9 MATERIALS AND METHODS Bacterial Strains Escherichia coli O157:H7 was provided by the Massachusetts Department of Public Health State Laboratory Institute (Jam aica Plain, MA), and was used as the target antigen in all specificity and sensitivity studi es. This environmental strain was recovered from taco meat distributed at a county fair which resulted in an outbreak of food poisoning in Massachusetts. The strain was received by our laboratory in December 1998 and was stored at -80C in sterile glycerol A green fluorescent pr otein expressing stock of Escherichia coli O157:H7 ATCC#35150 (GFPE. coli O157:H7) was used in all capture efficiency studies. The 5.4 kb GFP-encoded plasmid encoded ampicillin resistance and was regulated by an arab inose promoter which was activated by specialized media described in the Medi a and Culture Conditions section (36). Escherichia coli K-12 was purchased from the American Type Culture Collection (ATCC#23590). E. coli K-12 was used as a negative control in all specificity and sensitivity studies. Buffers Sodium phosphate-buffered saline, 0.1 M and pH 7.4, (PBS) contained 3.2 g NaH2PO4, 20.6 g Na2HPO4 and 8 g NaCl per liter of filter (0.22 m) sterilized water (Millipore; Billerica, MA). PBS with 0.05% Tween 20 (PBST) was used to remove any unbound reagents during rinsing steps.

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10 Media and Culture Conditions Stock Cultures Cultures of E. coli O157:H7 and E. coli K-12 were grown on tryptic soy agar (TSA) for 18 hours at 37C, then stored at 4C for up to two weeks before being used to prepare sample cultures. GFP expressing E. coli O157:H7 (GFPE. coli O157:H7) was maintained on Luria Bertani (LB) media containing 100 g/ml ampicillin (AMP) and 5 mg/ml arabinose (ARA) (LB+AMP+ARA). Af ter 18 hours of grow th at 37C the LB+AMP+ARA plates were inverted and stored at 4C for up to two weeks before being used to prepare sample broth cultures. All media was purchased from Becton Dickinson (Franklin Lakes, NJ) and was reconstituted and sterilized according to the manufacturers directions. Sample Cultures A single colony from a TSA plate was used to prep are a broth culture of E. coli O157:H7 or E. coli K-12 in tryptic soy broth (T SB). A single colony from an LB+AMP+ARA plate was used to pr epare a broth culture of GFPE. coli O157:H7 in LB+AMP+ARA broth. Broths were purchased from Becton Dickinson (Franklin Lakes, NJ) and were reconstituted and sterilized acco rding to the manufacturers directions. A broth culture used in an expe riment was grown in 10 ml of appropriate broth in a 50 ml conical tube for 18 hours at 37 C with shaking at 200 r.p.m. The culture was then diluted (1:100) in fresh broth and returned to the shaking incubator for 4-6 hours until an optical density at 600 nm (OD600) of 0.6-1.0 was reached. Optical densities were measured using a DU-64 spectrophotometer (Beckman, Fullerton, CA ). The sample culture was serially diluted in PBS for use in an assay. The bacterial dilution was maintained at 24C in PBS

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11 for one hour, and then it was vortexed for twen ty seconds using a Fi sher Vortex Genie 2 (Fisher Scientific; Suwanee, GA) to homoge nize the cell culture. Once a homogeneous solution was reached, the bacterial dilution was added to the assay. The cell concentration for each sample culture was determined using viable count. One hundred microliters of GFPE. coli O157:H7 was plated onto an LB+AMP+ARA plate, then incubated for 18 hours at 37C to allow grow th of colonies. E. coli O157:H7 and K-12 were similarly plated onto TSA to obtain viable counts. Sorbitol-MacConkey agar (SMAC) (Remel; Lenexa, KS) was used to recover and presumptively differentiate between E. coli O157:H7 and K-12 colonies. E. coli O157:H7 was unable to ferment sorbitol and produced colorless colonies on the SMAC plate. Alternatively, E. coli K-12 was able to ferment sorbitol and produced pink or purple colonies on the SMAC plate. Capture Matrix Proteins Albumins Ovalbumin (OVA) and human serum albu min (HSA) fraction V (96-99% purity by agarose gel electrophoresis) was purchased from Sigma-Aldrich (St. Louis, MO). Bovine serum albumin (BSA) was purchased from Fisher Scientific (Suwanee, GA). Lyophilized albumin crystals were rehydr ated in 50% (v/v) glycerol, and 40 l aliquots were stored at -20C in mi crofuge tubes. Working dilutions were prepared in PBS. Protein G and Streptavidin Native Protein G (PG) from S treptococcus species was purchased from Calbiochem (San Diego, CA). Streptavidin and PG lacking the albumin binding domain (recombinant PG) were purchased from SigmaAldrich. These lyophilized proteins were

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12 rehydrated to 1.0 mg/ml in PBS, and 40 l aliquots were stored at -20C in microfuge tubes. Working dilutions were prepared in PBS. Antibodies Lyophilized goat polyclonal antibody raised against E. coli O157:H7 and horse radish peroxidase labeled (HRP) labeled, biot in labeled or unlabele d (KPL; Gaithersburg, MD), was rehydrated in 50% (v /v) glycerol to 1.0 mg/ml. Mouse monoclonal antibodies, isotype IgG3, raised against E. coli O157:H7 were purchased from Abcam (Cambridge, MA), Biodesign Internationa l (Saco, ME), U.S. Biological (Swampscott, MA) and Fitzgerald (Concord, MA), and were dilu ted to 0.1 mg/ml in 50% (v/v) glycerol. Lyophilized mouse monoclonal antibody raised against rabbit or goat immunoglobulin; and HRP labeled, goat polyclonal antibody ra ised against mouse immunoglobulin; and rabbit polyclonal antibody ra ised against goat immunogl obulin; and HRP labeled or unlabeled rabbit (Jackson Imm uno Research; West Grove, PA ), was rehydrated in 50% (v/v) glycerol to 1.0 mg/ml. Lyophilized ra bbit polyclonal antibody raised against PG (Abcam; Cambridge, MA), and lyophilized ch icken polyclonal antibody raised against mouse immunoglobulin and HR P labeled (U.S. Biologica l; Swampscott, MA) was rehydrated in 50% (v/v) glyc erol to 1.0 mg/ml. To avoid protein degradation each antibody was divided into 40 l aliquots which were stored at -20C. Frozen antibody aliquots were used one tim e and never refrozen. Chicke n polyclonal antibody raised against mouse immunoglobulin and HRP labeled (Aves Labs; Tigard, OR) was stored at 4C.

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13 Antibody Labeling Cy5 Antibody Labeling Column A column, used to separate unbound dye from labeled antibody, was prepared twenty four hours before labeling. In a 50 ml conical tube 1.4 g Bi o-Gel P-10 fine (BioRad, Hercules, CA) was saturated with 20 ml PBS containing 0.02% (v/v) NaN3 (PBSNaN3). The gel was allowed to hydrate at 24 C for 4 hours. A 20 ml Econo Pac column (Bio-Rad) was secured to a column stand a nd the end was snapped off. The Bio-Gel mix was thinned by the addition of 40 ml PBS-NaN3. After inverting th e conical tube the slurry was transferred by pipette into the purification column. The gel was allowed to settle and excess buffer was drained. Once the gel settled, a frit was applied on top of the gel bed. A thin layer of PBS-NaN3 was applied to keep the frit moist. The column was then stored at 4C for twenty-four hours. Cy5 Antibody Labeling Procedure Antibody labeling was performed using a cyanine 5 dye (Cy5) labeling kit (FluorolinkCy5Reactive Dye 5-pack, Amer sham Life Sciences; Arlington Heights, IL). Lyophilized goat polycl onal antibody raised against E. coli O157:H7 was rehydrated in sodium carbonate buffer [0.1 M, pH 9.3] to 1.0 mg/ml. Th e antibody solution was transferred to a tube containing Cy5 reactive dye. The tube was capped and protected from light while it was incubated for one hour at 24C. The contents of the reaction tube were transferred by pipette onto the column frit. The contents were flushed from the frit by the addition of PBS, and clear liquid was collected as waste. The first of two blue bands observed moving through column was co llected in an amber microfuge tube as labeled antibody. The second blue band consisted of unbound Cy5, and was discarded as

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waste. The purification column was ri nsed for 30 minutes with PBS-NaN3, then secured and stored at 4C for reuse within three m onths. The concentrations of antibody and Cy5 dye in the labeled product were determin ed by measuring the absorbance at 280 nm (A280) and 650 nm (A650), respectively; and applying the Beer-Lambert Law, which explains the linear relationship between abso rbance and concentration of the absorbing substance. These concentrati ons were also used to determ ine the protein to dye ratio, which was the amount of dye particle s conjugated to each protein molecule. Absorbencies were measured using a DU-64 spectrophotometer (Beckman; Fullerton, CA). Concentration of Antibody (M) = (A 280 (0.05 x A 650 ))_ 1 cm x 170,000 M-1 cm-1 Concentration of Cy5 (M) = (A 650 ) 1 cm x 250,000 M-1 cm-1 Dye to Protein Ratio = [Antibody (M)] [Cy5 (M)] TABLE 2. Equations for Cy5 Labeling of Antibody DyLight Antibody Labeling Procedure Antibody labeling was performed using a DyLight antibody labeling kit (Pierce Biotechnology; Rockford, IL). The labeling buffer [50 mM sodium borate pH 8.8] was prepared by combining 925 l of PBS with 75 l of sodium borate buffer [67 mM]. Five hundred microliters of labeling buffer was a dded to 1.0 mg of lyophilized goat polyclonal 14

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antibody raised against E. coli O157:H7 to obtain a concen tration of 2.0 mg/ml. The rehydrated antibody was transferred to a vial containi ng DyLight reactive dye. The vial was gently inverted for ten seconds, and then centrifuged for thirty seconds to concentrate the protein at the bottom of the vial. The tube was protected from light and incubated for one hour at 24C Two purification spin colu mns were placed inside two collection tubes and four hundred microliters of pur ification resin was added to each of the spin columns. The spin columns were centrifuged for 45 s econds at 1,000 x g to remove excess storage buffer, then the colle ction tubes were replaced by new tubes to collect the purified protein. Labeled antibody was evenly divided into the two spin columns, and then the columns were centrif uged for 45 seconds at 1,000 x g to separate unbound fluorophore from labeled antibody. The c ontents of the tw o collection vials were combined, and the concentrations of antibody and dye in th e labeled product were determined by measuring the absorbance at 280 nm (A280) and 654 nm (A654), respectively. The Beer-Lambert Law was applied to these values. These concentrations were used to determine the protein to dye ratio, which was the amount of dye particles conjugated to each protein molecule. Ab sorbencies were measured using a DU-64 spectrophotometer (Bec kman; Fullerton, CA). Concentration of Antibody = (A 280 (0.0371 x A 654 ))_x Dilution Factor 210,000 M-1 cm-1 Dye to Protein Ratio = (A 654 ) x Dilution Factor [Antibody] x 250,000 M-1 cm-1 TABLE 3. Equations for DyLi ght Labeling of Antibody 15

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16 ELISAs PG Fc Binding Domain Specificity Assays One hundred microliter s of HSA [0.5 or 1.0 g/ml] was added to wells of a 96 well Nunc Immuno plate and allowed to incubate for 18 hours at 4C to determine the functionality and specificity of the Fc binding domain of PG. After incubation, the plate was washed three times with PBST using an ELx50 Auto Strip Washer (Bio-Tek Instruments, Inc.; Winooski, VT). One hundr ed microliters of native PG [0 2.5 g/ml] was added to albumin coated wells and allowe d to incubate for 60 minutes at 24C. The plate was then washed three times with PBST. For competitive ELISAs, mouse monoclonal or goat polyclonal antibodies raised against rabbit immunoglobulin and labeled with HRP was mixed in equal concentrations [0 1.0 g/ml] in a microfuge tube, with unlabeled mouse monoclonal or goat polyclonal antibody raised against E. coli O157:H7. One hundred microliters of the antibody combination was transferred into the ELISA well, and was incubated for 30 minutes at 24C. The plate was then washed three times with PBST. For indirect ELISAs, 100 l of primary antibody [1.0 g/ml], rabbit polyclonal antibody raised against goat im munoglobulin or goat polyclonal antibody raised against E. coli O157:H7, was added to the wells, and was incubated for 30 minutes at 24C. The plate was then washed three times with PBST One hundred microliters of mouse monoclonal antibody ra ised against rabbit or goat immunoglobulin and HRP labeled [0.1 or 0.5 g/ml], was added to the ELISA wells, and was incubated for 30 minutes at 24C. The plate was then washed three times with PBST. A QuantaBlu Fluorogenic Peroxidase Substrat e Kit (Pierce; Rockford, IL ) was used to activate the peroxidase activity of antibodies labeled with HRP. Signals were detected and quantified

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17 using a Spectra Max Gemini XS Microplate Fluorometer (Molecular Devices; Sunnyvale, CA) with excitation, emission, and cuto ff wavelengths set at 340nm, 470nm and 455, respectively. PG Albumin Binding Domain Func tionality and Specificity Assays One hundred microliters of HSA, ovalbumin (OVA) or bovine serum albumin (BSA) [0.5, 1 or 5 g/ml] was added to a 96 well N unc Immuno plate and allowed to incubate for 18 hours at 4C to determine the functionality and specificity of the albumin binding domain of PG. After incubation, the plate was washed three times with PBST using an ELx50 Auto Strip Washer (Bio-Tek Instruments, Inc.; Winooski, VT). One hundred microliters of native or recombinant PG [0-5 g/ml] was added to the albumin coated wells, and allowed to incubate for 8 or 60 minutes at 24C. The plate was then washed three times with PBST. One hundred microliters of primary antibody [1.0 g/ml], rabbit polyclonal antibody raised against PG or goat immunoglobulin was added to wells, and allowed to incubate for 30 minutes at 24 C. The plate was then washed three times with PBST. One hundred microliters of mous e monoclonal antibody ra ised against rabbit immunoglobulin and HRP labeled [0.1 or 0.5 g/ml] was added to wells, and was incubated for 30 minutes at 24C. The plate wa s then washed three times with PBST, and then analyzed for fluorescence using a Quan taBlu Fluorogenic Pe roxidase Substrate Kit (Pierce; Rockford, IL) to activate the peroxidase activity of antibodies labeled with HRP. Signals were detected and quantified using a Spectra Max Gemini XS Microplate Fluorometer (Molecular Devices; Sunnyvale, CA) with excitation, emission, and cutoff wavelengths set at 340nm, 470nm and 455, respectively.

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18 Direct Assays for Detection of E. coli O157:H7 One hundred microliters of HSA [0 80 g/ml] was added to a 96 well Nunc Immuno Plate (Fisher Scientific; Suwanee, GA), and was incubated for 18 hours at 4C to capture E. coli O157:H7 for detection by ELISA. Afte r incubation, the plate was washed three times with PBST using an ELx50 Auto Strip Washer (Bio-Tek Instruments, Inc.; Winooski, VT). Native or recombinant PG [0-10 g/ml] was added to HSA coated wells, and was incubated for 30 minutes at 24C. Th e plate was then washed three times with PBST. Capture antibody [0-5.0 g/ml], goat polyclonal antibody raised against E. coli O157:H7, was added to wells, and was incuba ted for 30 minutes at 24C. The plate was then washed three times with PBST. One hundred microliters of E. coli O157:H7 or K-12 was added to wells, and was incubated for 30 minutes at 24C; and then the plate was washed three times with PBST. One hundred microliters of mouse monoclonal or goat polyclonal antibody, raised against E. coli O157:H7 and HRP labeled [0.1 g/ml], was added to wells, and was incubated for 30 minut es at 24C. The plate was then washed three times with PBST, and analyzed for fluorescence using a QuantaBlu Fluorogenic Peroxidase Substrate Kit (Pier ce; Rockford, IL) to activate the peroxidase activity of antibodies labeled with HRP. Signals were detected and quantified using a Spectra Max Gemini XS Microplate Fluorometer (Molecul ar Devices; Sunnyvale, CA) with ex citation, emission, and cutoff wavelengths set at 340nm, 470nm and 455, respectively. Statistical Analysis A one-way ANOVA with Dunnett's post te st was performed using GraphPad InStat 3.00 (San Diego, CA) for all ELISAs. Pa ired data, in normal Gaussian distribution, was analyzed using a t test with a two tailed P value. Statistically significant P values (<

PAGE 31

19 0.05) were noted on the graphs to aid in the analysis of the data. Raw data was analyzed using Microsoft Excel. RAPTOR ASSAYS Coupon Preparation Four polystyrene waveguides, of approxi mately 38 mm in length, were sonicated in isopropanol for thirty seconds, and then rinsed in 250 ml of water that was filter (0.22 m), and ultraviolet light sterilized, using a Milli-Q Synthesis System (Millipore; Billerica, MA). The waveguides were allowed to dry for 30 minutes with optical heads facing down. Then the distal tip of each wavegui de was coated with matte black ink, to provide a light dump for the 635nm laser beam, and was dried for two hours at 24C. One waveguide was glued into each of the f our channels of the RAPTOR (Research International, Monroe, WA) coupon. The optical glue (Norland Produc ts, Inc; Cranbury, NJ) was dried for 30 minutes using a long-wave length ultraviolet lamp at 24C. Once the glue dried, the coupon was sealed in a small storage bag and stored at 24C for one to thirty days. Waveguide Preparation Twenty-four hours before a RAPTOR assay was performed 100 l of HSA [100 g/ml] was added to two of the waveguides in a coupon, while the other two waveguides in the coupon were coated with 100 l of streptavidin [100 g/ml]. The treated waveguides were incubated for 18 hours at 4C, and then an y excess protein was removed from the coupon by rinsing each waveguide three times with PBST. One hundred microliters of PG [50 g/ml] was added to the HSA treat ed waveguides and allowed to incubate for 15 minutes at 24C. Any unbound PG was aspirated from the waveguides,

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20 and fresh PG was added for a second 15 minut e incubation. HSA-PG and streptavidin coated waveguides were rinsed three times with PBST. Goat polyclonal antibody raised against E. coli O157:H7 [50 g/ml] was added to the HSAPG treated waveguides; and goat polyclonal antibody raised against E. coli O157:H7 and biotin labeled [50 g/ml] was added to streptavidin treated waveguide s. Antibody solutions were incubated for 30 minutes at 24C. Any unbound an tibody was aspirated from th e waveguides, and fresh antibody was added for a second 30 minute incubation. Each waveguide was rinsed three times with PBST. Once the waveguides in the coupon were treated with the appropriate capture matrix, the back side of the coupon was sealed with tape to prev ent fluid leakage, and to maintain vacuum pressure inside the individual channels. To operate the biosensor, each RAPTOR assay was an automated function defined by a unique recipe, which was encoded by a certain number ranging from 0 to 63. The biosensor determined which recipe to follow based on a recipe card th at was attached to the coupon, and marked with the appropriate recipe number. To avoid channe l-related bias in the data, the position of the differently treated waveguides was alternated for each assay replicate. RAPTOR Assay Procedure The RAPTOR biosensor was assembled by connecting a piece of tubing to the buffer inlet and placing the other end in a c ontainer of PBST. Detector antibody, 1.0 ml, was placed in each of four reagent vials, and then tubing was used to connect the reagent vials with the reagen t ports on the biosensor. A previously assembled coupon was placed securely into the biosensor, and the assay pr otocol was then commenced. A series of four blank samples consisting of 2.0 ml of PBS, injected into the sample port, were sequentially assayed to determine the bac kground signal. The thirty-two minute assay

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21 consisted of a 500 l sample pulsed twelve times (40 l of sample per pulse) over each of the four waveguides, with a two minut e incubation period between pulses. The waveguides were then rinsed for 30 seconds with PBST. Two hundred microliters of detector antibody was pumped into each of the four waveguide channels and allowed to incubate for two minutes. After the incuba tion, the reagent pump was reversed to withdraw the detector antibody back into th e reagent tubes. The waveguides were then rinsed twice for 30 seconds with PBST. Detection of the target was measur ed by fluorescence of the immune bound detector antibody, goat polyclonal antibody raised against E. coli O157:H7 and Cy5 or DyLight labeled [5.0 g/ml], for direct sandwich assa ys. After the baselines were determined, samples containing E. coli O157:H7 were interrogated by the capture matrix. Fluorescence emissions, within 100 1000 nm of the waveguide surface, were measured in picoamperes (pA) by a photodiode able to collect and quantitate emitted wavelengths above 650 nm. Blank samples were immediately followe d by bacterial samples consisting of E. coli O157:H7 [1 x 102-7 CFU/ml], beginning with the lowe st concentration, for limit of detection assays. Typically, thr ee to four bacterial sample concentrations were tested per RAPTOR coupon. For capture efficiency as says, blank samples were immediately followed by one bacterial sample consisting of GFPE. coli O157:H7 [1 x 106-8 CFU/ml]. Direct counts, using a Cellometer slide (N excelom Bioscience; Lawrence, MA), were used to determine sample concentrations (ce lls/ml) before the assays were performed. For data analysis, viable counts were performe d on TSA or LB+AMP+ARA respectively, to determine sample concentrations (CFU/ml) retroactively. All RAPTOR data was

PAGE 34

22 analyzed by Microsoft Excel. For capture e fficiency experiments, assayed coupons were sealed in a plastic bag and st ored at 4C for 24 to 72 hour s until analyzed by fluorescent microscopy. Determination of Capture Efficiencies Cells captured on the waveguide were manually counted usi ng a UIS2 LUCPlan FLM 20X long range objective mounted on an Olympus BX60 Epifluorescent microscope (Olympus America Inc.; Center Valley, PA). The optic al head of each waveguide was carefully removed using a sterile razor blade. The waveguide was then secured to a clean glass slid e using craft glue. After the glue dried (~2 minutes) photographs were taken using a SPOT Flex color CCD camera (Diagnostic Instruments Inc.;Sterling Heights, MI) and imaged using Adobe Photoshop Basic (Adobe Systems Inc; San Jose, CA). Cells were counted and averaged using three images per waveguide. Each viewable field represen ted 1 mm of waveguide length. Only 46 out of 360 degrees of the waveguide surface were viewable, so the average number of cells counted was multiplied by a correction factor (7 .8 = 360 / 46) to achieve the number of cells per mm of each waveguide. As depicted in Table 4, the number of cells counted per mm was multiplied by the length of the wave guide, 38mm, to produce the number of cells present on the surface of the entire waveguide.

PAGE 35

23 = (Number of Cells on Three WGs / 3) ( 360/46 Viewable Angl e) Length of WG = Average Number of Ce lls per mm 7.8 38 mm = Average Number of Cells per Waveguide Capture Efficiency = Average Number of Cells per Waveguide Sample Concentration (CFU/ml) TABLE 4. Capture Effici ency Calculations Detection of E. coli O157:H7 in Food Samples Ten grams of spinach or ground beef (20% fat) obtained from local grocers was placed in a Whirl-Pak filter bag (Nasco; Fo rt Atkinson, WI) and inoculated with 1.0 ml of E. coli O157:H7 in PBS, and stored for 18 hour s at 4C. Fifty milliliters of buffered peptone water (BPW) was added to the filter bag, and then the samples were homogenized for thirty seconds using a Pulsifier (Microbiol ogy International; Frederick, MD). The supernatant was removed and tran sferred to a 50 ml c onical tube. Serial dilutions of the supernatant we re prepared in BPW, and were used in the assay. The homogenized samples were assayed as de scribed in the RAPTOR Assay Procedure section. Data Analysis Signal above the limit of detection (SAL OD) values were calculated for each RAPTOR assay. The SALOD values were determined based on a method used to normalize the waveguides within each coupon. Each coupon contained four waveguides, and was considered an independent assay. Base lines were performed as described in the RAPTOR Assay Procedure secti on. Table 5 represents typi cal baseline values from a

PAGE 36

24 RAPTOR assay. The values in bold were th e lowest value for each baseline. Typically, the same waveguide produced all four of the lowest baseline values for each assay. Waveguide 1 Waveguide 2 Waveguide 3 Waveguide 4 Baseline 1 672.7 584.7 576.1 402.8 Baseline 2 703.4 594.4 617.9 433.6 Baseline 3 731.0 612.9 641.7 465.5 Baseline 4 763.8 623.1 675.8 489.6 TABLE 5. Baseline Values The four waveguide values were divided by the bolded value to achieve a normalization coefficient, for each baseline. Table 8 shows the normalization coefficients for the baseline values from Table 5. Waveguide 1 Waveguide 2 Waveguide 3 Waveguide 4 Baseline 1 1.7 1.5 1.4 1.0 Baseline 2 1.6 1.4 1.4 1.0 Baseline 3 1.6 1.3 1.4 1.0 Baseline 4 1.6 1.3 1.4 1.0 TABLE 6. Normalization Coefficients The normalization coefficients, from Table 6, were used to normalize the baseline values from Table 5. For each waveguide, th e baseline values from Table 5 were divided by the normalization coefficient for Baselin e 4 in Table 6. Typically, the normalization coefficients did not vary between the thir d and fourth baseline. Table 7 shows the normalized baseline values.

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25 Waveguide 1 Waveguide 2 Waveguide 3 Waveguide 4 Baseline 1 431.2 459.4 417.4 402.8 Baseline 2 450.9 467.0 447.7 433.6 Baseline 3 468.6 481.6 464.9 465.5 Baseline 4 489.6 489.6 489.6 489.6 TABLE 7. Normalized Baseline Values The normalized baselines, in Table 7, were evaluated for variability. For each waveguide, the four normalized baseline values were averaged and the standard deviation was calculated. A coefficient of variation (CoV) was determined by dividing the average normalized baseline value by the standard deviation. The CoV was then multiplied by 100 to achieve a percentage. Data from any waveguide with a CoV percentage greater than ten was not used for data analysis or graph construction. Table 8 shows that all of the waveguides had baseline values with minimal variability. Waveguide 1 Waveguide 2 Waveguide 3 Waveguide 4 Average 460.1 474.4 454.9 447.9 STDEV 24.9 13.7 30.4 37.8 CoV (%) 5.4 2.9 6.7 8.4 TABLE 8. Normalized Baseline Variability The limit of detection was determined to be the sum of the average normalized baseline plus three times the standard deviation, for each waveguide. Table 9 shows the level of detection for a typical RAPTOR assay.

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26 Waveguide 1 Waveguide 2 Waveguide 3 Waveguide 4 LOD 534.8 515.4 546.0 561.3 TABLE 9. Limit of Detection Once the LOD was calculated, the bacterial samples were assayed as described in the RAPTOR Assay Procedure section. Table 10 shows the sample values for a typical RAPTOR assay used to detect a series of E. coli O157:H7 concentrations. (CFU/ml) Waveguide 1 Waveguide 2 Waveguide 3 Waveguide 4 1.4 x 102 796.0 637.5 721.2 519.8 1.4 x 103 860.1 671.9 765.6 546.0 1.4 x 104 1286.5 956.0 1050.0 782.6 TABLE 10. Bacterial Sample Values The bacterial sample values were also normalized. For each waveguide, the values in Table 10 were divided by the normali zation coefficients for Baseline 4 in Table 6. Table 11 shows the bacterial samp le values after normalization. (CFU/ml) Waveguide 1 Waveguide 2 Waveguide 3 Waveguide 4 1.4 x 102 510.2 500.9 522.5 519.8 1.4 x 103 551.3 527.9 554.7 546.0 1.4 x 104 824.7 751.2 760.7 782.6 TABLE 11. Normalized Bact erial Sample Values

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27 The level of detection value from Tabl e 9 was subtracted from the bacterial sample value in Table 10 to determine if the normalized bacterial sample value was positive or negative for the detection of E. coli O157:H7, for each waveguide. (CFU/ml) Waveguide 1 Waveguide 2 Waveguide 3 Waveguide 4 1.4 x 102 -24.6 -14.5 -23.5 -41.5 1.4 x 103 16.5 12.5 8.7 -15.3 1.4 x 104 289.8 235.7 214.7 221.3 TABLE 12. Final Detection Values Table 12 shows the finalized va lues for the detection of E. coli O157:H7 by the RAPTOR biosensor. A positive remainder wa s considered a positiv e detection of the target bacterium. A negative remainder indi cated a negative detection of the target bacterium. For this example of a typical RAPTOR assay, the lowest concentration of E. coli O157:H7 detected was 1.4 x 103 CFU/ml for waveguide s 1, 2 and 3; while waveguide 4 detected E. coli O157:H7 at 1.4 x 104 CFU/ml. Statistical Analysis A one-way ANOVA with Dunnett's post te st was performed using GraphPad InStat 3.00 (San Diego, CA), for all RAPTOR assays. Paired data, in normal Gaussian distribution, was analyzed using a t test with a two tailed P value. Statistically significant P values (< 0.05) were noted on the graphs to aid in the an alysis of the data. Raw data was analyzed using Microsoft Excel.

PAGE 40

RESULTS AND ANALYSIS Functional Albumin Binding Domain Indirect ELISAs were performed as de scribed in the Materials and Methods section to verify the presence and functionali ty of the albumin binding domain in native PG, and a lack of albumin binding by recombinant PG. 0.0 g/ml HSA 0.5 g/ml HSA 1.0 g/ml HSA 0.0 g/ml HSA 0.5 g/ml HSA 1.0 g/ml HSA PG 0.0 g/mL 1 01 01 01 01 01 0 PG 0.25 g/m L 1.0 7.2 11.1 1.1 1.0 1.0 PG 0.5 g/mL 1.1 10.1 14.7 1.0 0.9 0.9 PG 1 g/mL 1.0 9.5 14.1 1.0 0.9 1.0 PG 2.5 g/mL 1.0 10.0 14.3 1.0 0.9 0.9 PG 5 g/mL 0.9 9.2 13.6 0.8 0.8 0.9 PG 10 g/mL 1.1 9.4 12.6 0.9 0.8 0.9 PG 20 g/mL 1.1 9.3 12.5 0.9 0.9 1.0 0 2 4 6 8 10 12 14 16 18 20Signal to Noise RatioNative Protein G Recombinant ProteinG FIGURE 4. Verification of a Functional Album in Binding Domain in Native PG Using ELISA Analysis. Rabbit polyclonal antibody [1.0 g/ml], raised against goat immunoglobulin, was used as the primary antibody; while mouse monoclonal antibody [0.1 g/ml], raised against rabbit immunoglobu lin, and HRP labeled, was used as the secondary antibody. HSA (shown on graph) wa s used to immobilize PG (shown on 28

PAGE 41

29 graph) before antibodies were added to the assay. Each column, and coinciding standard deviation bar, was calculated from the averag e of six data points; which were collected from three independent assays. A signal to noise ratio less than 2 wa s produced by all wells that contained recombinant PG (Figure 4). These low signals indicated the inability of recombinant PG to bind HSA. A signal to noise ratio greater than 2 was produced by all wells that contained native PG, which indicated th at PG was bound to HSA. Native PG was implemented in all future assays for albumin binding. Alternative Albumin Species HSA was compared to other species of albumin to determine if it was the optimal species to immobilize PG in the capture ma trix. Chicken ovalbumin (OVA) and bovine serum albumin (BSA) were tested as altern atives to HSA. Bindi ng to PG, by OVA, BSA and HSA, was measured using indirect ELI SAs as described in the Materials and Methods section.

PAGE 42

1 g/ml OVA5 g/ml OVA1 g/ml BSA5 g/ml BSA1 g/ml HSA5 g/ml HSA 0.25 g/ml PG 1.1 1.0 1.2 1.5 9.9 13.2 0.5 g/ml PG 1.1 1.1 1.2 1.9 11.414.7 1 g/ml PG 1.3 1.2 1.3 2.5 12.316.3 2.5 g/ml PG 1.2 1.2 1.6 3.2 12.417.1 5 g/ml PG 1.2 1.3 1.9 3.9 12.416.8 0 2 4 6 8 10 12 14 16 18 20Signal to Noise Ratio FIGURE 5. Verification of Specificity of Different Albumin Species for PG Using ELISA Analysis. Rabbit polyclonal antibody [1.0 g/ml], raised against PG, was used as the primary antibody; while m ouse monoclonal antibody [0.5 g/ml], raised against rabbit immunoglobulin, and HRP labeled, was used as the secondary antibody. OVA, BSA, or HSA (shown on graph) was used to immobilize PG (shown on graph) before antibodies were added to the assay. Each co lumn, and coinciding standard deviation bar, was calculated from the average of six data points; which were collected from three independent assays. A signal to noise ratio greater than 2 was produced only by wells that contained HSA at 1.0 and 5.0 g/ml, and for BSA at 5.0 g/ml (Figure 5). A signal to noise ratio less than 2 was produced by wells that contained OVA at 1.0 and 5.0 g/ml, and for BSA at 1.0 g/ml. The low signal to noise ratio pr oduced from OVA indicated a lack of binding between PG and OVA. A high concentr ation of BSA was required to produce a signal to noise ratio greater th an 2, when compared to HSA. These data indicated that 30

PAGE 43

BSA bound PG, but only at high BSA concentr ations. A minimal concentration of HSA produced high signal to noise ratio (> 8). The signal to noise ratio for the lowest concentration of HSA-PG (9.9) was nearly th ree times the signal to noise ratio for the highest concentration of BSAPG (3.9). These data indica ted that the albumin binding domain of PG had greater affinity for HSA. These results indicated that HSA was the optimal albumin species for use in the alternative capture matrix. HSA was implemented as the albumin used to immobilize PG in all future assays. Optimal Ratio of HSA to PG A direct sandwich ELISA was performe d as described in the Materials and Methods section to determine the optimal ra tio of HSA to PG for use in the capture matrix. E. coli O157:H7 was used to evaluate the capture of bacteria at the various concentrations of HSA and PG tested. 15.9 14.0 9.6 10.0 8.6 11.1 8.9 7.6 7.5 6.4 10.2 8.0 6.5 6.6 5.8 0 2 4 6 8 10 12 14 16 18 20 0.5 1.0 2.5 5.0 10.0Signal to Noise RatioProtein G ( g/ml) 1.0 g/ml HSA 5.0 g/ml HSA 10.0 g/ml HSAFIGURE 6. Verification of the Optimal Wo rking Ratio of HSA to PG Using ELISA Analysis. Goat polyclonal antibody [1.0 g/ml], raised against E. coli O157:H7, was used 31

PAGE 44

32 as the primary antibody. Goat polyclonal antibody [1.0 g/ml], raised against E. coli O157:H7 and HRP labeled, was used as the secondary antibody to detect captured E. coli O157:H7 at 8.1 x 107 CFU/ml. HSA (shown on graph) was used to immobilize PG (shown on graph) before antibodies were added to the assay. Each column, and coinciding standard deviation bar, was calcul ated from the average of two data points; which were collected from one assay. A signal to noise ratio great er than 2 was produced by all wells that contained HSA and PG (Figure 6). The signal to noise ra tio was greatest when the concentration of PG was 0.5 g/ml and HSA was 1.0 g/ml. The signal to noise ratio decreased as the concentration of both HSA a nd PG increased. These data suggested that the optimal working ratio of HSA to PG was 2:1. This ratio was implemented in all future assays. The Role of HSA in the Alternative Capture Matrix Indirect ELISAs were performed as de scribed in the Materials and Methods section to clarify the role of HSA in the ca pture matrix. The immobilization of antibodies by PG alone, or in combination with HSA, wa s compared to determine the importance of HSA in the alternative capture matrix.

PAGE 45

145.4 143.0 1820.3 0 500 1000 1500 2000 2500 1.0 g/ml HSA 1.0 g/ml PG 1.0 g/ml HSA and PGRaw Fluorescence (pA)----------P< 0.0001 ----------FIGURE 7. The Role of HSA in the Alternative Capture Matrix Clarified Using ELISA Analysis. Rabbit polyclonal antibody [1.0 g/ml], raised against goat immunoglobulin, was used as the primary antibody; while mouse monoclonal antibody [0.1 g/ml], raised against rabbit immunoglobulin a nd HRP labeled, was used as the secondary antibody. Each column, and coinciding standard deviatio n bar, was calculated from the average of six data points; which were collected from three independent assays. Minimal fluorescence was produced by we lls that contained HSA alone (145.4 pA) and PG alone (143.0 pA) (Figure 7). Thes e data indicated th at the primary or secondary antibodies did not bind to th e HSA alone or PG alone. The greatest fluorescence was produced by wells that cont ained HSA and PG (1820.3 pA) in complex. The combination of HSA and PG significan tly enhanced the fluorescence signal when compared to HSA alone and PG alone. The pr imary and secondary antibodies used in the assay were not specific for PG. However, these nonspecific antibodies produced the greatest fluorescence (1820.3 pA) when used to label PG, which was immobilized by HSA. This suggested that the PG Fc binding domain bound to the Fc domain of at least 33

PAGE 46

one of the antibodies used. As indicated by th ese data, an enhanced ability to immobilize antibodies existed when PG was oriented on a HSA coated surface. The Role of HSA in Capturing Bacteria Direct sandwich ELISAs were performe d as described in the Materials and Methods section to clarify the role of HSA in the capture of bacteria. E. coli O157:H7 was used to show the capture of bacteria by HSA, PG or a combination of HSA and PG. 172.1 361.5 2897.1 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 1 g/ml HSA 1 g/ml PG 1 g/ml HSA and 1 g/ml PG Raw Fluorescence (pA)----------P< 0.0001 --------------------P< 0.0001 -----------FIGURE 8. The Role of HSA in Capturing Bacteria Clarified Us ing ELISA Analysis. Goat polyclonal antibody [1.0 g/ml], raised against E. coli O157:H7, was used as the capture antibody and primary detector antibody. Mouse monoclonal antibody [0.1 g/ml], raised against goat immunoglobulin and HRP labe led, was used as the secondary detector antibody to detect captured E. coli O157:H7 at 4.7 x 106 CFU/ml. Each column, and coinciding standard deviation bar, was calculated from the average of six data points; which were collected from th ree independent assays. 34

PAGE 47

35 Minimal fluorescence (172.1 pA) was produc ed by wells that contained HSA alone, which indicated that bacteria was not captured by HSA (Figure 11). Significantly (P < 0.05) elevated fluorescence was produced by wells that contained PG alone (361.5 pA). The increased fluorescence by PG alone was minor, compared to the fluorescence produced by wells that contained the HS A and PG complex (2897.1 pA). PG, in combination with HSA, was able capture bacteria significantly (P < 0.05) better than HSA or PG alone. The HSA-PG combination wa s implemented in all future assays. Species Specificity of th e Fc Binding Domain The species specificity of the PG Fc binding domain was explored using competitive ELISAs as described in the Materials and Methods section. One species of antibody, labeled with HRP, was forced to compete for the Fc binding domain on PG with an equal concentration of unlabel led antibody from another species. This competition was performed to demonstrate th e strong affinity of the PG Fc binding domain for goat antibodies, and a lesser affi nity for mouse antibod ies, which had been shown in previous research (1, 4, 14,).

PAGE 48

7.0 7.0 13.0 13.7 15.2 15.2 0 2 4 6 8 10 12 14 16 18 20 0.00.51.00.00.51.0Signal to Noise RatioProtein G ( g/ml) 0.25 g/ml IgG 0.5 g/ml IgG 1.0 g/ml IgG HRP Goat Anti-Rabbit versus Mouse AntiE. coli O157:H7 HRP Mouse Anti-Rabbit versus Goat AntiE. coli O157:H7 FIGURE 9. Verification of Species Specificity for the Fc Binding Domain of PG Using ELISA Analysis. Goat polyclonal antibody [1.0 g/ml], raised against E. coli O157:H7 and unlabeled, or raised against rabbit im munoglobulin and HRP labeled, was used to compete with mouse monoclonal antibodies for the Fc binding domain of PG [1.0 g/ml], raised against rabbit immunoglobulin a nd HRP labeled, or raised against E. coli O157:H7 and unlabeled. HSA [1.0 g/ml] was used to immobilize PG before the antibodies were added. Each column, and coinciding standard deviation bar, was calculated from the average of six data points; which were coll ected from three independent assays. Signal to noise ratios greater than 2 were produced by a ll wells that contained PG and HRP labeled goat polycl onal antibody raised against rabbit immunoglobulin, with unlabeled mouse monoclona l antibody raised against E. coli O157:H7. This data indicated that the goat species antibody was the stronger competitor for the Fc binding domain of PG (Figure 9). Signal to noise ratios less than 2 were produced by all wells that contained HRP labeled mouse monoc lonal antibody raised against rabbit immunoglobulin, with unlabeled goat pol yclonal antibody raised against E. coli 36

PAGE 49

O157:H7. This data indicated th at the Fc binding domain of PG had minimal affinity for the mouse species antibody. These data corroborated the strong affinity of PG Fc binding domain for the goat antibodies, and the weak binding of mouse an tibodies to PG, as stated in previous studies (1, 4, 14). Optimal Capture Antibody Species The abilities of rabbit and goat polyclonal antibodies to bind to the Fc binding domain on PG were compared to determine the optimal species of capture antibody for use in the alternative capture matrix. Indirect ELISAs were performed as described in the Materials and Methods section. 1.0 1.0 1.0 1.0 1.0 1.0 1.0 3.1 6.7 1.2 4.7 8.0 0 2 4 6 8 10 12 14 16 18 20 0.0 0.5 1.0Signal to Noise RatioHuman Serum Albumin ( g/ml) 0.0 PG g/ml 0.0 PG g/ml 0.5 PG g/ml 0.5 PG g/ml--P < 0.01---P < 0.05-FIGURE 10. Determination of the Optimal Capture Antibody Species Using ELISA Analysis. Goat polyclonal antibody ( / ), raised against E. coli O157:H7, or rabbit polyclonal antibody ( / ) raised against goa t immunoglobulin, were used as primary antibodies [1.0 g/ml]. Mouse monoclonal antibodies raised against goat or 37

PAGE 50

38 rabbit immunoglobulin, and HRP labeled, were used as the secondary antibodies [0.1 g/ml], respectively. HSA (shown on graph) was used to immobilize PG (shown on graph) before antibodies were added to the assay. Each column, and coinciding standard deviation bar, was calculated from the averag e of six data points; which were collected from three independent assays. Signal to noise ratios greater than 2 were produced by a ll wells that contained PG and HSA. These data indicated that rabbit and goat polyclo nal antibodies bound to the Fc binding domain of PG in the alternative cap ture matrix (Figure 10). Goat polyclonal antibody raised against E. coli O157:H7 produced significan tly (P < 0.05) higher signal to noise ratios when compared to rabb it polyclonal antibody raised against goat immunoglobulin. These data suggested that goa t species antibody was the most effective capture antibody in the alterna tive capture matrix, when compared to rabbit species antibody. The rabbit species antibody bound to the Fc binding domain of PG, which suggested that it may be effective as a cap ture antibody if goat species antibody was not readily available for a particular target. Goat polyclonal antibody raised against E. coli O157:H7 was implemented as the ca pture antibody in further assays. Optimal Concentration of Detector Antibody Direct sandwich ELISAs were performe d as described in the Materials and Methods section. Goat polyclonal antibody, raised against E. coli O157:H7 and HRP labeled, was used to determine the optimal concentration of detector antibody for the alternative capture matrix.

PAGE 51

6.0 4.2 3.1 5.9 4.2 3.1 5.4 4.1 3.1 2.5 2.1 1.7 1.0 1.0 1.0 0.9 0.9 0.8 0 1 2 3 4 5 6 7 1.0 2.5 5.0Signal to Noise RatioDetector Antibody ( g/ml) O157:H7 3.0x10^8 O157:H7 3.0x10^7 O157:H7 3.0x10^6 O157:H7 3.0x10^5 No Cells K-12 1.5x10^7FIGURE 11. Determination of the Optimal Concentration of Detector Antibody Using ELISA Analysis. Goat polyclonal antibody [1.0 g/ml], raised against E. coli O157:H7, was used as the capture antibody. Goat polyclonal antibody [1.0, 2.5, 5.0 g/ml], raised against E. coli O157:H7 and HRP labeled, was used as the detector antibody to detect captured E. coli O157:H7 (CFU/ml shown on graph). E. coli K-12 [1.5 x 107 CFU/ml] was used as a negative control. HSA [1.0 g/ml] was used to immobilize PG [0.5 g/ml] before antibodies were added to the assa y. Each column, and coinciding standard deviation bar, was calculated from the averag e of six data points; which were collected from three independent assays. Signal to noise ratios great er than 2 were produced by all wells that contained E. coli O157:H7 at 3.0 x 106 through 3.0 x 108 CFU/ml (Figure 11). Signal to noise ratios decreased when the concentration of detect or antibody increased. These data suggested that saturation of PG began at, or before, 1.0 g/ml of detector antibody. Based on the greatest signal to noise ratio, the optimal concentration of detector antibody was 1.0 g/ml or less. 39

PAGE 52

Capture Antibody Displacement Direct sandwich ELISAs were performe d as described in the Materials and Methods section. The absence of E. coli O157:H7 was used to determine if the detector antibody, goat polyclonal antibody raised against E. coli O157:H7 and HRP labeled, had displaced the capture antibody, goat polyclonal antibody raised against E. coli O157:H7, for the Fc binding domain on PG. 88.3 2954.0 190.3 6259.9 286.4 7387.3 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 No Bacteria (PBS Only) E. coli O157:H7Raw Fluorescence (pA) 0.1 g/ml HRP IgG 0.5 g/ml HRP IgG 1.0 g/ml HRP IgGFIGURE 12. Capture Antibody Displacement Ev aluated Using ELISA Analysis. Goat polyclonal antibody [1.0 g/ml], raised against E. coli O157:H7, was used as the capture antibody. Goat polyclonal antibody [0.1, 0.5 or 1.0 g/ml], raised against E. coli O157:H7 and HRP labeled, was used as the detector antibody, to detect captured E. coli O157:H7 [9.7 x 106 CFU/ml]. The absence of bacteria was used as the negative control. HSA [1.0 g/ml] was used to immobilize PG [0.5 g/ml] before antibodies were added to the assay. Each column, and coinciding standa rd deviation bar, was calculated from the average of six data points; which were coll ected from three independent assays. 40 Fluorescence above 2000 pA was produ ced by all wells that contained E. coli O157:H7 (Figure 12). These data indicated that the alternative captu re matrix captured

PAGE 53

the targeted cells. In the presence of bact eria, fluorescence signals were not increased significantly (P < 0.05) when th e concentration of detector antibody was increased. This lack of signal increa se suggested that the antigen bindi ng sites were saturated by detector antibody at 0.5 g/ml. Minimal fluorescence (< 700 pA ) was produced by all wells that lacked bacteria. These data indicated that the detector antibody, at 0.5 and 1.0 g/ml, did not bind to PG. This lack of binding suggested that the dete ctor antibody di d not displace the capture antibody from the Fc binding domai n of PG. Goat polyc lonal antibody raised against E. coli O157:H7 and HRP labeled was implem ented as the detector antibody in all future ELISAs. Limit of Detection for Two Captur e Matrices Using ELISA Analysis Direct sandwich ELISAs were performe d as described in the Materials and Methods section. The streptavidin-biotin and HSA-PG capture matrices were compared to determine the limit of detection for E. coli O157:H7. 0.8 0.7 0.8 2.4 15.4 20.4 19.4 0.8 0.8 0.9 4.0 21.5 26.3 23.4 0 5 10 15 20 25 30 35 Signal to Noise RatioE. coli (CFU/ml) Streptavidin-Biotin-IgG HSA-Protein G-IgG-P < 0.05-P < 0.05-P < 0.005-P < 0.05-FIGURE 13. Determination of the Limit of Detection for Two Capture Matrices Using ELISA Analysis. Goat polyclonal antibodies [1.0 g/ml], raised against E. coli O157:H7 41

PAGE 54

42 and unlabeled or biotinylated, were used as the capture an tibody. Goat polyclonal antibody [1.0 g/ml], raised against E. coli O157:H7 and HRP labeled, was used as the detector antibody, to detect captured E. coli O157:H7 (CFU/ml shown on graph). E. coli K-12 (CFU/ml shown on graph) was used as the negative control. HSA [1.0 g/ml] was used to immobilize PG [0.5 g/ml], before antibodies were added to the HSA-PG matrix. Streptavidin [1.0 g/ml] was used to immobilize the bi otinylated capture antibody, before bacteria were added to the assay. Each co lumn, and coinciding standard deviation bar, was calculated from the average of six data points; which were collected from three independent assays. Signal to noise ratios less than 2 were produced by all wells that contained the negative control, E. coli K-12. These data indicated that E. coli K-12 was not detected by either capture matrix. Signal to noise ratios greater than 2 were produced by all wells that contained E. coli O157:H7, from 9.7 x 104 through 1 x 107 CFU/ml. This high signal to noise ratio indicated that the limit of detection for E. coli O157:H7 was 9.7 x 104 CFU/ml for both capture matrices using ELISA anal ysis. The HSA-PG capture matrix produced signal to noise ratios that were significantly higher than the streptavidin-biotin capture matrix, at every concentration of E. coli O157:H7. These data sugge sted that the HSA-PG capture matrix was a better method for the cap ture of target bacter ial cells using ELISA analysis.

PAGE 55

The Limit of Detection of Two Capture Matrices Using RAPTOR RAPTOR assays, using a sa ndwich assay format, were pe rformed as described in the Materials and Methods section. Experiment s were conducted to compare the detection limit of the streptavidin-biotin-IgG capture matrix to the HSA-PG-IgG capture matrix. GFPE. coli O157:H7 was used to show the capture of bacteria by these matrices. -21.0 11.4 184.4 2074.6 3777.2 -10.9 12.1 166.6 2476.1 4586.7 -1000 0 1000 2000 3000 4000 5000 6000 1.9x10^21.9x10^31.4x10^41.0x10^51.3x10^6Signal Above Limit of DetectionGFPE. coli O157:H7 (CFU/ml) Streptavidin-Biotin-IgG HSA-PG-IgG ----P < 0.05-------P < 0.05----FIGURE 14. Determination of the Limit of Detection for Two Capture Matrices Using RAPTOR Analysis. Goat polyclonal antibodies [50.0 g/ml], raised against E. coli O157:H7 and unlabeled or biotinylated, were used as the capture antibody. Goat polyclonal antibody [5.0 g/ml], raised against E. coli O157:H7 and Cy5 labeled, was used as the detector anti body, to detect captured GFPE. coli O157:H7 (CFU/ml shown on graph). HSA [100.0 g/ml] was used to immobilize PG [50.0 g/ml], before antibodies were added to the HS A-PG matrix. Streptavidin [100.0 g/ml] was used to immobilize the biotinylated cap ture antibody, before bacteria were added to the assay. Each column, and coinciding standard deviatio n bar, was calculated from the average of two data points from 1.0 x 105 and 1.0 x 106 CFU/ml, six data points from 1.4 x 104 43

PAGE 56

44 CFU/ml, and eight data points from 1.9 x 102 and 1.9 x 103 CFU/ml; which were collected from five independent assays. Positive SALOD values were produced by waveguides used to assay 1.9 x 103 through 1.3 x 106 CFU/ml of GFPE. coli O157:H7 (Figure 14). Thes e data indicated that the limit of detection fo r both matrices was 1.9 x 103 CFU/ml. No significant difference was observed between the two matrices at 1.9 x 102 through 1.4 x 104 CFU/ml of GFPE. coli O157:H7. These data indicated that both ma trices captured the target bacteria for detection below 1.0 x 105 CFU/ml, and produced similar SALOD values. The HSA-PG capture matrix produced significantly (P < 0.05) greater SALOD values at 1.0 x 105 through 1.3 x 106 CFU/ml; when compared to the st reptavidin-biotin capture matrix. These data suggested that the HSA-PG capture matrix was the better method for capture of target bacterial cells at high concentrations us ing the RAPTOR. The Limit of Detection for Ground Beef Homogenate Supernatant RAPTOR assays, using a sandwich format with direct detection, were performed as described in the Materials and Methods section. Ground beef homogenate supernatant fluid, containing E. coli O157:H7, was used to compare the HSA-PG and streptavidinbiotin capture matrices for use in an animal-based food sample.

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-23.0 -27.2 60.3 199.1 288.8 372.5 647.4 -200 0 200 400 600 800 1000 1200 1400 Streptavidin-Biotin HSA-PGSignal Above Limit of Detection O157:H7 7.0 x 10^4 O157:H7 5.4 x 10^5 O157:H7 5.4 x 10^6 O157:H7 3.8 x 10^7 FIGURE 15. Determination of the Limit of Detection for Two Capture Matrices in Homogenized Ground Beef Supernatant Fluid Using RAPTOR Analysis. Goat polyclonal antibodies [50.0 g/ml], raised against E. coli O157:H7 and unlabeled or biotinylated, was used as the capture antibody. Goat polyclonal antibody [5.0 g/ml], raised against E. coli O157:H7 and Cy5 labeled, was used as the detector antibody, to detect captured E. coli O157:H7 (CFU/ml shown on graph). HSA [100.0 g/ml] was used to immobilize PG [50.0 g/ml], before antibodies were added to the HSA-PG matrix. Streptavidin [100.0 g/ml] was used to immobilize the biotinylat ed capture antibody before bacteria were added to the assay. Each column, and coincidi ng standard deviation bar for the HSA-PG capture matrix, was calculated from the average of two data points for 7.0 x 104 CFU/ml, seven data points from 5.4 x 105 and 5.4 x 106 CFU/ml, and three da ta points for 3.8 x 107 CFU/ml; which were collected from three independent assays. Each column, and coinciding standard deviation bar for the streptavidin-biotin capture matrix, was calculated from the average of three data points; which were collected from three independent assays. The streptavidin-biotin cap ture matrix was not tested at the 7.0 x 104 CFU/ml sample concentration. 45

PAGE 58

Positive SALOD values were produced by all waveguides treated with the HSAPG and 5.4 x 105 CFU/ml of E. coli O157:H7 (Figure 15). All waveguides treated with streptavidin-biotin produced pos itive SALOD values at 5.4 x 106 CFU/ml of E. coli O157:H7; which was one log less sensitive when compared to the HSA-PG capture matrix. For each bacterial concentration, SALO D values were not significantly (P > 0.05) greater for the HSA-PG capture matrix, when compared to the streptavidin-biotin capture matrix. These data indicated that the HSA-PG capture matrix was the better method used to capture target bacteria in beef homogenized supernatan t fluid, using the RAPTOR for detection. The Limit of Detection for Spi nach Leaf Homogenate Supernatant RAPTOR assays, using a sandwich format with direct detection, were performed as described in the Materials and Methods s ection. Spinach leaf ho mogenate supernatant fluid containing E. coli O157:H7, was used to test the HS A-PG capture matrix in a plantbased food sample. 42.7 1541.4 1949.0 0 500 1000 1500 2000 2500 3000 2.9x10^5 2.9x10^6 2.9x10^7Signal Above Limit of DetectionE. coli O157:H7 (CFU/ml) FIGURE 16. Determination of the Limit of De tection for the HSA-PG Capture Matrix in 46

PAGE 59

47 Homogenized Spinach Leaf Supernatant Fluid Using RAPTOR Analysis. Goat polyclonal antibodies [50.0 g/ml], raised against E. coli O157:H7 and unlabeled, was used as the capture antibody. Goat polyclonal antibody [5.0 g/ml], raised against E. coli O157:H7 and Cy5 labeled, was used as th e detector antibody, to detect captured E. coli O157:H7 (CFU/ml shown on graph). HSA [100.0 g/ml] was used to immobilize PG [50.0 g/ml], before antibodies were added to the HSA-PG matrix. Each column, and coinciding standard deviation bar, was calcula ted from the average of four data points; which were collected from one assay. Positive SALOD values were produced for 2.9 x 105 CFU/ml E. coli O157:H7 (Figure 16). These data suggested that the limit of detection of E. coli O157:H7 in spinach homogenate supernatant fluid was 2.9 x 105 CFU/ml (Figure 16). SALOD values increased more than two logs when the bact erial concentration wa s increased by one log, to 2.9 x 106 CFU/ml. This unusual increase in si gnal suggested that the fluorescence detected may have been influenced by a component in the spinach homogenate supernatant fluid. These data suggested th at the SALOD values obtained were not accurately interpreted by the RAPTOR assay. The Capture Efficiency for Two Capture Matrices on Waveguide Surfaces The capture efficiency of the HSA-PG capture matrix was compared to the capture efficiency of the streptavidin-biotin capture matrix using GFPE. coli O157:H7. RAPTOR assays were performed using a direct sandwich assay format as described in the Materials and Methods s ection. After the RAPTOR assays were performed, the

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amount of bacterial cells captured on the su rfaces of the waveguide s was measured using fluorescent microscopy, as described in the Materials and Methods section. 3870 22279 36556 6751 27170 56934 0 10000 20000 30000 40000 50000 60000 70000 80000 3.7x10^6 5.3x10^7 1.7x10^8Cells Captured (n) on Waveguide GFPE. coli O157:H7 (CFU/ml) Streptavidin-Biotin HSA-PG ----P< 0.05 ----FIGURE 17. Average Numbers of Cells Capt ured on the Waveguide Surface After RAPTOR Analysis. Goat polyclonal antibodies [50.0 g/ml], raised against E. coli O157:H7 and unlabeled or biotinylated, were used as the capture antibody. Goat polyclonal antibody [5.0 g/ml], raised against E. coli O157:H7 and Cy5 labeled, was used as the detector anti body, to detect captured GFPE. coli O157:H7 (CFU/ml shown on graph). HSA [100.0 g/ml] was used to immobilize PG [50.0 g/ml], before antibodies were added to the HS A-PG matrix. Streptavidin [100.0 g/ml] was used to immobilize the biotinylated capture antibody be fore bacteria were added to the assay. Each column, and coinciding standard deviatio n bar, was calculated from the average of six data points for 3.7 x 106 CFU/ml, four data points for 5.3 x 107 CFU/ml; which were collected from six independent assays. Each column, and coinciding standard deviation bar, was calculated from the average of three data points for 1.7 x 108 CFU/ml using the 48

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HSA-PG matrix and one data point for 1.7 x 108 CFU/ml using the streptavidin biotin matrix; which were collected from three independent assays. The average number of cells manually counted in each viewable field on the surface of waveguides ranged from seven for sample concentrations of 3.7 x 106 CFU/ml, to over four hundred for sample concentrations of 1.7 x 108 CFU/ml. Waveguides treated with the HSA-PG capture matr ix retained twice the amount of cells, when compared to the streptavidin-biotin capture matrix, at 3.7 x 106 CFU/ml; which was a significantly (P < 0.05) greater amount (Figure 17 ). Waveguides treated with the HSA-PG capture matrix failed to significantly impr ove the amount of cells captu red on the waveguide surface, when compared to waveguides treated with th e streptavidin-biotin capture matrix from 5.3 x 107 through 1.7 x 108 CFU/ml. 0.37 0.10 0.04 0.72 0.13 0.07 -0.5 0.0 0.5 1.0 1.5 2.0 3.7x10^6 5.3x10^7 1.7x10^8Cells Captured (%) on WaveguideGFPE. coli O157:H7 (CFU/ml) Streptavidin-Biotin HSA-PG ----P< 0.05 ----FIGURE 18. Average Capture Efficiency on the Waveguide Surface After RAPTOR Analysis. Goat polyclonal antibodies [50.0 g/ml], raised against E. coli O157:H7 and 49

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50 unlabeled or biotinylated, were used as the capture antibody. Goat polyclonal antibody [5.0 g/ml], raised against E. coli O157:H7 and Cy5 labeled, was used as the detector antibody, to detect captured GFP-E. coli O157:H7 (CFU/ml s hown on graph). HSA [100.0 g/ml] was used to immobilize PG [50.0 g/ml], before antibodies were added to the HSA-PG matrix. Streptavidin [100.0 g/ml] was used to immobilize the biotinylated capture antibody before bacteria were added to the assay. Each co lumn, and coinciding standard deviation bar, was calculated from the average of six data points for 3.7 x 106 CFU/ml, four data points for 5.3 x 107 CFU/ml; which were collected from six independent assays. Each column, and coincidi ng standard deviation bar, was calculated from the average of three data points for 1.7 x 108 CFU/ml using the HSA-PG matrix and one data point for 1.7 x 108 CFU/ml using the streptavidin biotin matrix; which were collected from three independent assays. The capture efficiency ( %) was greatest at 3.7 x 106 CFU/ml, and decreased as the sample concentration increased, for both captu re matrices. The HSA-PG capture matrix showed significantly (P < 0.05) greater capture efficiency when compared to streptavidinbiotin for 3.7 x 106 CFU/ml. At sample concentrations from 5.3 x 107 through 1.7 x 108 CFU/ml, the waveguides treated with the HSAPG capture matrix failed to significantly increase the capture efficiencies of GFPE. coli O157:H7 on waveguide surfaces, when compared to waveguides treated with the streptavidin-biotin matrix.

PAGE 63

A Comparison of DyLight and Cy5 Labeled Detector Antibody Attainment of a lower limit of detecti on for the HSA-PG capture matrix was attempted by testing an alternative de tection fluorophore. The DyLight fluorophore has been reported by the manufacturer as being more photo stable than the Cy5 fluorophore. RAPTOR assays we re performed, using a direct sandwich format, as described in the Materials and Methods section, to compare detection limits of Cy5, and DyLight labeled detector antibodies. GFP-E. coli O157:H7 was used to show the detection of bacteria usin g waveguides treated with the HSA-PG capture matrix. -44.0 -12.4 52.9 -26.1 -0.3 53.1 -80 -60 -40 -20 0 20 40 60 80 100 6.0 x10^1 6.0x10^2 6.0x10^3Signal Above Limit of DetectionGFPE. coli O157:H7 (CFU/ml) DyLight649 Cy5FIGURE 19. A Comparison of DyLight and Cy5 Labeled Detector Antibody Using RAPTOR Analysis. Goat polyclonal antibodies [50.0 g/ml], raised against E. coli O157:H7 and unlabeled, was used as the capture antibody. Goat polyclonal antibodies [5.0 g/ml], raised against E. coli O157:H7 and DyLight or Cy5 labeled, was used as the detector antibody, to detect captured GFP-E. coli O157:H7 (CFU/ml shown on 51

PAGE 64

52 graph). HSA [100.0 g/ml] was used to immobilize PG [50.0 g/ml], before antibodies were added to the HSA-PG matrix. Each colu mn, and coinciding standard deviation bar, was calculated from the average of three da ta points; which were collected from two independent assays. A significant (P > 0.05) difference was not found between the SALOD values produced from DyLight and Cy5 labeled detector antibodies, for all sample concentrations tested (Figure 19). The limit of detection was the same for 6.0 x 103 CFU/ml, for both fluorophores tested. These da ta suggested that the use of DyLight labeled detector antibodies failed to increase the sensitivity of the RAPTOR assay, when compared to the use of Cy5 labeled detector antibodies, to detect E. coli O157:H7.

PAGE 65

53 DISCUSSION An alternative method for immobilizi ng capture antibody on an immunoassay surface was explored, after existing strategi es were researched (5, 6, 11, 15, 16, 19, 22, 23, 24, 31, 34, 35, 39, 40). One common strategy for securing capture antibodies to a polystyrene surface is to passively adsorb st reptavidin to the surface. The streptavidin coated surface is then used to bind biotinylated capture antibodies. This binding is incredibly strong (13), and re sists denaturation of the immo bilized antibody (39), which led to a broad acceptance for this method ( 6, 19, 23, 36) in the field of microbiology and immunology. Biotinylation of th e antibody is done by a covale nt interacti on targeted towards primary amines, which are located on the constant and variable regions of the antibody heavy chain. When the biotinylated antibody is incubated on the streptavidin coated surface the immobilizati on results in a random orientation of the antibodies (24, 31). This lack of uniformity, of the bioti nylated antibody may cause poor sensitivity (3, 24), which currently plagues immunoassays (6, 19, 23, 36). To test this reasoning, an alternative capture matrix was constructed and then compared to the streptavidin-biotin matrix. The most abundant form of streptoco ccal PG, available on the commercial market, is a recombinant PG, which lacks an albumin binding domain (14 kDa); which is located near the amine terminus. Recombin ant PG is commonly employed to remove antibodies from serum without nonspecifically binding to the albumin present. This common use of the recombinant form of PG le d to a minimal demand for the native form

PAGE 66

54 of PG. For this research, obtaining native PG with an intact and functional albumin binding domain was essential. This nati ve form of PG has limited commercial availability; and only one company, Calbiochem was located that sold the native protein, with the intact albumin binding domain. Validation of the pr esence and functionality of the albumin binding domain in this product was the first step performed to construct the alternative capture matrix. In the presence of HSA, Figure 4 shows the functionality of the albumin binding domain in native PG, by the production of signal to noise ratios greater than the signal to noise ratios produced by recombinant PG. These results indicated that native PG had a functional albumin binding domain able to bind HSA. These results also indicated that recombin ant PG did not have a functional albumin binding domain, and was not able to bind to HSA. The use of native PG was implemented for all future assays. Different species of albumin were tested to determine the species specificity of the albumin binding domain of PG. The album in species tested were chosen based on a previous study (25); which showed that huma n, rabbit and bovine se rum inhibited the Fc binding of radiolabeled human species antibodies to bacterial cells from group G and C streptococci. This binding inhibition suggest ed that the radiolabeled antibodies were displaced by competitive antibodies present in the serum. This inhibition did not occur when chicken, rat, dog and cat serum were tested (25). The Fc binding domain of PG was shown to bind to rabbit and human species antibody (4), but did not bind to chicken species antibody (14). This pattern of species specificity demonstrated by the Fc binding domain led to the reasoning that the specifi city may be transferrable to the albumin

PAGE 67

55 binding domain. To test this theory, PG was immobili zed by human serum albumin (HSA), bovine serum albumin (BSA ), or chicken ovalbumin (OVA). Minimal signal to noise ra tios were produced by wells that contained OVA, which indicated that OVA did not imm obilize PG (Figure 5). Signal to noise ratios greater than two were produced by wells that contained 5.0 g/ml of BSA, which indicated that high concentrations of BSA did immobilize PG Interaction between BSA and PG was important to consider because many laborat ory reagents, such as antibodies, contain nominal concentrations of BSA as a stabilizi ng agent. These results indicated that small amounts of BSA in the reagents did not interfere with the assays using the HSA-PG capture matrix. HSA showed the greatest binding to PG, even at minimal concentrations, which indicated that human was the optimal sp ecies of albumin for use in the alternative capture matrix. As theorized, the species sp ecificity shown by the Fc binding domain was also shared by the albumin binding domain. Further studies, using rabbit, goat, and mouse sera, are needed to explore the rela tionship between species specific domains on multi-functional proteins, and to find a supe rior albumin species to immobilize PG, to assay surfaces. A range of concentrations of PG and HS A were tested to determine the optimal working ratio of these two components in th e alternative capture matrix. Figures 4 and 5 compare the concentrations of HSA and PG without the presence of bacteria. Based on the greatest signal to noise ratios, Figure 4 shows the optim al concentration of HSA was 1.0 g/ml, and PG was 0.5 g/ml. Figure 5 shows the greatest signal to noise ratios at 5.0 g/ml for HSA, and 2.5 g/ml for PG. Higher concentratio ns showed greater signal to noise ratios, but the ratio of the components was identical, at 2.0 g/ml of HSA for every

PAGE 68

56 1.0 g/ml PG. E. coli O157:H7 was added to the experiment to verify that this ratio was optimal in the presence of bacteria and to better anticipate the activity of these components during a working immunoassay. Figure 6 shows the signal to noise ratios for 1.0 g/ml of HSA and 0.5 g/ml of PG. The 2:1 ratio pr oduced the greatest signal to noise ratios, using the lower concen trations of HSA and PG to capture E. coli O157:H7. This 2:1 ratio of HSA and PG was implemented for all future assays. In previous studies, fragments of PG or intact PG, have been used to immobilize capture antibodies on assay su rfaces in immunoassays (3, 1 8, 40). Methods to secure PG to the assay surface, e.g., amine or thiol reactive chemistry (3, 40), have improved antibody activity, when compared to direct adsorption. Covalent attachment of the albumin binding domain of PG to Fab frag ments has further improved antibody activity, by immobilizing the antibody on a HSA coated surface (18). In this study, the role of HSA in the alternative capture matrix was clarified by comparing antibody immobilization using HSA, PG or HSA-PG combined. Figure 7 shows antibodies immobilized by HSA alone, PG alone, and PG and HSA combined, in the absence of bacteria Minimal fluorescence was produced by HSA alone and PG alone which indicated that the antibodies were not immobilized. The combination of HSA and PG produced the grea test fluorescence, which was significantly (P < 0.05) greater than th e fluorescence produced by PG alone or HSA alone. The antibodies used were not specific for HSA or PG so any binding was via the Fc domain, which constituted non-immune binding; and not via the antibody paratopes, which are used for immune binding. This improved fluor escence indicated that a greater amount of primary antibody was immobilized by PG when PG was immobilized by HSA. As shown

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57 in a previous study, the greater density of immobilized antibody c ontributed to greater sensitivity of the assay when a target was introduced (24). E. coli O157:H7 was introduced into the experiment to test this theory using the alternative capture matrix. Figure 8 shows minimal fluorescence pr oduced by HSA alone, which indicated that E. coli O157:H7 was not captured by HSA alone Significantly greater fluorescence was produced by PG alone, which suggested that PG immobilized capture antibody and bacteria for detection. The greatest fluorescence was produced by HSA and PG combined, when compared to fluorescence prod uced by PG or HSA alone. When PG was immobilized by an albumin coated surface, th e Fc binding domain was available to bind antibodies in the environment. Previous study has shown that the struct ure of PG consists of structurally opposite Fc and albumin bi nding domains. PG was able to bind an antibody molecule and an albumin molecule at the same time, without allosteric modulation (7). This strict orientation of antibody, by the Fc binding domain, allowed for a greater incidence of antibody binding, when compared to PG passively adsorbed to a non-albumin coated surface; which resulted in random orientation of the PG (27). The greater availability of the Fc binding domain was character ized by a greater density of capture antibody; which resulted in the enhanced capture of E. coli O157:H7. The use of PG immobilized by HSA was implemented for all future assays. The optimal species of antibody for use as capture and detector antibody was determined for use in the alternative capture matrix. Figure 9 and 10 s how the results of a series of competitive ELISAs used to investig ate the species specificity of the Fc binding domain of PG. In Figure 9, competitive ELI SAs were used to compare goat and mouse antibodies. Signal to noise rati os greater than 6 were produce d by all wells that contained

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58 HRP labeled goat antibodies, and unlabeled m ouse antibodies. These results indicated that the goat antibodies were stronger compet itors for the Fc bindi ng domain of native PG. The success of goat antibody in compe ting for the Fc binding domain of PG suggested that goat species antibody was a prime candidate for use as the capture antibody in the alterna tive capture matrix. Signal to noise ratios less than 2 were produced by all wells that co ntained HRP labeled mouse antibodies and unlabeled goat antibodies. These results indicated that the HRP labeled mouse antibody failed to compete for Fc binding domain of PG. This failure suggested that mouse species antibody should not be implemen ted as a capture antibody in the alternative capture matrix. A previous study reported similar findi ngs as shown in Table 2 (14). Nearly five times the amount of mouse polyclonal anti body (1020 ng) was required to produce fifty percent inhibition of binding between the Fc binding domain of PG and rabbit polyclonal antibody, when compared to only 217 ng of goat polyclonal antib ody, required to produce the same inhibition. The greater requ irement for mouse antibody, compared to goat antibody, suggested that the Fc binding do main of PG had greater affinity for goat species antibody (14). These re sults led to the potential use of mouse antibody as a detector antibody; while goat species antibody was implemented as the capture antibody in the developing capture matrix. Further species specificity analysis of the Fc binding domain was warranted to investigate alternative capture antibodies for use in the alternative capture matrix. Table 2 shows the similar affinity of PG for rabbit and goat species antibody. Polyclonal goat and rabbit antibodies were compared using indirect ELISAs. To avoid competitive binding to PG, indirect detection was performed by HR P labeled mouse antibodies, which were

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59 raised against goat or rabbit antibodies. Figure 10 shows signal to noise ratios that were significantly (P < 0.05) greater for wells that contained goat antibodies, when compared to wells that contained rabbi t antibody. These results suggested that goat species antibody was the preferred candidate for use as a cap ture antibody. Rabbit antibodies showed the second highest affinity for the Fc binding dom ain of PG, and could therefore potentially be employed as an alternative capture antibody in the event that a goat species antibody is not readily available for a par ticular antigen. Since PG show ed high affinity for the goat polyclonal antibody raised against E. coli O157:H7, and since it is specific and sensitive for E. coli O157:H7, it was used as the capture antibody in all future assays. The species and concentration of the dete ctor antibody was important to consider when PG was used in an immunoassay form at. PG has previously shown a range of affinities for different species of antibody ( 1, 4, 14). However, PG has not shown a high affinity towards chicken (1, 14) or mouse ( 1, 4) antibodies, which suggested that they could be used as detector an tibodies. HRP labeled chicken and mouse antibodies, raised against E. coli O157:H7, were tested for use as dete ctor antibodies, but were found to be insensitive and non-specific for the target organism; when compared to goat antibodies raised against E. coli O157:H7 (data not shown). Goat polyclonal antibodies raised against E. coli O157:H7 were implemented as the cap ture and detector antibody for all future assays. Further investigation was required to avoid displacement of the goat species capture antibodies on PG by the goat species detector antibodies, which the Fc binding domain has similar affinity for. In Figure 11, 1.0 g/ml of detector antibody produced the greatest signal to noise ratios at all concentrations of E. coli O157:H7. Due to elevated

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60 background signals, the signal to noise ratios de creased as the concentration of detector antibody increased. These results suggested th at the excess detector antibody may have displaced the same-species capture antibody. To test for displacement of the capture antibody by the detector antibody fluorescence values produced by wells that contained bacteria were compared to fluorescence values produced wells that lacked bacteria. Figure 12 shows that when E. coli O157:H7 was absent from the environment, minimal fluorescence (< 300 pA) was produced, which i ndicated that the detector antibody did bind due to a lack of target. These results al so indicated that the detector antibody did not bind to the PG present in the capture matrix, and that the Fc binding domains were effectively saturated with the capture antibody. The lack of fluorescence further indicated that the capture antibodies at 1.0 g/ml was not displaced by the same-species detector antibodies, at 0.1 g/ml through to 1.0 g/ml. Goat species de tector antibody at 1.0 g/ml was implemented for all future assays using ELISA analysis. Sensitivity of the immunoassay was a major consideration during the development of the alternative capture matrix. The limit of detection was used as a measure of sensitivity, which was based on the concentration of bacteria captured and subsequently detected by the immunoassay. Fi gure 13 shows the limit of detection was 9.7 x 104 CFU/ml for E. coli O157:H7 by the streptavidin-b iotin and the HSA-PG capture matrices. The HSA-PG capture matrix produced signal to noise ratios significantly (P < 0.05) greater than the streptavid in-biotin capture matrix for all bacterial concentrations tested greater than 9.7 x 103 CFU/ml. The most significan t difference produced between the capture matrices was for the lo west sample concentration, 9.7 x 104 CFU/ml. As the concentration of bacteria increased, the difference in signal to noise ratios decreased

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61 between the two capture matrices. These result s suggested that the b acterial target began to saturate the antigen binding si tes for both matrices at 9.7 x 105 CFU/ml. The sensitivity of the two capture ma trices was compared using RAPTOR analysis. Figure 14 shows the same sensitivit y, or limit of detecti on, for the two capture matrices (streptavidin-biotin and HSA-PG) at 1.9 x 103 CFU/ml of E. coli O157:H7. At sample concentrations below 1.0 x 105 CFU/ml, a significant (P < 0.05) difference was not produced between the two capture matrices These results indicated that at lower concentrations of E. coli O157:H7 the sensitivity was not improved by the HSA-PG capture matrix. The HSA-PG capture matrix produced significantly (P < 0.05) greater SALOD values for 1.0 x 105 through 1.3 x 106 CFU/ml, when compared to the streptavidin-biotin capture matrix. The incr eased SALOD values suggested the enhanced capture of bacteria in samples with concentrations greater than 1.0 x 105 CFU/ml of E. coli O157:H7. The amount of bacteria captured on th e waveguide surface was quantified to compare the capture efficiency of the two capture matrices. Figure 17 shows that the greatest amount of GFPE. coli O157:H7 captured was by the HSA-PG matrix, when compared to the streptavidin-biotin matrix at all concentrations tested. The number of cells counted on the HSA-PG waveguides wa s significantly (P < 0.05) greater when compared to the streptavidin-biotin treate d waveguides, exclusiv ely at low bacterial concentrations (3.7 x 106 CFU/ml). At higher concen trations, the amount of cells captured by the two matrices was not signifi cantly (P < 0.05) different. Figure 18 shows the measured capture efficiency was 0.37% for the streptavidin-biotin capture matrix, and 0.72% for the HSA-PG capture matrix, when GFP-E. coli O157:H7 was assayed at 3.7 x

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62 106 CFU/ml in a 500 l sample volume. GFPE. coli O157:H7 was used in a previous study to measure the capture efficiency of th e streptavidin-biotin capture matrix, which was reported at 0.54% for 1.0 x 106 CFU/ml in an 800 l sample volume (36). This difference in capture efficiency for the strept avidin-biotin capture matrix could have been due to the change in sample volume, whic h was unavoidable due to a limitation in the equipment used. Capture efficiency was an invaluable tool used to examine the limitations of the capture matrices. Multiple strategies have been employed to investigate the lack of efficient capture : including sample volume, sample introduction and sample speed (36). More efficient target capture by capture matrices coul d lead to enhanced sensitivity by the immunoassay. These results indicated that when HSA and PG were used to uniformly orient the capture an tibody the capture efficiency was improved. Contaminated food samples were added to th e study to test the capabilities of the alternative capture matrix. Gr ound beef and spinach leaves were tested to determine if these food samples contained components that would interfere with the capture matrix. The supernatant of homogenized ground beef was tested by RAPTOR analysis. Figures 15 and 16 show the limit of detection of E. coli O157:H7 was 5.4 x 105 CFU/ml for the HSA-PG capture matrix, when homogenized beef and spinach supernatants were tested. The minimal baseline values produced by th e biosensor (Appendix B) suggested that components in the beef supernat ant did not interfere with the PG in the capture matrix. In contrast, baseline values pr oduced by the biosensor for th e spinach supernatant were extremely high (Appendix B). The inherent fluorescence from the chlorophyll (10), in spinach supernatants, could have enhanced th e detection by the biosensor; which resulted in false SALOD values and high background noise. The presence of ch lorophyll in a food

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63 product is important to consider when fluor escence-based assays are used to detect contamination. Figure 15 shows the limit of detection for the streptavidin-biotin capture matrix was 5.4 x 106 CFU/ml of E. coli O157:H7 in ground beef supernatant. For both capture matrices the limit of detection in the ground beef supernatant was less sensitive when compared to limit of detection in PBS (Figure 14). This decrease in sensitivity may be caused by unknown components in the food sample. Large fat or protein particles in the beef supernatant may block antigen binding sites on the waveguide surface, which may have led to decreased sensitivity of the assa y (6). Figure 15 shows greater sensitivity by the HSA-PG matrix when compared to strept avidin-biotin in ground beef supernatant. The difference in limits of detection between the capture matrices could be explained by the uniform orientation of th e antibodies by HSA-PG, and the random orientation of antibodies by the streptavidin-biotin captu re matrix. Uniform antibody orientation has previously shown greater antibody activity wh en compared to random orientation (24, 40). An alternative fluorophore was tested for enhanced sensitivity in the biosensor assay when the HSA-PG capture matrix was used. According to the manufacturer, Pierce Biotechnology (Rockford, IL), the DyLight fluorophore is more photo-stable, and produces more intense fluorescence when comp ared to the Cy5 fluorophore. Figure 19 shows the detection fluorophor es did not produce signifi cantly (P < 0.05) different SALOD values. These results suggested that the alternative fluorophore failed to enhance the sensitivity of the RAPTOR assay.

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64 Precision was used to measure the reproducibility of the RAPTOR assay, which was a critical characteristic for the i mmunoassay. Many parameters could diminish precision, e.g., approaching the detection limit of the assay. According to the manufacturer, Research Intern ational (Monroe, WA), the de tection limits of RAPTOR assays vary depending on the nature of the ta rget. Detection limits are lower for bacterial toxins (0.1 ng/ml to 1,000 ng/ml), as compared to whole bacterial cells (30 CFU/ml to 1.0 x 107 CFU/ml). In this study the detection limits of E. coli O157:H7 were compared to the precision of the assay. Ta ble 13 shows a total of 136 samp les that were assayed using the RAPTOR biosensor, to compare the capture of E. coli O157:H7 by streptavidin-biotin and HSA-PG. The precision of both capture matrices was nearly identical, with the exception of 1.0 x 102 CFU/ml. The precision of detection, at and below 1.0 x 103 CFU/ml of the target bacteria, was below 100%. This decrease in prec ision indicated that the limit of detection was surpassed, and that the results of the assa y were not reliable for sample concentrations below 1.0 x 103CFU/ml. Typically, 100% precision or reproducibility would be expected from an immunoassay when the health and safety of consumers is a priority. Table 13 shows the analysis of a total of 60 RAPTOR waveguides, which were performed to detect E. coli O157:H7 or GFPE. coli O157:H7 in PBS. Nine waveguides treated with the streptavidin-biotin capture matrix produced highly variable (CoV > 10%) baseline values. HSA-PG treated waveguide s were less variable, with only seven waveguides producing highly variab le baseline values. As described in the Materials and Methods section, the data produ ced by a waveguide with a hi ghly variable baseline was discarded and was not used for graph constr uction. Baseline variability could be caused

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65 by many factors, including a bowed waveguide (< 180) or a waveguide that was longer or shorter than 38mm, which were perhaps limitations of the ma nufacturing process. Figures 14 through 19 did not include data produced by waveguides with a baseline CoV greater than ten percent. Table 13 show s all the waveguides despite the baseline variability. E. coli CFU/ml HSA-PG Positive/Negative Assay Precision Streptavidin-Biotin Positive/Negative Assay Precision 101 0/2 0% 0/2 0% 102 3/9 25% 1/11 8% 103 8/6 57% 8/6 57% 104 10/0 100% 10/0 100% 105 6/0 100% 6/0 100% 106 12/0 100% 12/0 100% 107 6/0 100% 6/0 100% 108 6/0 100% 6/0 100% TABLE 13. Precision of Capture Matrices In conclusion, the alternative capture matr ix consisted of a 2 to 1 ratio of human serum albumin and streptococcal PG, which immobilized rabbit a nd goat antibody in a uniform orientation and captured targeted an tigen. This uniform antibody orientation led to significantly improved capture efficiency of E. coli O157:H7. Further study is required to improve reliable detection below 1.0 x 104 CFU/ml for the RAPTOR biosensor.

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68 18. Konig, T. and A. Skerra. 1998. Use of an albumin-binding domain for the selective immobilization of recombinant capture an tibody fragments on ELISA plates. J. Immun. Methods 218 :73-83. 19. Kramer, M. F. and D. V. Lim. 2004. A rapid and automated fiber optic-based biosensor assay for the detection of Salm onella in spent irrigation wate r used in the sprouting of sprout seeds. J. Food Protection 67:46-52. 20. Kraulis, P., P. Jonasson, P. Nygren, M. Uhln, L. Jendedberg, B. Nilsson and J. Krdel. 1996. The serum albumin-binding domain of streptococcal protein G is a threehelical bundle: a heteronuclear NMR study. FEBS Letters. 378:190-194. 21. Kronvall, G. 1973. A surface componenet in group A, C and G streptococci with nonimmune reactivity for immunoglobulin G. J. Immunol. 111:1401-1406. 22. Laitinen, O., H. Nordlund, V. Hytonen, and M. Kolumaa. 2007. Brave new (strept)avidins in biot echnology. Trends Biotech. 25:269-277. 23. Leskinen, S. D. and D. V. Lim. 2008. Rapid ultrafiltration c oncentration and biosensor detection of enterococci from large volumes of Florida recrea tional water. Appl. Environ. Microbiol. 74 :4792-4798. 24. Lu, B., M. Smyth and R. OKennedy. 1996. Oriented immobilization of antibodies and its applications in immunoassays and immunosensors. Analyst. 121:29R-32R. 25. Myhre, E. and G. Kronvall. 1977. Heterogeneity of noni mmune immunoglobulin Fc reactivity among Gram-positive cocci: Descripti on of three major types of receptors for human immunoglobulin G. Infection and Immun. 17:475-482. 26. Nanninga, N. 1998. Morphogenesis of Escherichia coli. Micro. Mol. Bio. Reviews. 62:110-129.

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70 34. Shriver-Lake, L., B. Donner, R. Edelstein, K. Breslin, S. K. Bhatia and F. S. Ligler. 1997. Antibody immobilization using heterobif unctional crosslinkers. Biosensors and Bioelectronics 12:1101-1106. 35. Shriver-Lake, L., S. Turner and C. R. Taitt. 2007. Rapid detection of E. coli O157:H7 spiked into food matrices. Analy. Chim. Acta. 584:66-71. 36. Simpson-Stroot, J., E. Kearns, P. St root, S. Magana and D. V. Lim. 2008. Monitoring biosensor capture efficiencies: Developm ent of a model using GFP-expressing Escherichia coli O157:H7. J. Mircro. Methods 72 :29-37. 37. Sjbring, U., C. Falkenberg, E. Nielsen, B. kerstrm and L. Bjrck. 1988. Isolation and characterization of a 14-kDa albumin-binding fragment of streptococcal protein G. J. Immunol. 140:1595-1599. 38. Sugio, S., A. Kashima, S. Mochizuki, M. Noda and K. Kobayashi. 1999. Crystal structure of human serum albumin at 2.5 resolution. Prot. Engin. 12:439-446. 39. Suter, M., J. E. Butler and J. H. Peterman. 1989. The immunochemistry of sandwich ELISAs-III. The stoichiometry and efficacy of the protein-avidin-biotin capture (PABC) system. Mol. Immun. 23 :221-230. 40. Vijayendran, R. and D. Leckband. 2001. A quantitative assessment of heterogeneity for surface-immobilized proteins. Anal. Chem. 73 :471-480.

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71 APPENDICES

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Appendix A: ELISA Analysis TABLE 14. Functional Albumi n Binding Domain, HSA Role in Capture Matrix HSA in Columns ( g/ml) 0.00.00.50.51.01.00.00.00.50.51.01.0 0.0 g/ml PG 121.5125.7120.1130.7138.3131.5 151.8129.5140.3140.7134.4130.4 0.25 g/ml PG 121.2134.2967.91044.41545.51569.1 160.5131.7138.3140.7134.5130.5 0.5 g/ml PG 132.2127.11172.51231.91848.11864.9 138.2125.3131.7131.5134.0133.2 1.0 g/ml PG 119.8130.11216.31344.51943.51907.6 136.9123.3133.4143.7128.9129.7 2.5 g/ml PG 122.5134.21380.91336.11991.11997.7 141.1136.8132.5138.1132.2132.6 5.0 g/ml PG 110.7153.91555.31518.72147.52132.7 138.5120.8127.3128.1132.0126.0 10.0 g/ml PG 117.1148.81605.51570.82212.62140.0 158.1136.9136.6139.2134.8137.7 20.0 g/ml PG 139.2157.51583.21567.12180.52222.2 142.6135.6147.9133.7139.5137.3 0.0 g/ml PG 148.7168.1158.5158.6151.4156.8 185.8170.3169.2173.6163.6144.0 0.25 g/ml PG 139.8180.0985.0997.91488.41485.6 221.3197.3166.1186.1147.3150.1 0.5 g/ml PG 153.2176.01565.41372.82207.22119.1 205.8156.8141.0173.5141.4138.5 1.0 g/ml PG 153.6157.81362.41650.22038.42318.9 257.4163.2143.3154.9150.5139.9 2.5 g/ml PG 125.4160.41343.01335.41666.31891.0 148.7158.8141.6149.8139.2155.1 5.0 g/ml PG 119.9135.41270.41267.81750.91763.3 137.6133.7131.9126.4138.6135.8 10.0 g/ml PG 127.0176.01284.01294.71741.91715.2 143.7137.4126.4120.7129.0127.4 20.0 g/ml PG 148.1169.61264.11255.61609.31641.3 140.1143.4145.3140.7137.4139.5 0.0 g/ml PG 134.6150.6146.2148.4144.2150.1 160.8162.6166.4167.8153.8159.1 0.25 g/ml PG 144.6157.21054.01144.21751.61788.0 171.6174.2164.6171.0147.9168.1 0.5 g/ml PG 150.9176.11698.51698.22433.02393.8 159.9162.0142.1152.1138.2147.4 1.0 g/ml PG 135.3161.51257.51348.02045.42037.5 155.7171.5146.8143.8141.8158.3 2.5 g/ml PG 137.1170.51599.61573.72423.72467.9 168.3165.8162.2155.5144.3134.8 5.0 g/ml PG 112.7137.01125.01046.91910.62060.7 127.5129.4130.1131.8134.2123.1 10.0 g/ml PG 133.7203.21107.91107.01542.31569.7 143.6147.4133.1142.1134.7133.3 20.0 g/ml PG 139.2146.71024.81119.41533.81590.1 140.5144.8141.5147.2141.8141.2 No Bold Indicates Native PG Bold Indicates Recombinant PG TABLE 15. Alternativ e Albumin Species Albumin in columns (g/ml) 1 OVA1 OVA5 OVA5 OVA1 BSA1 BSA5 BSA5 BSA1 HSA1 HSA5 HSA5 HSA 0 g/ml PG283.9206.0187.9185.4258.7279.2188.8217.8253.0261.4173.7215.6 0.25 g/ml PG318.6273.7185.2182.3303.0291.8327.4354.02615.22601.92781.52889.5 0.5 g/ml PG289.4255.1186.9187.3320.5319.2453.2383.03056.63079.63245.53389.8 1 g/ml PG317.2285.3186.1188.6382.3357.4549.1591.03269.93368.23524.33692.3 2.5 g/ml PG224.1272.1197.0192.5434.5465.4705.6709.13353.63380.73959.23934.0 5 g/ml PG237.0348.8245.5211.4466.3488.8915.1864.93381.73363.53903.63926.4 No PG 181.3244.7244.9154.1258.3254.2250.6242.7300.4258.0209.7234.9 PG in columns (g/ml) 0.00.00.250.250.50.51.01.02.52.55.05.0 No Albumin341.4432.3552.3639.8754.2804.2838.91227.01375.81471.71786.31869.4Albumin in columns (g/ml) 1 OVA1 OVA5 OVA5 OVA1 BSA1 BSA5 BSA5 BSA1 HSA1 HSA5 HSA5 HSA 0 g/ml PG256.7287.9221.5272.7278. 7266.1182.9258.9269. 6288.0236.2240.5 0.25 g/ml PG264.6322.7279.8261.3353.2327 .8343.6312.22745.5273 2.93043.33024.1 0.5 g/ml PG287.2282.1304. 9293.8348.7344.1543.2383.03 188.73121.83413.93413.0 1 g/ml PG318.5360.6304.3290.1391.4382 .9594.5531.23447.2355 2.83670.13761.8 2.5 g/ml PG292.8342.3333. 6262.4447.7469.9763.7748.43 471.63482.83856.73975.0 5 g/ml PG292.4367.4338.5327.7503.3564 .2870.9892.03460.0344 2.93847.03935.9 No PG 261.3221.0277.4210.3278.32 69.9206.2216.6305. 6242.2228.9255.8 PG in columns (g/ml) 0.00.00.250.250.50.5 1.01.02.52.55.05.0 No Albumin482.5575.4882.6637.6796.7900 .8825.11086.61536.8144 5.11753.22252.4 72

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Appendix A: (Continued) TABLE 15. Alternative Albu min Species (Continued) Albumin in columns (g/ml) 1 OVA1 OVA5 OVA5 OVA1 BSA1 BSA5 BSA5 BSA1 HSA1 HSA5 HSA5 HSA 0 g/ml PG243.6278.9285.8264.6294.2370.9291.9285.1291.8351.8263.4294.8 0.25 g/ml PG280.6322.8270.6269.1383.2418.3388.5395.13122.73117.13304.23608.9 0.5 g/ml PG356.0277.9290.6277.9499.7348.5438.5476.33569.93534.53695.13447.3 1 g/ml PG417.7313.3313.4370.2454.9366.0587.4576.93669.63775.54012.54225.8 2.5 g/ml PG313.5376.7385.7329.1490.8478.9753.2764.03733.53753.34010.94091.0 5 g/ml PG332.0360.2405.2473.7648.5598.2931.7999.63681.53771.93870.93870.8 No PG 209.2265.1276.0216.5309.1272.3248.0235.6247.3313.8261.9245.5 PG in columns (g/ml) 0.00.00.250.250.50.51.01.02.52.55.05.0 No Albumin490.6717.7739.01021.0825.1798.41214.2929.51129.41123.8909.71119.3 TABLE 16. HSA Role in Capturing Bacteria PG in Columns ( g/ml) 0.00.00.250.250.50.5 1.01.02.52.55.05.0 No Bacteria142.4156.4162.0179.6176. 5187.7174.2182.0174.7181.7197.5190.9 4.70E+071524.21239.72741.12778.82924.728 10.52883.32866.02855.72792.62844.22856.5 4.70E+06449.9373.12431.22563.42551.72630.72814.72529.52835.82845.22797.32862.5 4.70E+05165.0155.61599.61755.51877.22027.42201.02081.72235.82217.22288.32310.1 4.70E+04136.8134.71287.01284.81476.11643.31888.51733.41817.31766.81961.61979.6 4.70E+03119.9116.01015.01156.61273.91393.91617.61503.81742.01628.11691.91842.3 4.70E+02112.7112.1951.11023.71337.41292.51408.81419.41443.91481.41512.51540.9 4.70E+01105.3115.8799.9930.81279.11235.31349.61339.61434.81401.51473.91500.4 No Bacteria145.5146.4175.5184.3178. 6185.7169.1175.4179.3180.7197.5193.0 4.70E+071344.21264.43102.73152.63280.232 40.13270.63266.13218.93233.43192.53189.9 4.70E+06392.8303.03150.13066.33102.62839.12830.73002.72935.22937.83159.73173.7 4.70E+05154.5138.62039.22100.12151.42208.62240.32365.52350.22427.02479.22448.0 4.70E+04141.2125.41859.51859.32029.21980.42133.22192.62286.92182.52329.62367.2 4.70E+03116.8123.31730.81673.51863.01862.22145.31963.92233.52336.72165.52363.7 4.70E+02123.0108.71509.91524.91695.31636.01884.61764.51890.91928.51904.52009.8 4.70E+01111.9123.11475.31475.71706.61668.91772.11813.61815.01848.41916.01965.5 No Bacteria133.6156.3146.7158.6152. 2160.2162.1169.6190.7201.8189.5194.7 4.70E+071343.11064.53012.33162.53290.132 44.03267.63265.03154.13238.53162.33191.9 4.70E+06370.5279.42824.82843.52911.13040.83050.03155.03311.83112.63135.13105.0 4.70E+05177.6137.22188.72128.92267.72262.92380.52481.72613.22480.92444.52382.7 4.70E+04124.1126.41816.61825.32032.32010.31990.02026.32123.81911.41939.71772.2 4.70E+03118.6113.51699.61849.71917.41836.01792.61787.71884.61773.51830.11738.4 4.70E+02119.1119.71660.81658.01791.31603.71709.51702.61881.51768.61758.61726.1 4.70E+01114.5129.91610.21463.81642.91615.41757.31702.01779.41737.21678.01651.2 E. coli O157:H7 in Rows (CFU/ml) TABLE 17. Optimal Ratio of HSA to PG PG in columns ( g/ml) 0.00.51.02.55.010.00.00.51.02.55.010.0 0 g/ml HSA1746.13675.85101.35678.25961.16329.3 354.8399.8723.4403.2557.5760.0 0 g/ml HSA2823.33974.75264.75754.95948.06246.4 326.2433.7456.9430.9550.5622.3 HSA in columns ( g/ml) 1.01.05.05.010.010.01.01.05.05.010.010.0 0.0 g/ml PG1124.81026.1968.01050.01070.11056.1 114.7103.6111.1119.9156.2127.9 0.5 g/ml PG6233.96542.36769.86716.66725.16490.1 402.6401.8585.5625.8634.9656.0 1.0 g/ml PG6309.16267.56723.46504.46702.46499.8 437.9462.8740.7741.0765.1875.4 2.5 g/ml PG6187.06294.26578.06400.96549.46304.0 641.8654.6849.3863.3967.51007.4 5.0 g/ml PG6102.86207.86581.16433.46511.96461.5 607.5625.1857.6882.3962.21012.7 10.0 g/ml PG6123.56386.56562.16527.96574.46597.5 717.8745.01035.6994.81106.91147.5 No Bold Indicates 8.1x107 CFU/ml E. coli O157:H7 Bold indicates no bacteria 73

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Appendix A: (Continued) TABLE 18. Species Specific Fc Binding Domain PG in Columns ( g/ml) 0.00.00.50.51.01.0 0 g/ml IgG 90.195.293.592.694.597.3 0.25 g/ml IgG 92.992.2692.9693.9643.1672.2 0.5 g/ml IgG 97.494.6994.8993.21387.01402.0 1.0 g/ml IgG 93.391.41391.81407.71499.21501.3 0 g/ml IgG 92.088.390.692.789.291.1 0.25 g/ml IgG 99.584.588.788.388.192.8 0.5 g/ml IgG90.385.788.288.788.989.9 1.0 g/ml IgG 88.188.189.087.189.691.9 0 g/ml IgG 88.490.289.489.091.195.6 0.25 g/ml IgG 93.298.0602.2593.8653.1772.2 0.5 g/ml IgG 94.790.51394.81393.11388.11472.0 1.0 g/ml IgG 98.595.91491.81411.81497.51521.4 0 g/ml IgG 88.185.988.889.687.488.6 0.25 g/ml IgG 90.293.492.191.694.0103.1 0.5 g/ml IgG96.591.788.888.181.685.2 1.0 g/ml IgG 94.093.296.193.194.491.1 0 g/ml IgG 94.395.393.493.894.4100.5 0.25 g/ml IgG 91.898.6640.3632.2646.0631.8 0.5 g/ml IgG 100.5109.81181.01192.31088.21085.0 1.0 g/ml IgG 103.2119.71372.31304.41367.01313.2 0 g/ml IgG 97.691.592.789.490.693.9 0.25 g/ml IgG 130.5111.4105.7109.0104.6101.8 0.5 g/ml IgG108.0112.4100.7112.4102.4119.3 1.0 g/ml IgG 109.9101.6134.9112.3102.9103.6 Bold Indicates HRP goat antirabbit and mouse antiE. coli O157:H7 No Bold Indicates HRP mouse antirabbit and goat antiE. coli O157:H7 TABLE 19. Optimal Capture Antibody Species HSA in Columns ( g/ml) 0.00.00.50.51.01.00.00.00.50.51.01.0 0.0 g/ml PG 93.395.6111.9109.5124.8107.4 132.8137.8115.9113.5121.2106.6 0.5 g/ml PG 108.7103.1340.6334.8603.5672.0 152.2147.5523.6552.9819.1810.1 1.0 g/ml PG 117.3131.2397.0405.0738.5742.9 149.6143.9940.7945.8921.11011.2 2.5 g/ml PG 107.7107.2446.4489.4849.5904.5 193.1212.01014.7989.61156.71488.4 0.0 g/ml PG 119.1101.9105.1111.3121.8134.7 166.8182.7139.8159.5140.8143.8 0.5 g/ml PG 126.8116.9116.5129.5109.6114.0 157.3157.8145.1162.7145.1146.4 1.0 g/ml PG 109.7129.7112.8114.3146.0111.5 207.6169.8136.5160.8154.2164.0 2.5 g/ml PG 125.9114.2127.7125.7130.6137.8 171.8155.5163.3168.2156.6156.7 0.0 g/ml PG 120.3105.992.6120.0131.5119.2 128.8146.3117.4106.5126.0114.7 0.5 g/ml PG 108.6112.1464.3476.3886.2828.7 166.5164.4586.1564.0996.1970.9 1.0 g/ml PG 112.8109.4657.7659.81030.4910.4 142.4145.9671.1674.91163.31439.9 2.5 g/ml PG 124.3136.7644.6712.91293.01117.3 183.0134.8712.3848.91344.31502.8 0.0 g/ml PG 105.8134.393.4102.0135.9118.2 210.4128.2115.3126.6139.3129.9 0.5 g/ml PG 93.0105.8109.0119.7113.1131.9 185.3143.4125.6131.5128.4134.1 1.0 g/ml PG 91.6101.0115.8132.493.2104.9 247.1193.9124.6143.7130.2152.2 2.5 g/ml PG 113.0106.1118.0130.7140.5152.4 192.9207.9172.5167.6152.2170.4 0.0 g/ml PG 104.1118.499.4365.893.2110.5 137.2163.3121.7119.2111.5117.9 0.5 g/ml PG 106.3115.8395.9410.1785.3812.8 171.1172.2527.4528.21021.4995.6 1.0 g/ml PG 111.698.7510.2462.6939.2937.2 135.8166.9647.4663.11136.61411.8 2.5 g/ml PG 97.7135.1593.7555.31019.41047.1 174.8159.8787.3859.91342.51703.2 0.0 g/ml PG 129.7103.5129.1101.4101.7101.1 189.0154.3125.9134.5140.2149.5 0.5 g/ml PG 120.4115.2114.5126.3147.4171.0 197.3148.0137.6123.3149.7146.3 1.0 g/ml PG 110.7109.9130.1118.2103.7101.8 204.2227.6151.7150.7140.5152.2 2.5 g/ml PG 97.0119.0132.9121.6122.5153.8 213.5212.1176.1165.2164.3169.0 Bold indicates PG lacking ABD No Bold Indicates Rabbit antiGoat and Mouse antiRabbit Italics indicate Goat antiE. coli O157:H7 and Mouse antiGoat 74

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Appendix A: (Continued) TABLE 20. Optimal Concentra tion of Detector Antibody HRP goat anti E. coli O157:H7 in Columns ( g/ml) 1.01.02.52.55.05.0 3.0E+087454.38396.78158 .97386.77414.37830.8 3.0E+077246.47924.87819 .17454.57467.17337.9 3.0E+065887.16487.07122 .46892.77150.17117.9 3.0E+052113.92357.23008 .62865.53557.03459.8 3.0E+041191.31318.71646 .91604.32147.92218.6 3.0E+031041.31116.61526 .61470.12161.91979.5 1.5E+071030.21118.81530 .21376.51933.91862.0 No Bacteria1257.11372.31784.71656.72229.42229.1 3.0E+088570.08511.28420 .48491.28603.58335.1 3.0E+078326.08585.68786 .38824.78528.18279.8 3.0E+067950.38187.48607 .08670.48747.88623.5 3.0E+054006.44006.64842 .54463.95277.05029.3 3.0E+041768.81908.42281 .12288.12591.52547.3 3.0E+031466.31495.81925 .21841.82446.42445.9 1.5E+071437.41325.71760 .91727.62437.22070.2 No Bacteria1478.71370.91981.02128.82886.92545.5 3.0E+086850.56956.67310 .97229.67441.97269.4 3.0E+076941.17097.37393 .77128.77474.17365.0 3.0E+066699.56677.47307 .07054.47364.77335.9 3.0E+053323.83247.14093 .64023.14741.94593.5 3.0E+041283.61355.01667 .31661.72042.72041.9 3.0E+031092.31166.31609 .71656.42070.92067.4 1.5E+071055.81103.21617 .11630.42064.62035.6 No Bacteria1149.71138.01828.61798.22625.52697.2 Bold Indicates E. coli K-12 (CFU/ml) No Bold Indicates E. coli O157:H7 (CFU/ml) TABLE 21. The Limit of Detection Usi ng ELISA, Capture Antibody Displacement Concentration of HRP goat anti E. coli O157:H7 ( g/ml) 0.10.10.50.51.01.00.10.10.50.51.01.0 No Bacteria89.686.1155.3218.5329.2297.8 96.996.8203.6207.8247.9235.4 K-12 (6.0x10^6 ) 85.484.4137.8150.3284.6208.4 95.594.0198.5196.7230.9229.1 O157 (9.7x10^281.383.6132.9154.0210.4223.9 86.188.0161.9132.7237.2260.4 O157 (9.7x10^396.799.4146.7182.2300.3272.3 98.697.7106.8107.9217.4247.5 O157 (9.7x10^ 4 321.3317.1650.9773.91027.01089.0 349.8317.6605.1617.41070.91063.9 O157 (9.7x10^51591.91399.84494.93945.15143.75388.6 2534.42527.45969.65451.66958.36994.6 O157 (9.7x10^ 6 2461.72353.15819.75947.27047.76940.5 3737.03735.96250.26274.67906.07936.7 O157 (9.7x10^71697.21735.85084.44868.86241.96216.4 2396.02446.95913.95884.76862.26674.8 No Bacteria95.196.3168.1227.9356.8376.5 81.480.4166.9212.5264.7326.7 K-12 (6.0x10^6 ) 99.195.7142.3161.5297.3261.3 79.181.1138.0124.8240.2177.7 O157 (9.7x10^289.086.2141.9190.0201.4354.2 84.293.1156.2139.1254.8285.2 O157 (9.7x10^393.796.1131.4203.1233.3334.6 102.297.5166.9184.8278.0301.8 O157 (9.7x10^ 4 375.1338.1620.2641.4646.7688.8 381.8349.7880.0878.21181.31203.4 O157 (9.7x10^51744.91706.74213.04107.05188.05232.0 1728.81625.24348.34386.85607.55492.9 O157 (9.7x10^ 6 2840.12781.46257.06202.96861.56915.5 2694.02536.56199.06282.97092.16921.8 O157 (9.7x10^72476.72472.36068.05893.26985.66734.7 1766.81792.15139.64703.56232.16136.6 No Bacteria92.295.7269.9224.8335.3376.4 84.190.5147.0204.0287.7355.8 K-12 (6.0x10^6 ) 90.299.5247.2244.0264.1283.5 77.482.9121.2138.7208.5220.2 O157 (9.7x10^299.396.8141.1118.4196.3197.9 82.286.5169.9129.0190.6193.8 O157 (9.7x10^392.491.8178.2147.8230.2242.9 100.2103.8315.2171.0229.6234.6 O157 (9.7x10^ 4 367.5398.5607.9629.5687.3697.8 354.9347.9826.1813.31100.31146.0 O157 (9.7x10^51827.01466.14290.34267.65570.85237.6 1631.91552.44268.24270.95454.75417.0 O157 (9.7x10^ 6 3017.52330.46816.96480.97163.77080.6 2487.62533.16255.46297.57230.07237.2 O157 (9.7x10^72706.42121.46476.56352.57078.56895.3 1827.71759.65241.35260.06831.96892.4 No Bold Indicates Streptavidin-Biotin-IgG Bold Indicates HSA-PG-IgG 75

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Appendix B: RAPTOR Analysis TABLE 22. The Limit of Detection of Two Ca pture Matrices Using RAPTOR Analysis Strept-BiotinHSA-PGHSA-PGStrept-Biotin Baseline Signals Baseline 1 691.9520.5535.3822.1 Baseline 2 799.2558.5645.3864.2 Baseline 3 913.2590.3747.5949.9 Baseline 4 1013.1622.6820.81115.6 Normalization Coefficients B1/ LC = B1N 1.31.01.0 1.6 B2/ LC = B2N 1.41.01.2 1.5 B3/ LC = B3N 1.51.01.3 1.6 B4/ LC = B4N 1.61.01.3 1.8 Normalized Baselines B1/ B4N 425.2520.5406.0458.8 B2/B4N 491.1558.5489.5482.3 B3/B4N 561.2590.3567.0530.1 B4/B4N 622.6622.6622.6622.6 Average 525.0573.0521.3523.5 STDEV 85.543.794.272.5 %CoV 16.37.618.113.8 E. coli O157:H7 Signals 10^-6 1139.7651.9917.31169.5 10^-5 1228.2685.2965.91303.5 10^-4 1357.3728.31099.61333.7 Normalized Ec Signals 10^-6/B4N 700.4651.9695.8652.7 10^-5/B4N 754.8685.2732.7727.5 10^-4/B4N 834.1728.3834.1744.3 Limit of Detection LOD 781.6704.0804.0740.8 E. coli O157:H7 SALOD 4.0E+01 -81.2-52.1-108.2-88.1 4.0E+02 -26.8-18.8-71.4-13.4 4.0E+03 52.524.330.1 3.5 76

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Appendix B: (Continued) TABLE 22. The Limit of Detection of Two Ca pture Matrices Using RAPTOR Analysis (Continued) Strept-BiotinStrept-BiotinHSA-PGHSA-PG Baseline Signals Baseline 1 479.5454.9501.8579.9 Baseline 2 504.5478.0562.7607.6 Baseline 3 534.7503.6617.4630.7 Baseline 4 570.1521.6665.4649.7 Normalization Coefficients B1/ LC = B1N 1.1 1.01.11.3 B2/ LC = B2N 1.1 1.01.21.3 B3/ LC = B3N 1.1 1.01.21.3 B4/ LC = B4N 1.1 1.01.31.2 Normalized Baselines B1/ B4N 438.7454.9393.4465.6 B2/B4N 461.6478.0441.1487.8 B3/B4N 489.2503.6484.0506.3 B4/B4N 521.6521.6521.6521.6 Average 477.8489.5460.0495.3 STDEV 35.829.255.324.2 %CoV 7.5 6.012.04.9 E. coli O157:H7 Signals 10^-6 600.8543.6664.4668.7 10^-5 653.4568.3735.1693.8 10^-4 2008.61467.82447.51803.3 Normalized Ec Signals 10^-6/B4N 549.7543.6520.8536.9 10^-5/B4N 597.8568.3576.2557.0 10^-4/B4N 1837.71467.81918.61447.7 Limit of Detection LOD 585.1577.1625.9567.9 E. coli O157:H7 SALOD 1.3E+02 -35.4-33.5-105.0-31.0 1.3E+03 12.7-8.8-49.6-10.9 1.3E+06 1252.6890.71292.7879.9 HSA-PGHSA-PGStrept-BiotinStrept-Biotin Baseline Signals Baseline 1 645.8613.9 558434.6 Baseline 2 660.5627.2 567450.8 Baseline 3 661636.1569.5 456 Baseline 4 672.5652 577 466 Normalization Coefficients B1/ LC = B1N 1.51.4 1.3 1.0 B2/ LC = B2N 1.51.4 1.3 1.0 B3/ LC = B3N 1.41.4 1.2 1.0 B4/ LC = B4N 1.41.4 1.2 1.0 Normalized Baselines B1/ B4N 447.5438.8450.7434.6 B2/B4N 457.7448.3457.9450.8 B3/B4N 458.0454.6459.9456.0 B4/B4N 466.0466.0466.0466.0 Average 457.3451.9458.6451.9 STDEV 7.611.4 6.3 13.1 %CoV 1.72.5 1.4 2.9 E. coli O157:H7 Signals 10^-6 761.7685.2581.8478.8 10^-5 802.7702.3 621492.9 10^-4 862.7721.1641.2521.9 Normalized Ec Signals 10^-6/B4N 527.8489.7469.9478.8 10^-5/B4N 556.2502.0501.5492.9 10^-4/B4N 597.8515.4517.8521.9 Limit of Detection LOD 480.0486.2477.6491.2 E. coli O157:H7 SALOD 2.1E+02 47.83.5 -7.7-12.4 2.1E+03 76.215.7 23.9 1.7 2.1E+04 117.729.2 40.2 30.7 77

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Appendix B: (Continued) TABLE 22. The Limit of Detection of Two Ca pture Matrices Using RAPTOR Analysis (Continued) Strept-BiotinStrept-BiotinHSA-PGHSA-PG Baseline Signals Baseline 1 672.7584.7576.1402.8 Baseline 2 703.4594.4617.9433.6 Baseline 3 731.0612.9641.7465.5 Baseline 4 763.8623.1675.8489.6 Normalization Coefficients B1/ LC = B1N 1.7 1.51.41.0 B2/ LC = B2N 1.6 1.41.41.0 B3/ LC = B3N 1.6 1.31.41.0 B4/ LC = B4N 1.6 1.31.41.0 Normalized Baselines B1/ B4N 431.2459.4417.4402.8 B2/B4N 450.9467.0447.7433.6 B3/B4N 468.6481.6464.9465.5 B4/B4N 489.6489.6489.6489.6 Average 460.1474.4454.9447.9 STDEV 24.9 13.730.437.8 %CoV 5.4 2.96.78.4 E. coli O157:H7 Signals 10^-5 796.0637.5721.2519.8 10^-4 860.1671.9765.6546.0 10^-3 1286.5956.01050.0782.6 Normalized Ec Signals 10^-5/B4N 510.2500.9522.5519.8 10^-4/B4N 551.3527.9554.7546.0 10^-3/B4N 824.7751.2760.7782.6 Limit of Detection LOD 534.8515.4546.0561.3 E. coli O157:H7 SALOD 1.0E+02 -24.6-14.5-23.5-41.5 1.0E+03 16.5 12.58.7-15.3 1.0E+04 289.8235.7214.7221.3 HSA-PGHSA-PGStrept-BiotinStrept-Biotin Baseline Signals Baseline 1 618.0 519.0 615.4 476.2 Baseline 2 636.7 502.6 657.2 463.4 Baseline 3 668.0 506.7 694.9 479.8 Baseline 4 695.5 525.7 739.8 491.1 Normalization Coefficients B1/ LC = B1N 1.3 1.1 1.3 1.0 B2/ LC = B2N 1.4 1.1 1.4 1.0 B3/ LC = B3N 1.4 1.1 1.4 1.0 B4/ LC = B4N 1.4 1.1 1.5 1.0 Normalized Baselines B1/ B4N 436.4 484.8 408.5 476.2 B2/B4N 449.6 469.5 436.3 463.4 B3/B4N 471.7 473.4 461.3 479.8 B4/B4N 491.1 491.1 491.1 491.1 Average 462.2 479.7 449.3 477.6 STDEV 24.2 10.0 35.2 11.4 %CoV 5.2 2.1 7.8 2.4 E. coli O157:H7 Signals 10^-5 722.6 546.7 759.9 522.9 10^-4 742.4 554.9 813.6 559.4 10^-3 1069.4 755.51163.6 804.1 10^-2 3811.52678.54411.03091.0 10^-1 6385.54378.07381.05340.5 Normalized Ec Signals 10^-5/B4N 510.2 510.7 504.4 522.9 10^-4/B4N 524.2 518.4 540.1 559.4 10^-3/B4N 755.1 705.8 772.4 804.1 10^-2/B4N 2691.32502.22928.13091.0 10^1/B4N 4508.94089.94899.75340.5 Limit of Detection LOD 534.7 509.7 555.0 511.9 E. coli O157:H7 SALOD 1.0E+02 -24.4 1.0 -50.5 11.0 1.0E+03 -10.5 8.7 -14.9 47.5 1.0E+04 220.4 196.1 217.4 292.2 1.0E+05 2156.71992.52373.22579.1 1.0E+06 3974.23580.14344.74828.6 78

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79 Appendix B: (Continued) TABLE 23.The Limit of Detection in Ground Beef Homogenate Supernatant Fluid HSA-PGHSA-PGHSA-PGHSA-PG Baseline SignalsBaseline 1 474.7397.5322.6301.4 Baseline 2 498.2321.5367.0363.8 Baseline 3 456.1394.0325.2345.4 Baseline 4 465.4372.0301.5315.6 Normalization Coefficients B1/LC = B1N1.61.31.11.0 B2/LC = B2N1.51.01.11.1 B3/LC = B3N1.41.21.01.1 B4/LC = B4N1.51.21.01.0 Normalized Baselines B1/ B4N 307.5322.2322.6287.9 B2/B4N 322.7260.6367.0347.5 B3/B4N 295.5319.3325.2330.0 B4/B4N 301.5301.5301.5301.5 Average 306.8300.9329.1316.7 STDEV 11.728.427.427.0 %CoV 3.89.48.38.5 E. coli O157:H7 Signals 10^-2 559.7488.5351.6350.5 10^-1 913.7598.5456.4474.1 10^0 1313.6864.5551.9508.0 Normalized Ec Signals 10^-2/B4N 362.6395.9351.6334.8 10^-1/B4N 591.9485.1456.4452.9 10^0/B4N 851.0700.7551.9485.3 Limit of Detection LOD 341.9386.1411.3397.7 E. coli O157:H7 SALOD 5.7E+04 20.79.8-59.7-62.9 5.7E+05 250.099.045.155.2 5.7E+06 509.1314.6140.687.6 Strept-BiotinStrept-BiotinHSA-PGHSA-PG Baseline SignalsBaseline 1 380.6337.0339.1351.0 Baseline 2 335.3280.5336.1321.2 Baseline 3 319.1257.0295.8300.2 Baseline 4 306.6270.3292.2299.2 Normalization Coefficients B1/LC = B1N 1.1 1.01.01.0 B2/LC = B2N 1.2 1.01.21.1 B3/LC = B3N 1.2 1.01.21.2 B4/LC = B4N 1.1 1.01.11.1 Normalized Baselines B1/ B4N 335.5337.0313.7317.1 B2/B4N 295.6280.5310.9290.2 B3/B4N 281.3257.0273.6271.2 B4/B4N 270.3270.3270.3270.3 Average 295.7286.2292.1287.2 STDEV 28.5 35.223.421.9 %CoV 9.6 12.38.07.6 E. coli O157:H7 Signals 10^-2 366.5396.5352.1346.9 10^-1 646.6815.5646.3664.0 10^0 835.1850.9726.3855.0 Normalized Ec Signals 10^-2/B4N 323.1396.5325.7313.4 10^-1/B4N 570.0815.5597.9599.9 10^0/B4N 736.2850.9671.9772.4 Limit of Detection LOD 381.2391.8362.2353.0 E. coli O157:H7 SALOD 5.4E+05 -58.1 4.7-36.5-39.6 5.4E+06 188.8423.7235.7246.8 5.4E+07 355.0459.1309.7419.4

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Appendix B: (Continued) TABLE 23.The Limit of Detection in Ground Beef Homogenate Supernatant Fluid Strept-BiotinStrept-BiotinHSA-PGHSA-PG Baseline SignalsBaseline 1 323.8 453.9331.9330.5 Baseline 2 305.1 553.0296.3315.1 Baseline 3 307.7 605.0288.6316.1 Baseline 4 316.0 662.5298.9314.1 Normalization Coefficients B1/LC = B1N 1.0 1.41.01.0 B2/LC = B2N 1.0 1.91.01.1 B3/LC = B3N 1.1 2.11.01.1 B4/LC = B4N 1.1 2.21.01.1 Normalized Baselines B1/ B4N 306.3 204.8331.9314.5 B2/B4N 288.6 249.5296.3299.9 B3/B4N 291.0 273.0288.6300.8 B4/B4N 298.9 298.9298.9298.9 Average 296.2 256.5303.9303.5 STDEV 8.0 40.019.27.4 %CoV 2.7 15.66.32.4 E. coli O157:H7 Signals 10^-2 390.5 693.0323.7357.1 10^-1 853.81086.5446.5682.0 10^0 1621.22254.5617.9873.9 Normalized Ec Signals 10^-2/B4N 369.4 312.7323.7339.8 10^-1/B4N 807.6 490.2446.5649.0 10^0/B4N 1533.51017.2617.9831.6 Limit of Detection LOD 320.3 376.4361.4325.6 E. coli O157:H7 SALOD 1.5E+05 49.1 -63.8-37.714.2 1.5E+06 487.3 113.885.1323.4 1.5E+071213.2 640.7256.5506.0 TABLE 24. The Limit of Detection in Sp inach Homogenate Supernatant Fluid HSA-PGHSA-PGHSA-PGHSA-PG Baseline SignalsBaseline 1 891.3929.8844.0805.2 Baseline 2 1163.11028.31036.11142.3 Baseline 3 1349.51056.51149.11242.9 Baseline 4 1518.2989.91195.11308.1 Normalization Coefficients B1/LC = B1N 1.11.21.01.0 B2/LC = B2N 1.11.01.01.1 B3/LC = B3N 1.31.01.11.2 B4/LC = B4N 1.51.01.21.3 Normalized Baselines B1/ B4N 581.1929.8699.1609.3 B2/B4N 758.41028.3858.2864.4 B3/B4N 879.91056.5951.8940.6 B4/B4N 989.9989.9989.9989.9 Average 802.31001.1874.7851.1 STDEV 175.254.8129.5169.2 %CoV 21.85.514.819.9 E. coli O157:H7 Signals 10^-2 2077.31313.51647.31657.0 10^-1 4603.52123.04312.03415.5 10^0 4785.03096.54862.03525.5 Normalized Ec Signals 10^-2/B4N 1354.41313.51364.51253.9 10^-1/B4N 3001.62123.03571.62584.7 10^0/B4N 3119.93096.54027.22667.9 Limit of Detection LOD 1327.81165.61263.31358.7 E. coli O157:H7 SALOD 2.9E+05 26.6147.9101.1-104.8 2.9E+06 1673.7957.42308.31226.0 2.9E+07 1792.11930.92763.91309.2 80

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Appendix B: (Continued) TABLE 25. Cells Capture on Waveguide Surface, Capture Efficiency HSA-PGStrept-BiotinStrept-BiotinHSA-PG Baseline SignalsBaseline 1 502.9 346.2 375.8429.5 Baseline 2 533.1 378 458.9449.5 Baseline 3 602.9 387.7 516.8462.4 Baseline 4 633.6 407.7 552.4478.8 Normalization Coefficients B1/LC = B1N1.5 1.0 1.11.2 B2/LC = B2N1.4 1.0 1.21.2 B3/LC = B3N1.6 1.0 1.31.2 B4/LC = B4N1.6 1.0 1.41.2 Normalized Baselines B1/ B4N 323.6 346.2 277.4365.7 B2/B4N 343.0 378.0 338.7382.8 B3/B4N 387.9 387.7 381.4393.7 B4/B4N 407.7 407.7 407.7407.7 Average 365.6 379.9 351.3387.5 STDEV 38.9 25.6 56.917.7 %CoV 10.6 6.8 16.24.6 E. coli O157:H7 Signals 10^0 22881323.71837.31513.8 Normalized Ec Signals 10^0 1472.31323.71356.01289.0 Limit of Detection LOD 482.3 456.8 522.0440.7 E. coli O157:H7 SALOD 1.7E+08 989.9 866.9 834.0848.3 HSA-PGHSA-PGStrept-BiotinStrept-Biotin Baseline SignalsBaseline 1 657.5547.2 690.2 673.8 Baseline 2 783626.2 995.3 824.7 Baseline 3 887.6705.71205.41002.7 Baseline 4 885.5760.11358.71038.7 Normalization Coefficients B1/LC = B1N1.21.0 1.3 1.2 B2/LC = B2N1.31.0 1.6 1.3 B3/LC = B3N1.31.0 1.7 1.4 B4/LC = B4N1.21.0 1.8 1.4 Normalized Baselines B1/ B4N 564.4547.2 386.1 493.1 B2/B4N 672.1626.2 556.8 603.5 B3/B4N 761.9705.7 674.3 733.8 B4/B4N 760.1760.1 760.1 760.1 Average 689.6659.8 594.3 647.6 STDEV 93.493.0 161.9 123.7 %CoV 13.514.1 27.2 19.1 E. coli O157:H7 Signals 10^-1 32462315.54559.5 2904 Normalized Ec Signals 10^-1 2786.32315.52550.72125.1 Limit of Detection LOD 969.9938.91080.11018.7 E. coli O157:H7 SALOD 1.7E+07 1816.41376.61470.71106.4 81

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Appendix B: (Continued) TABLE 25. Cells Capture on Waveguide Surf ace, Capture Efficiency (Continued) HSA-PGStrept-BiotinStrept-BiotinHSA-PG Baseline SignalsBaseline 1 345.2 344.1 343.8389.0 Baseline 2 580.5 388.2 421.8536.3 Baseline 3 652.9 419.0 470.8603.0 Baseline 4 737.2 443.6 511.6648.7 Normalization Coefficients B1/LC = B1N1.0 1.0 1.01.1 B2/LC = B2N1.5 1.0 1.11.4 B3/LC = B3N1.6 1.0 1.11.4 B4/LC = B4N1.7 1.0 1.21.5 Normalized Baselines B1/ B4N 207.7 344.1 298.1266.0 B2/B4N 349.3 388.2 365.7366.7 B3/B4N 392.9 419.0 408.2412.3 B4/B4N 443.6 443.6 443.6443.6 Average 348.4 398.7 378.9372.2 STDEV 101.4 42.9 62.677.5 %CoV 29.1 10.8 16.520.8 E. coli O157:H7 Signals 10^0 2000.81549.42073.71037.1 Normalized Ec Signals 10^0 1204.01549.41798.1709.2 Limit of Detection LOD 652.5 527.4 566.6604.7 E. coli O157:H7 SALOD 4.3E+08 551.41022.01231.4104.5 Strept-BiotinStrept-BiotinHSA-PGHSA-PG Baseline SignalsBaseline 1 441 362.1373.2498.3 Baseline 2 475.9 389.8404.7644.4 Baseline 3 517 412.9420.2663.7 Baseline 4 529.5 431.3444.5693.3 Normalization Coefficients B1/LC = B1N 1.2 1.01.01.4 B2/LC = B2N 1.2 1.01.01.7 B3/LC = B3N 1.3 1.01.01.6 B4/LC = B4N 1.2 1.01.01.6 Normalized Baselines B1/ B4N 359.2 362.1362.1310.0 B2/B4N 387.6 389.8392.7400.9 B3/B4N 421.1 412.9407.7412.9 B4/B4N 431.3 431.3431.3431.3 Average 399.8 399.0398.5388.8 STDEV 32.9 29.929.054.0 %CoV 8.2 7.57.313.9 E. coli O157:H7 Signals 10^-1 2172.51622.72568.53575.7 Normalized Ec Signals 10^-1 1769.61622.72492.22224.4 Limit of Detection LOD 498.4 488.7485.4550.7 E. coli O157:H7 SALOD 2.8E+07 1271.21134.02006.81673.7 82

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Appendix B: (Continued) TABLE 25. Cells Capture on Waveguide Surf ace, Capture Efficiency (Continued) Strept-BiotinHSA-PGHSA-PGStrept-Biotin Baseline SignalsBaseline 1 470.6422.1464.1 459.3 Baseline 2 492.5432.4497.6 481.9 Baseline 3 510.2442.6535.3 516.8 Baseline 4 530456.5568.4 540.9 Normalization Coefficients B1/LC = B1N 1.11.01.1 1.1 B2/LC = B2N 1.11.01.2 1.1 B3/LC = B3N 1.21.01.2 1.2 B4/LC = B4N 1.21.01.2 1.2 Normalized Baselines B1/ B4N 405.3422.1372.7 387.6 B2/B4N 424.2432.4399.6 406.7 B3/B4N 439.4442.6429.9 436.2 B4/B4N 456.5456.5456.5 456.5 Average 431.4438.4414.7 421.7 STDEV 21.814.736.4 30.6 %CoV 5.13.38.8 7.3 E. coli O157:H7 Signals 10^-2 2114.82234.12867.5 2222 Normalized Ec Signals 10^-2 1821.52234.12303.01875.3 Limit of Detection LOD 496.8482.5523.8 513.5 E. coli O157:H7 SALOD 1.1E+06 1324.71751.61779.21361.8 Strept-BiotinHSA-PGHSA-PGStrept-Biotin Baseline SignalsBaseline 1 467371.8356.7 394.1 Baseline 2 518422.6409.9 490.6 Baseline 3 550.8447.7457.9 502.9 Baseline 4 565.4446.7485.8 544.5 Normalization Coefficients B1/LC = B1N 1.31.01.0 1.1 B2/LC = B2N 1.31.01.0 1.2 B3/LC = B3N 1.21.01.0 1.1 B4/LC = B4N 1.31.01.1 1.2 Normalized Baselines B1/ B4N 369.0371.8328.0 323.3 B2/B4N 409.3422.6376.9 402.5 B3/B4N 435.2447.7421.0 412.6 B4/B4N 446.7446.7446.7 446.7 Average 415.0422.2393.2 396.3 STDEV 34.535.552.1 52.2 %CoV 8.38.413.3 13.2 E. coli O157:H7 Signals 10^0 2821.514751537.8 1538 Normalized Ec Signals 10^0 2229.21475.01414.01261.8 Limit of Detection LOD 518.4528.8549.6 552.8 E. coli O157:H7 SALOD 1.8E+08 1710.7946.2864.5 708.9 83

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Appendix B: (Continued) TABLE 25. Cells Capture on Waveguide Surf ace, Capture Efficiency (Continued) HSA-PGStrept-BiotinStrept-BiotinHSA-PG Baseline SignalsBaseline 1 647.1 404.6 538.4530.1 Baseline 2 693.5 422.6 593.2549.6 Baseline 3 741.4 453.9 624.1584 Baseline 4 762.7 463.1 639.1607.1 Normalization Coefficients B1/LC = B1N1.6 1.0 1.31.3 B2/LC = B2N1.6 1.0 1.41.3 B3/LC = B3N1.6 1.0 1.41.3 B4/LC = B4N1.6 1.0 1.41.3 Normalized Baselines B1/ B4N 392.9 404.6 390.1404.4 B2/B4N 421.1 422.6 429.8419.2 B3/B4N 450.2 453.9 452.2445.5 B4/B4N 463.1 463.1 463.1463.1 Average 431.8 436.1 433.8433.0 STDEV 31.3 27.2 32.326.3 %CoV 7.3 6.2 7.46.1 E. coli O157:H7 Signals 10^-1 6583.53657.55626.54653 Normalized Ec Signals 10^-1 3997.43657.54077.03549.3 Limit of Detection LOD 525.8 517.7 530.6511.9 E. coli O157:H7 SALOD 7.8E+07 3471.63139.83546.43037.5 HSA-PGHSA-PGStrept-BiotinStrept-Biotin Baseline SignalsBaseline 1 594503.6 478.5 449 Baseline 2 613.8533.9 503.3 474.1 Baseline 3 653.4562.6 519.3 490.6 Baseline 4 679.4587.2 526 503.9 Normalization Coefficients B1/LC = B1N1.31.1 1.1 1.0 B2/LC = B2N1.31.1 1.1 1.0 B3/LC = B3N1.31.1 1.1 1.0 B4/LC = B4N1.31.2 1.0 1.0 Normalized Baselines B1/ B4N 440.6432.2 458.4 449.0 B2/B4N 455.2458.2 482.2 474.1 B3/B4N 484.6482.8 497.5 490.6 B4/B4N 503.9503.9 503.9 503.9 Average 471.1469.3 485.5 479.4 STDEV 28.531.0 20.2 23.6 %CoV 6.16.6 4.2 4.9 E. coli O157:H7 Signals 10^-2 4240.52832.5 3388 3080 Normalized Ec Signals 10^-2 3145.12430.73245.73080.0 Limit of Detection LOD 556.7562.2 546.2 550.3 E. coli O157:H7 SALOD 1.1E+06 2588.41868.42699.52529.7 84

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85 Appendix B: (Continued) TABLE 26. A Comparison of DyLight and Cy5 DyLightCy5Cy5DyLightCy5DyLightDyLightCy5 Baseline SignalsBaseline 1 916.3541.6465.15873230.5542.1750.61742.7 Baseline 2 1029.8575.4488.4634.83377.0585.7764.01742.2 Baseline 3 1127.2600.6519.3689.23899.5592.4798.61764.3 Baseline 4 1202.7632.4549.8724.63993.0611.9819.31776.6 Normalization Coefficients B1/LC = B1N 1.71.00.91.16.01.01.43.2 B2/LC = B2N 1.81.00.81.15.81.01.33.0 B3/LC = B3N 1.91.00.91.16.61.01.33.0 B4/LC = B4N 1.91.00.91.16.51.01.32.9 Normalized Baselines B1/ B4N 481.8541.6535.0512.3495.1542.1560.6600.2 B2/B4N 541.5575.4561.8554.0517.5585.7570.6600.1 B3/B4N 592.7600.6597.3601.5597.6592.4596.4607.7 B4/B4N 632.4632.4632.4632.4611.9611.9611.9611.9 Average 562.1587.5581.6575.1555.5583.0584.9605.0 STDEV 65.238.542.452.857.929.523.55.8 %CoV 11.66.57.39.210.45.14.01.0 E. coli O157:H7 Signals 10^-6 1287659583.3768.73212.0620.6852.81817.7 10^-5 1357.3703.2615.9805.23932.5643.7906.01804.8 10^-4 1477.6761.6691.2887.34020.5716.0975.71849.0 Normalized Ec Signals 10^-6/B4N 676.7659.0670.9670.9492.2620.6636.9626.1 10^-5/B4N 713.7703.2708.4702.7602.6643.7676.7621.6 10^-4/B4N 776.9761.6795.0774.4616.1716.0728.7636.8 Limit of Detection LOD 757.7702.9708.8733.5729.1671.4655.4622.5 E. coli O157:H7 SALOD 6.0E+01 -81.0-43.9-37.9-62.6-236.9-50.8-18.53.6 6.0E+02 -44.00.3-0.4-30 .8-126.5-27.721.3-0.8 6.0E+03 19.358.786.240.9-113.044.673.314.4


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McCabe, Christie Renee.
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A novel antibody based capture matrix utilizing human serum albumin and streptococcal Protein G to increase capture efficiency of bacteria
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by Christie Renee McCabe.
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[Tampa, Fla] :
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2009.
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Thesis (M.S.)--University of South Florida, 2009.
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Text (Electronic thesis) in PDF format.
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ABSTRACT: A novel capture matrix utilizing human serum albumin (HSA) and streptococcal Protein G (PG), which possesses an albumin binding domain (ABD), was used to immobilize antibodies for improved bacterial capture efficiency in immunoassays. Enzyme linked immunosorbent assays (ELISA) were used to characterize and optimize a specific protocol for the HSA-PG capture matrix; which revealed several critical factors that should be considered. The Fc binding domain, on PG, should have high affinity for the species of capture antibody used in the assay. Goat and rabbit species antibodies bound strongly to the Fc binding domain of PG. Displacement of the capture antibody, by the detector antibody should be avoided to reduce background signals. The Fc binding domain on PG should have equivalent or lower affinity for the detector antibody, when compared to the capture antibody. Goat species antibody, used as a detector antibody, did not displace the same-species capture antibody. ELISA analysis showed detection of Escherichia coli O157:H7 cells at 1.0 x 10 CFU/ml using HSA-PG and goat antibody raised against Escherichia coli O157:H7; unlabeled antibody was used for capture while HRP labeled antibody was used for detection. Studies were performed on an automated fiber optic biosensor, RAPTOR, which was used for the rapid detection of pathogens. Biosensor assays showed detection of E. coli O157:H7 at 1.0 x 10 CFU/ml in PBS and 1.0 x 10 CFU/ml in homogenized ground beef supernatant. Capture efficiency of the HSA-PG capture matrix was studied using the biosensor and GFP-E. coli O157:H7. The amount of cells captured was less than one percent of the sample concentration. This limit of detection and capture efficiency was comparable to the streptavidin-biotin capture matrix.
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Advisor: Daniel V. Lim, Ph.D.
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ELISA
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