Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1993 Latex agglutination tests for selected Escherichia coli enzymes Mark John Wolcott Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Analytical Chemistry Commons, and the Microbiology Commons Recommended Citation Wolcott, Mark John, "Latex agglutination tests for selected Escherichia coli enzymes " (1993). Retrospective Theses and Dissertations. 10288. https://lib.dr.iastate.edu/rtd/10288 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. 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Ann Arbor, MI 48106 Latex agglutination tests for selected Escherichia coli enzymes by Mark John Wolcott A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department: Microbiology, Immunology and Preventive Medicine Major: Microbiology Approved; Signature was redacted for privacy. In Charge of Major Work Signature was redacted for privacy. For the Major Program Signature was redacted for privacy. Signature was redacted for privacy. Graduate College Iowa State University Ames, Iowa 1993 ii TABLE OF CONTENTS GENERAL INTRODUCTION 1 Statement of the Problem 1 Dissertation Format 3 PARTI. LITERATURE REVIEW 4 INTRODUCTION 5 Tiie Food Problem 5 Indicator Organisms 5 Criteria for indicator or index organisms 6 Indicator organisms 6 Rapid Methods 7 Reasons for rapid methods 7 Types of microbiological rapid methods 8 IMMUNOASSAYS 9 History 9 Basic Concepts and Principles of Immunoassays 9 Antibodies 10 Antigens 10 Types of Serological Reactions 10 Precipitation 10 Agglutination 11 Radioimmunoassay 11 Enzyme immunoassay 11 Affinity chromatography 12 Application to Food and Water 12 Obstacles In Using Immunoassays 13 LATEX TESTS 14 Fabrication of Latex 14 Preparation of Latex Agglutination Assay Reagents 15 Cleaning latex 16 Binding of ligand 17 Adsorption 17 Covalent coupling 20 Optimization 21 Uses of Latex Particles 22 Changes in performance 22 Automated agglutination assays 23 Filter based particle assays 24 Latex chromatography 25 Magnetic particles 25 Radioimmunoassay 26 iii Nucleic acid assays 26 High density particles 26 Fluorescent microparticles 27 THE ENZYMES 28 Glucuronidase 28 Properties 28 Methods of detection 28 Bacterial identification 32 Glutamate Decarboxylase 36 Properties 36 Methods of detection 36 Bacterial identification 39 Tryptophanase 40 Properties of the enzyme 40 Methods of detection 41 Bacterial identification 42 LITERATURE CITED 44 PART 2. PRODUCTION AND CHARACTERIZATION OF POLYCLONAL ANTIBODIES TO SELECTED E. CO//ENZYMES 69 INTRODUCTION 70 MATERIALS AND METHODS 71 Antibody Production 71 Antibody Purification 72 Protein A antibody purification 73 immunoPure® gentle antibody 73 Antigen-specific affinity chromatography antibody purification 74 DEAE Affi-Gel® Biue antibody purification 75 Dialysis and storage 75 Characterization of Antibodies 75 Polyacryiamide gei electrophoresis 75 Ouchteriony immunodiffusion analysis 76 Western blot analysis 77 EIA analysis 78 RESULTS AND DISCUSSION 81 Antibody Production and Purification 81 Electrophoretic analysis of antibody proteins 82 Nondenaturing conditions 83 Denaturing conditions 84 Ouchteriony 86 Tryptophanase 87 Glucuronidase 87 iv Glutamate decarboxyiase 88 Western Blotting 89 Anti-tryptophanase primary antibody 89 Antl-glutamate decarboxylase primary antibody 91 Anti-glucuronidase primary antibody 92 Absorbed anti-giutamate decarboxylase primary antibody 93 Anti-BSA cross-reactivity 94 EIA analysis 95 SUMMARY AND CONCLUSIONS 97 LITERATURE CITED 100 PART 3. LATEX PREPARATION FOR SENSITIZATION WITH IMMUNOGLOBULINS AND ADSORPTION OF IMMUNOGLOBULINS 141 INTRODUCTION 142 MATERIALS AND METHODS 143 Common Procedures Used in this Project 143 Cleaning Latex for Use 143 Latex Bound Protein Determinations 143 Performance of Agglutination Assays 144 Effects of Saline on Unsensitized Latex 145 Sensitization of Latex to Determine of the Effects of Different Brands of Latex and Different Buffers on Protein Adsorption 145 Sensitization of Latex to Determine Maximal Protein Binding 146 Differences in Non-specific Agglutination of Sensitized Latex Resulting from Immunoglobulins 147 Sensitization of latex to determine the effects of antibody purification methods on nonspecific agglutination 147 Sensitization of latex to determine nonspecific agglutination of commercial anti-glucuronidase 148 Sensitization of latex with irrelevant immunoglobulins to determine immunoglobulin-induced nonspecific agglutination 149 RESULTS AND DISCUSSION 150 Effects of Saline on Unsensitized Latex 150 Protein Adsorption to Latex 151 Comparison of latex brands 151 Comparison of binding buffers 152 Determination of Maximum Protein Binding 153 Differences in Non-specific Agglutination of Sensitized Latex Resulting from Immunoglobulins 153 Effects of antibody purification methods on nonspecific agglutination 154 Nonspecific agglutination of commercial anti-glucuronidase 155 V General immunoglobulin induced nonspecific agglutination 157 SUMMARY AND CONCLUSIONS 159 LITERATURE CITED 161 PART 4. EFFECTS OF BUFFERS AND BLOCKING AGENTS ON SENSITIZED LATEX 172 INTRODUCTION 173 MATERIALS AND METHODS 175 Common Procedures Used In thiis Project 175 Stocl< Cultures and Media 175 Cell lysis 175 Sensitization of Latex to Determine the Effects of Assay Conditions on Nonspecific Agglutination 176 Sensitization of anti-glucuronidase latex for testing the effects of different assay buffers on nonspecific agglutination 176 Sensitization of anti-glucuronidase latex for testing the effects of saline on nonspecific agglutination 177 Sensitization of anti glutamate decarboxylase latex for testing the effects of saline on nonspecific agglutination 178 Preparation of Anti-glutamate Decarboxylase Latex with Multiple Blocking Agents . 179 Preparation of Sensitized Latex to Determine the Effects of Sodium Salicylate on Nonspecific Agglutination 180 RESULTS AND DISCUSSION 182 Effects of Assay Conditions on Nonspecific Agglutination 182 Nonspecific agglutination of anti-glucuronidase latex in different assay buffers 182 Nonspecific agglutination of anti-glucuronidase latex in saline 183 Nonspecific agglutination of anti-glutamate decarboxylase latex in saline .. 184 Anti-glutamate decarboxylase sensitized latex with multiple blocking agents 185 Determination of the Effects of Sodium Salicylate on Nonspecific Agglutination .... 186 Lysis buffers 187 SUMMARY AND CONCLUSIONS 189 LITERATURE CITED 190 PART 5. LATEX AGGLUTINATION TESTS WITH CELL CULTURE LYSATES 206 INTRODUCTION 207 vi MATERIALS AND METHODS 208 Preparation of Sensitized Latex 208 Testing of tfie Latex Reagents 209 Calculation of Sensitivity, Specificity, and Efficiency of the Latex Agglutination Assays 210 RESULTS AND DISCUSSION 210 Culture Lysate Results 211 Results With Commercial Enzymes 212 SUMMARY AND CONCLUSIONS 215 LITERATURE CITED 216 GENERAL SUMMARY AND CONCLUSIONS 221 LITERATURE CITED 224 ACKNOWLEDGMENTS 225 1 GENERAL INTRODUCTION Statement of the Problem The association between food and water quaiity and the public health of a society has been well established. To date, however, totally safe food and water supplies do not exist. Food and water supplies require continual monitoring to ensure that they are safe because many are contaminated with potentially pathogenic microorganisms and/or toxic chemicals. On the basis of sheer numbers, microbiological diseases are by far the most prominent food and waterborne threat. The cost in terms