Methods in Virology (eBook)

Volume VII

Hilary Koprowski, Karl Maramorosch (Herausgeber)

eBook Download: PDF

2014 | 1. Auflage
348 Seiten
Elsevier Science (Verlag)
978-1-4832-6950-4 (ISBN)

Infos

Methods in Virology, Volume VII focuses on the methods used in virology, including radioimmunoassays, microscopy, hybridization, and mutagenesis. The selection first elaborates on monoclonal antibody techniques applied to viruses; competition radioimmunoassays for characterization of antibody reactions to viral antigens; and enzyme immunosorbent assays in plant virology. Discussions focus on the principles of enzyme immunosorbent assay, choice of enzyme and preparation of conjugate, determination of immunoglobulin class, and maintenance and specificity testing of hybridomas. The text then elaborates on electron microscopy for the identification of plant viruses in in vitro preparations and cloning and expression of viral antigens in Escherichia coli and other microorganisms, including influenza virus, expression of foreign coding sequences in Escherichia coli, hepatitis B virus, electron microscope, immunoelectron microscopy, and imaging of nucleic acids. The manuscript takes a look at the detection and characterization of subgenomic RNA in plant viruses; exploring the gene organization of baculoviruses; and spot hybridization for detection of viroids and viruses. Topics include application to viral diseases, mapping mutuations of baculoviruses, transcriptional mapping of baculovirus genomes, and genetic mapping by blot hybridization. The selection is a valuable source of information for researchers interested in the methods employed in virology.

Inhalt

Front Cover 1
Methods in Virology 4
Copyright Page 5
Table of Contents 6
Contributors 10
Preface 12
Contents of Previous Volumes 14
Chapter 1. Monoclonal Antibody Techniques Applied to Viruses 20
I. Introduction 20
II. Immunization Protocol 22
III. Preparation of Cells 25
IV. Hybridization 26
V. Maintenance and Specificity Testing of Hybridomas 26
VI. Cloning of Hybridomas 29
VII. Determination of Immunoglobulin Class 30
VIII. Preparation of Ascites Fluid 30
IX. Concentration and Purification of Immunoglobulins 31
X. Characterization of Viral Antigens Using Monoclonal Antibodies 31
XI. Prospectus 33
XII. Appendix: Preparation of Reagents 34
REFERENCES 36
Chapter 2. Competition Radioimmunoassays for 
 
38 
I. Introduction 38
II. Preparation and Characterization of Reagents 39
III. Basic Methodologies 43
IV. Applicability to Identification of Antibody Populations 53
V. Applicability to Characterization of Viral Antigens 57
VI. Biological Tracking of Viral Proteins 64
VII. Concluding Comments 65
ACKNOWLEDGMENT 66
REFERENCES 66
Chapter 3. Enzyme Immunosorbent Assays in Plant Virology 70
I. Introduction 70
II. Principles of Enzyme Immunosorbent Assay 72
III. Preparation of Sample 74
IV. Production of Antiserum 76
V. Choice of Enzyme and Preparation of Conjugate 82
VI. Substrates 86
VII. Choice of Solid Phase 89
VIII. Types of ELISA Procedure 89
IX. Evaluation of Reagents and Analysis of Results 94
X. Conclusions 101
XI. Appendix: Buffer Formulations 101
REFERENCES 102
Chapter 4. Electron Microscopy for the 
 
106 
I. Introduction 107
II. The Electron Microscope 107
III. Support Films 108
IV. Calibration of Magnification 109
V. Negative Staining 111
VI. Metal Shadowing 126
VII. Immunoelectron Microscopy 127
VIII. Imaging of Nucleic Acids 131
ACKNOWLEDGMENTS 135
REFERENCES 135
Chapter 5. Cloning and Expression of Viral 
 
140 
I. Introduction 141
II. Expression of Foreign Coding Sequences in Escherichia coli 142
III. Hepatitis B Virus 146
IV. Foot-and-Mouth Disease Virus 155
V. Poliovirus: Purification of Polio RNA and Cloning of Polio cDNA 163
VI. Influenza Virus 166
VII. Rabies Virus 169
VIII. Expression of Viral Antigens in Bacillus subtilis 176
IX. Expression of Viral Antigens in Saccharomyces cerevisiae 182
X. Conclusion 185
ACKNOWLEDGMENTS 185
REFERENCES 185
Chapter 6. Spot Hybridization for Detection of Viroids and Viruses 192
I. Introduction 192
II. PSTV Diagnosis by Nucleic Acid Spot Hybridization 194
III. Application to Other Viroid Diseases 200
IV. Application to Viral Diseases 201
V. Supplementary Methodologies 202
REFERENCES 205
Chapter 7. Detection of Viral Nucleic Acids by in Situ Hybridization 208
I. Introduction 208
II. Materials and Methods 209
III. Applications 233
ACKNOWLEDGMENTS 242
REFERENCES 243
Chapter 8. Exploring the Gene Organization of Baculoviruses 246
I. Introduction 246
II. Establishing the Physical Map of a Baculovirus 248
III. Mapping Mutuations of Baculoviruses 253
IV. Correlating Proteins with the Physical Map 260
V. Transcriptional Mapping of Baculovirus Genomes 268
VI. DNA Sequence Homology to Facilitate Mapping of Baculoviruses Other than AcNPV 271
VII. Site-Directed Mutagenesis and Allelic Replacement 272
VIII. Summary 273
IX. Addendum 274
ACKNOWLEDGMENTS 275
REFERENCES 275
Chapter 9. Detection and Characterization of Subgenomic RNA in Plant Viruses 278
I. Introduction 278
II. Detection and Characterization of Encapsidated Viral Subgenomic RNAs 279
III. Detection and Characterization of Subgenomic Viral RNAs in Extracts of Infected Tissues 302
IV. Genetic Mapping by Blot Hybridization 327
V. Concluding Remarks 328
ACKNOWLEDGMENTS 330
REFERENCES 330
Index 338

Produkt-Details

• Erscheint lt. Verlag: 28.6.2014
• Sprache: englisch
• Themenwelt: Medizin / Pharmazie ► Medizinische Fachgebiete
• Themenwelt: Studium ► Querschnittsbereiche ► Infektiologie / Immunologie
• Themenwelt: Naturwissenschaften ► Biologie ► Evolution
• Themenwelt: Technik
• ISBN-10: 1-4832-6950-7 / 1483269507
• ISBN-13: 978-1-4832-6950-4 / 9781483269504