Maramorosch / Koprowski Methods in Virology

Volume VII
1. Auflage 2014
ISBN: 978-1-4832-6950-4
Verlag: Elsevier Science & Techn.
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, 348 Seiten, Web PDF

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.

Autoren/Hrsg.

Maramorosch, Karl

Koprowski, Hilary

Inhaltsverzeichnis

1;Front Cover;1
2;Methods in Virology;4
3;Copyright Page;5
4;Table of Contents;6
5;Contributors;10
6;Preface;12
7;Contents of Previous Volumes;14
8;Chapter 1. Monoclonal Antibody Techniques Applied to Viruses;20
8.1;I. Introduction;20
8.2;II. Immunization Protocol;22
8.3;III. Preparation of Cells;25
8.4;IV. Hybridization;26
8.5;V. Maintenance and Specificity Testing of Hybridomas;26
8.6;VI. Cloning of Hybridomas;29
8.7;VII. Determination of Immunoglobulin Class;30
8.8;VIII. Preparation of Ascites Fluid;30
8.9;IX. Concentration and Purification of Immunoglobulins;31
8.10;X. Characterization of Viral Antigens Using Monoclonal Antibodies;31
8.11;XI. Prospectus;33
8.12;XII. Appendix: Preparation of Reagents;34
8.13;REFERENCES;36
9;Chapter 2. Competition Radioimmunoassays for
Characterization of Antibody Reactions
to Viral Antigens;38
9.1;I. Introduction;38
9.2;II. Preparation and Characterization of Reagents;39
9.3;III. Basic Methodologies;43
9.4;IV. Applicability to Identification of Antibody Populations;53
9.5;V. Applicability to Characterization of Viral Antigens;57
9.6;VI. Biological Tracking of Viral Proteins;64
9.7;VII. Concluding Comments;65
9.8;ACKNOWLEDGMENT;66
9.9;REFERENCES;66
10;Chapter 3. Enzyme Immunosorbent Assays in Plant Virology;70
10.1;I. Introduction;70
10.2;II. Principles of Enzyme Immunosorbent Assay;72
10.3;III. Preparation of Sample;74
10.4;IV. Production of Antiserum;76
10.5;V. Choice of Enzyme and Preparation of Conjugate;82
10.6;VI. Substrates;86
10.7;VII. Choice of Solid Phase;89
10.8;VIII. Types of ELISA Procedure;89
10.9;IX. Evaluation of Reagents and Analysis of Results;94
10.10;X. Conclusions;101
10.11;XI. Appendix: Buffer Formulations;101
10.12;REFERENCES;102
11;Chapter 4. Electron Microscopy for the
Identification of Plant Viruses in
in Vitro Preparations;106
11.1;I. Introduction;107
11.2;II. The Electron Microscope;107
11.3;III. Support Films;108
11.4;IV. Calibration of Magnification;109
11.5;V. Negative Staining;111
11.6;VI. Metal Shadowing;126
11.7;VII. Immunoelectron Microscopy;127
11.8;VIII. Imaging of Nucleic Acids;131
11.9;ACKNOWLEDGMENTS;135
11.10;REFERENCES;135
12;Chapter 5. Cloning and Expression of Viral
Antigens in Escherichia coli and
Other Microorganisms;140
12.1;I. Introduction;141
12.2;II. Expression of Foreign Coding Sequences in Escherichia coli;142
12.3;III. Hepatitis B Virus;146
12.4;IV. Foot-and-Mouth Disease Virus;155
12.5;V. Poliovirus: Purification of Polio RNA and Cloning of Polio cDNA;163
12.6;VI. Influenza Virus;166
12.7;VII. Rabies Virus;169
12.8;VIII. Expression of Viral Antigens in Bacillus subtilis;176
12.9;IX. Expression of Viral Antigens in Saccharomyces cerevisiae;182
12.10;X. Conclusion;185
12.11;ACKNOWLEDGMENTS;185
12.12;REFERENCES;185
13;Chapter 6. Spot Hybridization for Detection of Viroids and Viruses;192
13.1;I. Introduction;192
13.2;II. PSTV Diagnosis by Nucleic Acid Spot Hybridization;194
13.3;III. Application to Other Viroid Diseases;200
13.4;IV. Application to Viral Diseases;201
13.5;V. Supplementary Methodologies;202
13.6;REFERENCES;205
14;Chapter 7. Detection of Viral Nucleic Acids by in Situ Hybridization;208
14.1;I. Introduction;208
14.2;II. Materials and Methods;209
14.3;III. Applications;233
14.4;ACKNOWLEDGMENTS;242
14.5;REFERENCES;243
15;Chapter 8. Exploring the Gene Organization of Baculoviruses;246
15.1;I. Introduction;246
15.2;II. Establishing the Physical Map of a Baculovirus;248
15.3;III. Mapping Mutuations of Baculoviruses;253
15.4;IV. Correlating Proteins with the Physical Map;260
15.5;V. Transcriptional Mapping of Baculovirus Genomes;268
15.6;VI. DNA Sequence Homology to Facilitate Mapping of Baculoviruses Other than AcNPV;271
15.7;VII. Site-Directed Mutagenesis and Allelic Replacement;272
15.8;VIII. Summary;273
15.9;IX. Addendum;274
15.10;ACKNOWLEDGMENTS;275
15.11;REFERENCES;275
16;Chapter 9. Detection and Characterization of Subgenomic RNA in Plant Viruses;278
16.1;I. Introduction;278
16.2;II. Detection and Characterization of Encapsidated Viral Subgenomic RNAs;279
16.3;III. Detection and Characterization of Subgenomic Viral RNAs in Extracts of Infected Tissues;302
16.4;IV. Genetic Mapping by Blot Hybridization;327
16.5;V. Concluding Remarks;328
16.6;ACKNOWLEDGMENTS;330
16.7;REFERENCES;330
17;Index;338