Basic Methods in Antibody Production and Characterization

Written for researchers and professionals in the fields of biomedical research, immunology, biochemistry, molecular biology, pathology, and biotechnology, Basic Methods in Antibody Production and Characterization uses a cookbook approach to presenting the methods for the production, characterization, and use of antibodies. Antibodies described include polyclonal and monoclonal and those made by the phage display. Basic Methods in Antibody Production and Characterization focuses on specific aspects of antibodies and takes readers on a step-by-step process from antigen preparation to immunizations, adjuvants, screening, purification, storage, and applications. Introductory material accompanies each chapter, giving readers a better understanding of the methods and applications. Ample references arm researchers with many applications pertaining to the particular technology.

Table of Contents:
STARTING OUT, Delia R. Bethell and Gary C. Howard Monoclonal vs. Polyclonal Antibodies Using This Manual Laboratory Safety ANIMAL WELFARE AND REGULATORY ISSUES, Sigmund T. Rich Introduction Legal Aspects Compliance Problems Compliance Solutions Synopsis of the Animal Welfare Act ANTIGENS, Donald H. Lewis Introduction Haptens, Immunospecificity, and Immunogen-Antibody Interactions Specificity and Cross-Reactivity Peptides As Immunogens Potential Pitfalls ADJUVANTS, Jory R. Baldridge and Michael J. Lacy Introduction General Guidelines and Precautions for Working with Adjuvants Protocols for Using Freund's Adjuvants Protocol for Using Titermax7 Use of Ribi Adjuvant System POLYCLONAL ANTIBODIES, Eric S. Bean Introduction Selection of Host Injection Protocols Dose of Antigen Choice of Adjuvant Route of Immunization Bleeding Testing Serum Samples Post-Production Purification MONOCLONAL ANTIBODY PRODUCTION, Sandy J. Stewart Introduction Cell Lines and Media Immunization Hybridization Protocol Culture and Propagation Cloning and Cryopreservation SCREENING TECHNIQUES, Gamal E. Osman and Warren E. Ladiges Introduction Antibody-Captured ELISA to Detect Specific Antibodies Double Antibody-Sandwich ELISA Antibody Capture on Cell Surface ANTIBODY PRODUCTION BY ASCITES GROWTH, Kristi R. Harkins Introduction Ascites Production in Mice (Monoclonal Antibody) Ascites Production in Mice (Polyclonal Antibody) Ascites Production in Rats (Monoclonal Antibody) GROWTH OF CELL LINES IN BIOREACTORS, Beverly J. Norris, Michael J. Gramer, and Mark Hirschel Introduction Common Bioreactor Themes Batch Culture Fed Batch Culture Perfusion Culture Hollow-Fiber Bioreactor Summary: Choosing a Technology PHAGE DISPLAY, Peter Amersdorfer and James D. Marks Introduction Generation of Human and Murine scFv Gene Repertoires Library Construction Panning Identification of Antigen Binding Clones Expression and Purification of Native Antibody Fragments ANTIBODY PURIFICATION METHODS, Kristi R. Harkins Introduction Preliminary Steps Purification IgG Purification of IgM Purification of IgY (Chicken Yolk) Other Methods Final Product MOLECULAR CHARACTERIZATION OF IMMUNOGLOBULIN GENES, Warren Ladiges and Gamal E. Osman Introduction Cloning immunoglobulin V Regions Using PCR with Universal Primers Cloning Ig V Regions Using Linkered-Ligated PCR ANTIBODY STORAGE, Kathryn Elwell and Delia R. Bethell Introduction Storage of Serum, Ascites, and Tissue-Culture Supernatant Storage of Purified Antibodies Storage of Conjugated Antibodies CHEMICALLY MODIFYING ANTIBODIES, Kathryn Elwell and Gary C. Howard Basic Considerations Sites for Modification Adding a Fluorescent Label Adding Biotin to Amino Groups Activating Carboxyl Groups with EDAC Adding Biotin to the Carbohydrate Group Radioiodination by the Bolton-Hunter Procedure Radioiodination by the Chloramine T Procedure Bifunctional Reagents and Conjugations Cross-Linking with Glutaraldehyde Preparing Antibody-Enzyme Conjugates with SPDP Preparing Antibody-Enzyme Conjugates with MBS Preparing Antibody-Enzyme Conjugates with SMCC Preparing Horseradish Peroxidase-Antibody Conjugates Preparing Antibody-Phycobiliprotein Conjugates WESTERN BLOTTING, Lee Bendickson and Marit Nilsen-Hamilton Introduction Transferring Proteins Antigen Detection Artifacts and Troubleshooting Clinical Applications Conclusion Western Blot Protocol IMMUNOHISTOCHEMICAL METHODS, Kendrick J. Morrell, Neil M. Hand, and Nicholas R. Griffin Introduction Tissue Preparation Enzyme Labels Achieving Specificity Pretreatment Techniques Immunoenzyme Techniques The Indirect Peroxidase Technique The Labeled Streptavidin/Biotin (LSAB) and the Streptavidin Biotin Complex (ABC) Techniques The Alkaline Phosphatase Anti-Alkaline Phosphatase (APAAP) Technique Immunoflourescence Techniques The Direct Immunoflourescence Technique Specialized Techniques