B4931 Sigma

Basic Methods in Antibody Production and Characterization

  •  ISBN-10 0-8493-9445-7

  •  ISBN-13 978-0-8493-9445-4

Properties

publication info   G.C. Howard and D.R. Bethell, ed., CRC Press, 2001, 288 pp., hard cover

Description

General description

This manual 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. It 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. Ample references provide researchers with many applications pertaining to the technology.

Table of Contents

STARTING OUT
Monoclonal vs. Polyclonal Antibodies
Using This Manual
Laboratory Safety
ANIMAL WELFARE AND REGULATORY ISSUES
Introduction Legal Aspects Compliance Problems Compliance Solutions Synopsis of the Animal Welfare Act
ANTIGENS,
Introduction Haptens, Immunospecificity, and Immunogen-Antibody Interactions Specificity and Cross-Reactivity Peptides As Immunogens Potential Pitfalls
ADJUVANTS,
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,
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,
Introduction Cell Lines and Media Immunization Hybridization Protocol Culture and Propagation Cloning and Cryopreservation
SCREENING TECHNIQUES,
Introduction Antibody-Captured ELISA to Detect Specific Antibodies Double Antibody-Sandwich ELISA Antibody Capture on Cell Surface
ANTIBODY PRODUCTION BY ASCITES GROWTH,
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,
Introduction Common Bioreactor Themes Batch Culture Fed Batch Culture Perfusion Culture Hollow-Fiber Bioreactor Summary: Choosing a Technology
PHAGE DISPLAY,
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,
Introduction Preliminary Steps Purification IgG Purification of IgM Purification of IgY (Chicken Yolk) Other Methods Final Product
MOLECULAR CHARACTERIZATION OF IMMUNOGLOBULIN GENES,
Introduction Cloning immunoglobulin V Regions Using PCR with Universal Primers Cloning Ig V Regions Using Linkered-Ligated PCR
ANTIBODY STORAGE,
Introduction Storage of Serum, Ascites, and Tissue-Culture Supernatant Storage of Purified Antibodies Storage of Conjugated Antibodies
CHEMICALLY MODIFYING ANTIBODIES,
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,
Introduction Transferring Proteins Antigen Detection Artifacts and Troubleshooting Clinical Applications Conclusion Western Blot Protocol
IMMUNOHISTOCHEMICAL METHODS,
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

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