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