Preface

Immune Response

A simple review of the immune response...definitions of standard terms...Specific interactions between host proteins and foreign molecules control the strength and effectiveness of an immune response. Selective expansion or deletion of antigen-specific lymphocytes is the cellular basis of the response

Antibody Molecules

Structure of the antibody molecule...generation of a functional immunoglobulin heavy- or light-chain gene....Specific mechanisms have evolved to allow the production of a vast repertoire of antigen recognition sites. This repertoire allows an organism to respond to an extensive array of foreign molecules

Antibody--Antigen Interactions

Structure of antibody--antigen interactions...affinity...avidity....Antibodies and antigens are held by a series of noncovalent bonds. The strength of the individual interactions and the overall stability of an antibody--antigen complex determines the ultimate success of every immunochemical test

Antibody Response

Molecular and cellular development of an antibody response...multiple steps of a primary or secondary antibody response....The generation of a strong antibody response relies on cell-to-cell communication among B cells, helper T cells, and antigen presenting cells. Manipulating these interactions allows the tailoring of a response to a chosen antigen

Immunizations

Many molecules can be used as successful immunogens to raise useful antibodies....In many cases, even poor immunogens can be altered to produce better responses

Immunogenicity

Sources of Antigen

Pure Antigens

Purifying Antigens from Polyacrylamide Gels

Locating the Antigen after Electrophoresis

Processing of the Gel Fragments for Immunization

Haptens

Synthetic Peptides

Designing the Peptide

Coupling Peptides to Carrier Proteins

Preparing Antigens from Bacterial Overexpression Vectors

Immunizing Animals

Choice of Animal

Adjuvants

Dose of the Antigen

Form of the Antigen

Routes of Injection

Subcutaneous Injections

Intramuscular Injections

Intradermal Injections

Intravenous Injections

Intraperitoneal Injections

Injections into Lymphoid Organs

Boosts

Sampling Serum

Test Bleeds

Serum Preparation

Exsanguination

Inducing Ascites Fluid in Mice

Making Weak Antigens Strong

Modifying Antigens

Coupling Antigens

Immune Complexes as Antigens

Monoclonal Antibodies

Allelic exclusion ensures that a clonal population of cells arising from an individual B cell will secrete identical antibodies with a unique antigen recognition site. Techniques of cell fusion allow individual B cells to be converted into permanent antibody-secreting cell lines. These monoclonal antibodies can be used to test for the presence of a particular epitope

Production of Monoclonal Antibodies

Stages of Hybridoma Production

Immunizing Mice

Dose and Form of the Antigen

Soluble Proteins

Particulate Proteins

Proteins Produced by Overexpression

Synthetic Peptides

Live Cells

Nucleic Acids

Carbohydrates

Route of Inoculation

Identifying Individual Mice

Test Bleeds

Deciding to Boost Again or to Fuse

Developing the Screening Method

Screening Strategies

Antibody Capture Assays

Antigen Capture Assays

Functional Assays

Producing Hybridomas

Preparation for Fusions

Drug Selections

Final Boost

Preparing the Parental Cells for Fusions

Fusions

Feeding Hybridomas

Screening

Expanding and Freezing Positive Clones

Single-Cell Cloning

Unstable Lines

Contamination

Classing and Subclassing of Monoclonal Antibodies

Selecting Class-Switch Variants

Interspecies Hybridomas

Human Hybridomas

Future Trends

Growing Hybridomas

Hybridomas and myelomas can be grown under standard mammalian tissue culture conditions, and monoclonal antibodies can be collected as spent media or following the induction of ascites in animals

Growing Hybridomas and Myelomas

Tissue Culture

Long-Term Storage of Cell Lines

Contamination by Bacteria or Fungi

Contamination by Mycoplasma

Producing and Storing Monoclonal Antibodies

Drug Selection

Storing and Purifying Antibodies

Antibodies are relatively stable proteins that can be stored easily and purified by a large number of common protein chemistry techniques

Storing Antibodies

Purifying Antibodies

Conventional Methods

Purification on Protein A Beads

Immunoaffinity Purification of Antibodies

Labeling Antibodies

When purified antibodies are labeled with an easily detectable ``tag,'' they can be used to identify specific antigens even when displayed in a complicated mixture of other molecules

Direct Versus Indirect Detection

Choice of Label

Labeling Antibodies with Iodine

Iodinations Using Chemical Oxidation

Iodinations Using Enzymatic Oxidation

Iodinations Using Bolton--Hunter Reagent

Labeling Antibodies with Biotin

Labeling Antibodies with Enzymes

Coupling Antibodies to Horseradish Peroxidase

Coupling Antibodies to Alkaline Phosphatase

Coupling Antibodies to β-Galactosidase

Labeling Antibodies with Fluorochromes

Labeling Monoclonal Antibodies by Biosynthesis

Cell Staining

When labeled antibodies are used to stain cells or tissues, they can be used to determine not only the presence of an antigen but also its localization

Major Constraints

Choice of Antibody

Cell Staining with Polyclonal Antibodies

Cell Staining with Monoclonal Antibodies

Cell Staining with Pooled Monoclonal Antibodies

Protocols for Cell Staining

Preparation of Cells and Tissues

Adherent Cells

Suspension Cells

Yeast Cells

Tissue Sections

Fixation

Attached Cells

Suspension Cells

Yeast Cells

Antibody Binding

Detection

Detecting Enzyme-Labeled Reagents

Detecting Fluorochrome-Labeled Reagents

Detecting Gold-Labeled Reagents

Detecting Iodine-Labeled Reagents

Mounting

Photographing the Samples

Immunoprecipitation

Antibody-antigen complexes can be purified by collection on matrices that specifically bind antibodies. This is a versatile technique for determining many properties of soluble antigens

Major Constraints

Choice of Antibody

Immunoprecipitations Using Polyclonal Antibodies

Immunoprecipitations Using Monoclonal Antibodies

Immunoprecipitations Using Pooled Monoclonal Antibodies

Immunoprecipitation Protocols

Labeling Protein Antigens

Labeling Cells in Tissue Culture

Labeling Yeast Cells

Labeling Bacteria

Iodinating Immunoprecipitated Proteins

Lysing Cells

Lysis of Tissue Culture Cells

Lysis of Yeast Cells

Lysis of Bacteria

Denaturing Lysis

Preclearing the Lysate

Forming the Immune Complexes

Purifying the Immune Complexes

Immunoblotting

Many antigens are easiest to study on immunoblots. Because the antigens are resolved prior to immunochemical detection, antibody binding is not limited to soluble molecules and can be used to detect and quantitate antigens from a wide variety of sources

Major Constraints

Choice of Antibody

Immunoblots Using Polyclonal Antibodies

Immunoblots Using Monoclonal Antibodies

Immunoblots Using Pooled Monoclonal Antibodies

Immunoblotting Protocols

Sample Preparation

Gel Electrophoresis

Transfer of Proteins from Gels to Membranes

Staining the Blot for Total Protein (Optional)

Blocking Nonspecific Binding Sites on the Blot

Addition of Antibody

Detection

Detection with Radiolabeled Reagents

Detection with Enzyme-Labeled Reagents

Immunoaffinity Purification

When antibodies are covalently attached to a solid matrix, they can be used to purify large amounts of a particular antigen. Because of the specificity of the antibody-antigen interaction, these techniques provide excellent results, exceeding all other single-column methods in yield and purity

Major Constraints

Choice of Antibody

Immunoaffinity Purification Using Polyclonal Antibodies

Immunoaffinity Purification Using Monoclonal Antibodies

Immunoaffinity Purification Using Pooled Monoclonal Antibodies

Protocols for Immunoaffinity Purification

Preparing Antibody Affinity Columns

Coupling Antibodies to Protein A Beads

Coupling Antibodies to Activated Beads

Preparing Antibody-Affinity Columns with Activated Antibodies

Binding Antigens to Immunoaffinity Columns

Eluting Antigens from Immunoaffinity Columns

Eluting the Antigen

Strategies for Testing Elution Conditions

Immunoassay

A wide variety of immunoassays can be used to detect and quantitate antigens and antibodies, often well beyond the sensitivity of conventional methods. These assays are particularly useful when a large number of samples need to be analyzed or when extreme sensitivity is required

Types of Immunoassays

Deciding Where to Start

Detecting and Quantitating Antigens

Detecting and Quantitating Antibodies

Protocols for Immunoassays

Antibody Capture Assays

Two-Antibody Sandwich Assays

Antigen Capture Assays

Detection

Iodine-Labeled Antigens, Antibodies, or Secondary Reagents

Biotin-Labeled Antibodies, Antigens, or Secondary Reagents

Enzyme-Labeled Antigens, Antibodies, or Secondary Reagents

Designing Immunoassays

Assay Geometry

Solid-Phase Matrices for Immunoassays

Alternative Detection Methods

Reagents

Bacterial Cell Wall Proteins That Bind Antibodies

Protein A

Preparing S. aureus for Collecting Immune Complexes

Protein G

Anti-Immunoglobulin Antibodies

Preparing Anti-immunoglobulin Antibodies

Proteolytic Fragments of Antibodies

Adsorption to Remove Nonspecific Binding

Preparing Acetone Powders

Appendix I Electrophoresis

Appendix II Protein Techniques

Appendix III General Information

Appendix IV Bacterial Expression

References

Index