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9780879693145

Antibodies: A Laboratory Manual

by ;
  • ISBN13:

    9780879693145

  • ISBN10:

    0879693142

  • Edition: 1st
  • Format: Paperback
  • Copyright: 1988-01-01
  • Publisher: Cold Spring Harbor Laboratory Press
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List Price: $92.80

Summary

Introduction to immunochemistry for molecular biologists and other nonspecialists. Spiral.

Table of Contents

Preface xi
Immune Response
1(6)
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
7(16)
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
23(14)
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
37(16)
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
53(86)
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
55(4)
Sources of Antigen
59(1)
Pure Antigens
60(1)
Purifying Antigens from Polyacrylamide Gels
61(11)
Locating the Antigen after Electrophoresis
61(6)
Processing of the Gel Fragments for Immunization
67(5)
Haptens
72(1)
Synthetic Peptides
72(16)
Designing the Peptide
75(3)
Coupling Peptides to Carrier Proteins
78(10)
Preparing Antigens from Bacterial Overexpression Vectors
88(4)
Immunizing Animals
92(1)
Choice of Animal
93(3)
Adjuvants
96(4)
Dose of the Antigen
100(1)
Form of the Antigen
100(3)
Routes of Injection
103(11)
Subcutaneous Injections
104(2)
Intramuscular Injections
106(2)
Intradermal Injections
108(2)
Intravenous Injections
110(2)
Intraperitoneal Injections
112(1)
Injections into Lymphoid Organs
112(2)
Boosts
114(2)
Sampling Serum
116(1)
Test Bleeds
116(3)
Serum Preparation
119(1)
Exsanguination
120(1)
Inducing Ascites Fluid in Mice
121(3)
Making Weak Antigens Strong
124(1)
Modifying Antigens
124(4)
Coupling Antigens
128(7)
Immune Complexes as Antigens
135(4)
Monoclonal Antibodies
139(106)
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
148(1)
Stages of Hybridoma Production
148(2)
Immunizing Mice
150(1)
Dose and Form of the Antigen
151(4)
Soluble Proteins
151(2)
Particulate Proteins
153(1)
Proteins Produced by Overexpression
153(1)
Synthetic Peptides
153(1)
Live Cells
153(1)
Nucleic Acids
154(1)
Carbohydrates
154(1)
Route of Inoculation
155(16)
Identifying Individual Mice
171(1)
Test Bleeds
171(2)
Deciding to Boost Again or to Fuse
173(1)
Developing the Screening Method
174(1)
Screening Strategies
175(1)
Antibody Capture Assays
175(13)
Antigen Capture Assays
188(7)
Functional Assays
195(1)
Producing Hybridomas
196(1)
Preparation for Fusions
197(6)
Drug Selections
203(4)
Final Boost
207(1)
Preparing the Parental Cells for Fusions
207(3)
Fusions
210(4)
Feeding Hybridomas
214(2)
Screening
216(2)
Expanding and Freezing Positive Clones
218(1)
Single-Cell Cloning
219(9)
Unstable Lines
228(1)
Contamination
228(3)
Classing and Subclassing of Monoclonal Antibodies
231(7)
Selecting Class-Switch Variants
238(2)
Interspecies Hybridomas
240(1)
Human Hybridomas
241(1)
Future Trends
242(3)
Growing Hybridomas
245(38)
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
247(1)
Tissue Culture
247(10)
Long-Term Storage of Cell Lines
257(4)
Contamination by Bacteria or Fungi
261(4)
Contamination by Mycoplasma
265(6)
Producing and Storing Monoclonal Antibodies
271(6)
Drug Selection
277(6)
Storing and Purifying Antibodies
283(36)
Antibodies are relatively stable proteins that can be stored easily and purified by a large number of common protein chemistry techniques
Storing Antibodies
285(3)
Purifying Antibodies
288(1)
Conventional Methods
289(20)
Purification on Protein A Beads
309(3)
Immunoaffinity Purification of Antibodies
312(7)
Labeling Antibodies
319(40)
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
321(1)
Choice of Label
321(3)
Labeling Antibodies with Iodine
324(3)
Iodinations Using Chemical Oxidation
327(7)
Iodinations Using Enzymatic Oxidation
334(4)
Iodinations Using Bolton--Hunter Reagent
338(2)
Labeling Antibodies with Biotin
340(2)
Labeling Antibodies with Enzymes
342(2)
Coupling Antibodies to Horseradish Peroxidase
344(5)
Coupling Antibodies to Alkaline Phosphatase
349(1)
Coupling Antibodies to β-Galactosidase
350(3)
Labeling Antibodies with Fluorochromes
353(5)
Labeling Monoclonal Antibodies by Biosynthesis
358(1)
Cell Staining
359(62)
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
363(1)
Choice of Antibody
364(1)
Cell Staining with Polyclonal Antibodies
364(1)
Cell Staining with Monoclonal Antibodies
365(1)
Cell Staining with Pooled Monoclonal Antibodies
365(2)
Protocols for Cell Staining
367(1)
Preparation of Cells and Tissues
367(17)
Adherent Cells
367(3)
Suspension Cells
370(4)
Yeast Cells
374(2)
Tissue Sections
376(8)
Fixation
384(6)
Attached Cells
385(3)
Suspension Cells
388(1)
Yeast Cells
389(1)
Antibody Binding
390(6)
Detection
396(20)
Detecting Enzyme-Labeled Reagents
400(9)
Detecting Fluorochrome-Labeled Reagents
409(3)
Detecting Gold-Labeled Reagents
412(2)
Detecting Iodine-Labeled Reagents
414(2)
Mounting
416(3)
Photographing the Samples
419(2)
Immunoprecipitation
421(50)
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
424(1)
Choice of Antibody
425(1)
Immunoprecipitations Using Polyclonal Antibodies
425(1)
Immunoprecipitations Using Monoclonal Antibodies
426(1)
Immunoprecipitations Using Pooled Monoclonal Antibodies
427(2)
Immunoprecipitation Protocols
429(1)
Labeling Protein Antigens
429(17)
Labeling Cells in Tissue Culture
430(8)
Labeling Yeast Cells
438(4)
Labeling Bacteria
442(3)
Iodinating Immunoprecipitated Proteins
445(1)
Lysing Cells
446(15)
Lysis of Tissue Culture Cells
448(4)
Lysis of Yeast Cells
452(5)
Lysis of Bacteria
457(3)
Denaturing Lysis
460(1)
Preclearing the Lysate
461(3)
Forming the Immune Complexes
464(2)
Purifying the Immune Complexes
466(5)
Immunoblotting
471(40)
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
474(1)
Choice of Antibody
475(1)
Immunoblots Using Polyclonal Antibodies
475(1)
Immunoblots Using Monoclonal Antibodies
476(1)
Immunoblots Using Pooled Monoclonal Antibodies
477(2)
Immunoblotting Protocols
479(1)
Sample Preparation
480(4)
Gel Electrophoresis
484(2)
Transfer of Proteins from Gels to Membranes
486(7)
Staining the Blot for Total Protein (Optional)
493(4)
Blocking Nonspecific Binding Sites on the Blot
497(2)
Addition of Antibody
499(3)
Detection
502(9)
Detection with Radiolabeled Reagents
503(1)
Detection with Enzyme-Labeled Reagents
504(7)
Immunoaffinity Purification
511(42)
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
514(2)
Choice of Antibody
516(1)
Immunoaffinity Purification Using Polyclonal Antibodies
516(1)
Immunoaffinity Purification Using Monoclonal Antibodies
517(1)
Immunoaffinity Purification Using Pooled Monoclonal Antibodies
517(2)
Protocols for Immunoaffinity Purification
519(1)
Preparing Antibody Affinity Columns
519(22)
Coupling Antibodies to Protein A Beads
521(7)
Coupling Antibodies to Activated Beads
528(10)
Preparing Antibody-Affinity Columns with Activated Antibodies
538(3)
Binding Antigens to Immunoaffinity Columns
541(6)
Eluting Antigens from Immunoaffinity Columns
547(6)
Eluting the Antigen
550(1)
Strategies for Testing Elution Conditions
551(2)
Immunoassay
553(60)
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
555(2)
Deciding Where to Start
557(2)
Detecting and Quantitating Antigens
559(1)
Detecting and Quantitating Antibodies
560(1)
Protocols for Immunoassays
561(2)
Antibody Capture Assays
563(16)
Two-Antibody Sandwich Assays
579(6)
Antigen Capture Assays
585(6)
Detection
591(8)
Iodine-Labeled Antigens, Antibodies, or Secondary Reagents
591(1)
Biotin-Labeled Antibodies, Antigens, or Secondary Reagents
591(1)
Enzyme-Labeled Antigens, Antibodies, or Secondary Reagents
592(7)
Designing Immunoassays
599(1)
Assay Geometry
600(5)
Solid-Phase Matrices for Immunoassays
605(7)
Alternative Detection Methods
612(1)
Reagents
613(22)
Bacterial Cell Wall Proteins That Bind Antibodies
615(1)
Protein A
616(6)
Preparing S. aureus for Collecting Immune Complexes
620(2)
Protein G
622(1)
Anti-Immunoglobulin Antibodies
622(4)
Preparing Anti-immunoglobulin Antibodies
624(2)
Proteolytic Fragments of Antibodies
626(6)
Adsorption to Remove Nonspecific Binding
632(3)
Preparing Acetone Powders
633(2)
Appendix I Electrophoresis 635(23)
Appendix II Protein Techniques 658(24)
Appendix III General Information 682(8)
Appendix IV Bacterial Expression 690(7)
References 697(14)
Index 711

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