Immunological Methods (eBook)

Front Cover 1
Immunological Methods 4
Copyright Page 5
Table of Contents 6
Contributors 14
Preface 18
Contents of Previous Volumes 20
Abbreviations List 24
Chapter 1. Methods in Molecular Immunology: Chromosomal Walking in the Major Histocompatibility Complex 30
I. INTRODUCTION 31
II. MATERIALS 32
III. CONSTRUCTION OF COSMID LIBRARIES 34
IV. SCREENING OF COSMID LIBRARIES 40
V. CHARACTERIZATION OF COSMID CLONES 43
VI. APPRAISAL 46
REFERENCES 47
Chapter 2. DNA-Mediated Tranformation of Lymphoid Cells 50
I. INTRODUCTION 50
II. DOMINANT SELECTABLE MARKERS 51
III. TECHNIQUES OF STABLE GENE TRANSFER 52
IV. OTHER TECHNIQUES OF STABLE GENE TRANSFER 57
REFERENCES 57
Chapter 3. Cloning of cDNAs with Vectors Permitting Expression in Host Cells 60
I. INTRODUCTION 60
II. GENERAL STRATEGIES AND THEIR PROBLEMS 61
III. EXPERIMENTAL PROCEDURES 63
IV. SOME COMMENTS 79
REFERENCES 82
Chapter 4. Construction of Vectors for Immunoglobulin Reverse Genetics 84
I. INTRODUCTION 84
II. GENERAL STRATEGY 85
III. MATERIAL AND STOCK SOLUTIONS 86
IV. EXPERIMENTAL PROCEDURES 87
V. EXAMPLES OF CONSTRUCTIONS 93
REFERENCES 95
Chapter 5. Strategies of Oligonucleotide Synthesis 98
I. INTRODUCTION 98
II. STRATEGIES 99
III. EXPERIMENTAL SECTION 107
IV. REAGENTS AND SOLVENTS THAT MUST BE PURIFIED OR PREPARED 111
REFERENCES 112
Chapter 6. High-performance Liquid Chromatography of Biologically Active Proteins in the Nanogram (Picomole) Range 114
I. INTRODUCTION 115
II. MATERIALS AND METHODS 116
III. SEPARATION BY SIZE: GEL PERMEATION 117
IV. SEPARATION BY CHARGE: ION EXCHANGE AND HYDROXYAPATITE 120
V. SEPARATION BY HYDROPHOBICITY: REVERSE PHASE 124
VI. QUANTITATION AND LIMITS OF DETECTION 132
VII. PROTEIN PURIFICATION 136
REFERENCES 138
Chapter 7. The HPLC of Immunoglobulins 140
I. INTRODUCTION 140
II. HPLC: THEORY, PRACTICE, AND EQUIPMENT 141
III. IMMUNOGLOBULINS ON HPLC 145
IV. DISCUSSION 152
REFERENCES 153
Chapter 8. Preparation of Liposomes Carrying Lymphocyte Membrane Proteins 154
I. INTRODUCTION 154
II. ISOLATION OF MEMBRANE PROTEINS 155
III. PREPARATION OF LIPOSOMES 158
IV. APPLICATIONS 161
REFERENCES 161
Chapter 9. Detection of Glycolipid Antigens with Monoclonal Antibodies 162
I. INTRODUCTION 162
II. PRELIMINARY TESTS 163
III. LIPID EXTRACTION 164
IV. SOLID PHASE RIA 165
V. HAPTEN INHIBITION AND HAPTEN BINDING 168
VI. THIN-LAYER CHROMATOGRAPHY 169
VII. CRITICAL APPRAISAL 173
REFERENCES 173
Chapter 10. Mapping of Novel B Lymphocyte Differentiation Antigens 176
I. INTRODUCTION 176
II. DEFINITION OF B CELL DIFFERENTIATION ANTIGENS BY NEGATIVE SELECTION ASSAY 178
III. TWO-DIMENSIONAL GEL ANALYSIS OF B CELL DIFFERENTIATION ANTIGENS 186
IV. APPENDIX 189
SUGGESTED READING 190
REFERENCES 191
Chapter 11. Use of Large-Scale Two-Dimensional ISODALT Gel Electrophoresis System in Immunology 192
I. OBJECTIVE 193
II. MATERIALS 193
III. METHODS 203
IV. CRITICAL APPRAISAL 211
REFERENCES 214
Chapter 12. Tissue Typing Using Biosynthetically Labeled Monoclonal Antibodies 216
I. INTRODUCTION 216
II. BIOSYNTHETIC LABELING 217
III. PURIFICATION 219
IV. ACTIVITY AND STABILITY 223
V. BINDING ASSAY 223
VI. APPLICATIONS AND LIMITATIONS 225
REFERENCES 227
Chapter 13. Assays for Lymphokines Supporting B Cell Growth 230
I. INTRODUCTION 230
II. MATERIALS 231
III. PREACTIVATION CONDITIONS 233
IV. RECULTIVATION CONDITIONS 235
V. MEASUREMENT OF PROLIFERATION 235
REFERENCES 236
Chapter 14. Establishment of Cellular Microenvironments for in Vitro B Cell Progenitor Differentiation 238
I. INTRODUCTION 238
II. PRINCIPLE OF THE METHOD 240
III. MATERIALS 240
IV. PROCEDURE 242
V. COMMENTS 247
REFERENCES 248
Chapter 15. Development of B Cell Progenitors in Semisolid Agar Cultures 250
I. INTRODUCTION 250
II. EXPERIMENTAL METHODS 251
III. SUMMARY OF RESULTS 260
REFERENCES 261
Chapter 16. Isolation of Human Alloreactive T Helper Clones and Their Use as Polyclonal Activators of B Cells 264
I. OBJECTIVE AND PRINCIPLE OF THE METHOD 264
II. MATERIALS 265
III. PROCEDURE 268
IV. CRITICAL APPRAISAL 270
REFERENCES 273
Chapter 17. Cytolytic T Cell Clones and Hybridomas 274
I. INTRODUCTION 275
II. MATERIALS 275
III. METHODS 278
IV. APPLICATION 285
REFERENCES 291
Chapter 18. Conditions for the Establishment in Vitro of Interleukin 3-Dependent Murine B Cell Precursor Lines 294
I. INTRODUCTION 294
II. MATERIALS 295
III. WEHI-3 SUPERNATANTS 297
IV. ASSAY FOR IL-3 ACTIVITY 297
V. PREPARATION OF CELLS 299
VI. ESTABLISHMENT OF IL-3-DEPENDENT B CELL PRECURSOR LINES 300
VII. CLONING IL-3-DEPENDENT B CELL PRECURSORS 305
VIII. CHARACTERIZING THE CELL LINES 305
IX. CONCLUDING REMARKS 306
REFERENCES 307
Chapter 19. Assays for the Detection of Rheumatoid Factor-Producing Cells 308
I. INTRODUCTION 309
II. HEMOLYSIS ASSAY 309
III. PLAQUE ASSAY 312
IV. RADIOIMMUNOASSAY 314
REFERENCES 317
Chapter 20. Fluorescence Cell Sorter Techniques in Immunology 318
I. INTRODUCTION 319
II. DESCRIPTION OF A CELL SORTER 319
III. PREPARATION OF REAGENTS 323
IV. SAMPLE PREPARATION 325
V. CELL SEPARATION 327
VI. DATA ACQUISITION 329
VII. SINGLE-PARAMETER DATA ANALYSIS AND INTERPRETATION 335
VIII. EXAMPLES 340
ACKNOWLEDGMENTS 344
SUGGESTED READING 344
Chapter 21. Tables for Evaluating Limiting Diluting Experiments 346
I. INTRODUCTION 347
II. PERCENTAGE OF SINGLE CLONES 347
III. CONFIDENCE LIMITS 350
IV. TEST FOR INDEPENDENCE IN 2 x 2 CONTINGENCY TABLES 360
REFERENCES 401
Chapter 22. Development of an Avian Lymphoid Chimera 404
I. INTRODUCTION 404
II. PRINCIPLE OF THE METHOD 405
III. DETAILS OF THE PROCEDURE 405
IV. EVALUATION OF CHIMERAS 407
V. COMMENTS 412
REFERENCES 413
Chapter 23. Avian Embryos in Immunology 414
I. INTRODUCTION 414
II. MATERIALS 415
III. METHODS 417
REFERENCES 430
Chapter 24. Sheep as an Experimental Model for Immunology: Immunological Techniques in Vitro and in Vivo 432
I. INTRODUCTION 432
II. MAINTENANCE OF EXPERIMENTAL SHEEP 434
III. IN VIVO PROCEDURES 435
IV. IN VITRO PROCEDURES 445
ACKNOWLEDGMENTS 451
REFERENCES 451
Chapter 25. Methods Used to Study the Immune System of Xenopus (Amphibia, Anura) 454
I. INTRODUCTION: THE IMMUNE SYSTEM OF XENOPUS 455
II. STRAINS AND SPECIES AVAILABLE 461
III. OPERATING ON XENOPUS 463
IV. PREPARING LYMPHOCYTES FOR CELL CULTURE 469
V. MAKING REAGENTS 473
VI. ANALYZING THE IMMUNE SYSTEM 475
VII. FUNCTIONAL ASSAYS ANALYZING IMMUNE RESPONSES 482
VIII. SOMATIC CELL HYBRID BETWEEN FROG LYMPHOCYTES AND MOUSE MYELOMA CELLS 488
IX. CELL MARKERS, PLOIDY MARKERS 489
X. APPENDIX 491
REFERENCES 492
Index 496