Immunological Methods a compendium of basic research techniques being used in one of the largest immunology research institutes, the Basel Institute for Immunology, with particular emphasis given to new methodology. The procedures have been described by individuals judged to be highly expert in their specialties. In many instances the methods developed or adapted to unique uses by the contributors have not previously been described in detail. The book contains 34 chapters covering techniques for detection, isolation, and purification of antibodies (including dansylation, two-dimensional chromatography, isoelectric focusing, polyacrylamide gel electrophoresis, and isotachophoresis); measurement of equilibrium constants (equilibrium dialysis, filtration, and sedimentation); and isotope and fluorescent labeling and detection of cell-surface components. Techniques such as isotope laboratory maintenance; chemical modification of proteins, haptens, and solid supports, and haptenation of viable biological carriers; production of antisera against allotypes and histocompatibility antigens and production of antibody with clonai dominance; histocompatibility and MLR testing; and cell separation by haptenated gels and by velocity sedimentation of rosette-forming cells are also discussed. Other chapters cover detection of antibody-secreting and alloantigen-binding cells; immune responses in vitro and their analysis by limiting dilution; production of T-cell factors; hybridoma production by cell fusion; maintenance of cell lines and cloning in semisolid media; and the mathematical analysis of immunological data.
Front Cover 1
Immunological Methods 4
Copyright Page 5
Table of Contents 6
List of Contributors 16
Preface 18
Abbreviations List 22
Chapter 1. The Quality of Antibodies and Cellular Receptors 26
I. INTRODUCTION 26
II. SIMPLE EQUILIBRIA 27
III. COMPETITIVE EQUILIBRIA 55
REFERENCES 66
Chapter 2. The Isolation and Characterization of Immunoglobulins, Antibodies, and Their Constituent Polypeptide
68
I. INTRODUCTION 69
II. FRACTIONATION
69
III. PURIFICATION OF Ig's 70
IV. FRACTIONATION BY GEL FILTRATION
72
V. ELECTROPHORETIC SEPARATION ON A SOLID
73
VI.
76
VII. IMMUNOADSORBENTS WITH INSOLUBILIZED
83
VIII. SEPARATION OF POLYPEPTIDE CHAINS 85
IX. USE OF PROTEIN A FROM Staphylococcus aureus AS AN IMMUNOADSORBENT FOR THE
87
ACKNOWLEDGMENTS 91
REFERENCES 91
Chapter 3. Peptide Mapping at the Nanomole Level 94
I. OBJECTIVE 94
II. PRINCIPLE OF THE METHOD 95
III. MATERIALS 95
IV. PROCEDURE 97
V. CRITICAL APPRAISAL 100
VI. AN EXAMPLE OF THE APPLICATION OF THE METHOD TO ANTIGENIC VARIANTS OF INFLUENZA-A
102
REFERENCES 104
Chapter 4. Electrophoresis of Proteins in Polyacrylamide Slab
106
I. INTRODUCTION 106
II. PROCEDURES FOR POLYACRYLAMIDE GEL
107
III. CONCLUSION 129
REFERENCES 129
Chapter 5. Resolution of Immunoglobulin Patterns
132
I. INTRODUCTION 132
II. PRINCIPLE OF THE METHOD 133
III. MATERIALS 133
IV. PROCEDURES 134
V. APPLICATION, SENSITIVITY, AND REPRODUCIBILITY
142
REFERENCES 145
Chapter 6. Isolation of Monoclonal Antibody by Preparative Isoelectric Focusing in Horizontal
148
I. INTRODUCTION 148
II. PRINCIPLE OF THE METHOD 149
III. MATERIAL 150
IV. PROCEDURE 151
V. APPLICATIONS 153
VI. LIMITATIONS 153
VII. DEGREE OF PURIFICATION AND SENSITIVITY 155
VIII.
155
ACKNOWLEDGMENT 155
REFERENCES 155
Chapter 7. Isotachophoresìs of Immunoglobulins 156
1. INTRODUCTION 156
II. PROCEDURE 157
III. DISCUSSION 160
REFERENCES 161
Chapter 8. The Chemical Modification of Proteins, Haptens, and Solid
162
I. INTRODUCTION 162
II. THEORETICAL BACKGROUND 163
III. EXPERIMENTATION 170
SUGGESTED READING 175
REFERENCES 175
Chapter 9. Reagents for Immunofluorescence and Their Use for Studying Lymphoid
176
I. INTRODUCTION 176
II. REAGENTS FOR IMMUNOFLUORESCENCE 178
III. STAINING PROCEDURES 185
IV. GENERAL COMMENTS 190
SUGGESTED READING 191
REFERENCES 191
Chapter 10. Radiolab eling and Immunoprecipitation
194
I. INTRODUCTION 194
II. LABELING PROCEDURES 195
III. LYSIS OF LABELED CELLS 200
IV. SPECIFIC PURIFICATION OF LABELED CELL-SURFACE
201
REFERENCES 203
Chapter 11. Haptenation of Viable Biological Carriers 206
I. INTRODUCTION 206
II. PREPARATION OF ONS ESTERS 210
III. HAPTENATION OF CARRIERS 214
IV. CML
215
V. OBSERVATIONS ON CML RESPONSES
219
ACKNOWLEDGMENT 219
REFERENCES 219
Chapter 12. Production and Assay of Murine
222
1. PRODUCTION OF ANTI-ALLOTYPE SERUM 224
II. QUANTITATION OF ANTI-ALLOTYPE SERUM 226
III. APPLICATIONS 231
REFERENCES 231
Chapter 13. Preparation of Mouse Antisera against
232
I. OBJECTIVE 232
II. PRINCIPLE OF THE METHOD 233
III. MATERIALS AND PROCEDURE 233
IV. CONTROLS 239
V. CRITICAL APPRAISAL 240
REFERENCES 241
Chapter 14. Technique of HLA Typing by
242
I. INTRODUCTION 242
II.
243
III. DETAILS OF THE TEST 244
IV. FAMILY STUDIES 247
V. SOME COMMENTS ON THE CYTOTOXICITY TEST 247
VI. TECHNIQUE FOR DETECTING B-CELL ANTIGENS OF
250
REFERENCES 251
Chapter 15. The MLR Test in the Mouse 252
I. THE CONVENTIONAL PRIMARY MLR 252
II. IN VITRO SECONDARY MLR 260
III. CRITICAL COMMENTS 262
ACKNOWLEDGMENT 263
REFERENCES 263
Chapter 16. Sensitive Method for the Separation of
266
I. OBJECTIVE 267
II. PRINCIPLE OF THE METHOD 267
III. FORMATION OF ROSETTES 267
IV. CELL FRACTIONATION 269
V. RECOVERY, DEPLETION, AND ENRICHMENT 275
VI. APPLICATIONS, SENSITIVITY, AND LIMITATIONS 277
VII. CONCLUSION 283
ACKNOWLEDGMENTS 283
REFERENCES 284
Chapter 17. The Use
286
I. INTRODUCTION 286
II. PRINCIPLE OF THE METHOD 287
III. MATERIALS 287
IV. PROCEDURES 288
V. CONTROLS 290
VI. CRITICAL ASPECTS 290
VII. APPLICATIONS 291
REFERENCES 292
Chapter 18. Hapten-Gelatin Gels Used as Adsorbents for Separation of Hapten-Specific B
294
I. PRINCIPLE 294
II. DESCRIPTION OF THE TECHNIQUE 295
III. APPLICATIONS 299
IV. LIMITATIONS 300
REFERENCES 300
Chapter 19. Assay for Plaque-Forming Cells 302
I. OBJECTIVE 302
II. PRINCIPLE OF THE METHOD 302
III. MATERIAL 303
IV. PREPARATION OF CELL SUSPENSIONS 304
V. PLAQUING PROCEDURES 305
VI. CALCULATIONS 307
VII. CRITICAL FACTORS 307
REFERENCES 309
Chapter 20. Plaquing and Recovery of Individual
312
I. OBJECTIVE 312
II. MATERIALS 313
III. PROCEDURE 313
REFERENCES 315
Chapter 21. Assay for Specific Alloantigen-Binding T Cells Activated in the Mixed Lymphocyte
316
I. OBJECTIVE 317
II. PRINCIPLE OF THE METHOD 317
III. CELLS AND ALLOANTISERA 317
IV. DETECTION OF T-CELL MARKERS AND STIMULATOR ANTIGENS ON RESPONDER
321
V. ASSAY FOR ALLOANTIGEN-BINDING CELLS 325
VI. CRITICAL APPRAISAL: APPLICATIONS AND
327
VII. CONCLUSION 332
ACKNOWLEDGMENTS 332
REFERENCES 333
Chapter 22. Assay for Antigen-Specific T-Cell
334
I. OBJECTIVE 334
II. PRINCIPLE OF THE METHOD 335
III. MATERIALS 335
IV. PROCEDURE 336
V. CRITICAL APPRAISAL 339
ACKNOWLEDGMENT 340
REFERENCES 340
NOTE ADDED IN PROOF 340
Chapter 23. Antigen-Specific Helper T-Cell Factor
342
I. INTRODUCTION 342
II. PRINCIPLE OF THE METHOD 343
III. MATERIALS 344
IV. PROCEDURE 344
V. CALCULATIONS 347
VI. CRITICAL APPRAISAL 347
REFERENCES 350
Chapter 24. In Vitro Immunization of Dissociated
352
I. OBJECTIVE 352
II. PRINCIPLE OF THE METHOD 353
III. MATERIALS 353
IV. PROCEDURE 355
V. CRITICAL APPRAISAL 357
REFERENCES 359
Chapter 25. Induction of a Secondary Antibody Response in Vitro with Rabbit Peripheral Blood
360
I. OBJECTIVE 360
II. PRINCIPLE OF THE METHOD 361
III. MATERIALS 361
IV. PROCEDURE 363
V. COMMENTS 367
REFERENCES 368
Chapter 26. Induction of Immune Responses with Clonal Dominance at High
370
I. INTRODUCTION 370
II.
372
III. VACCINE PREPARATION 372
IV. IMMUNIZATION 373
V. SERIAL TRANSFER OF LIMITED SPLEEN CELL
376
VI. CRITICAL APPRAISAL 377
SUGGESTED READING 377
REFERENCES 378
Chapter 27. Limiting Dilution Analysis 380
I. OBJECTIVE 381
II. PRINCIPLE OF THE METHOD 381
III. MATERIALS 381
IV. METHODS (FIG. 3, FLOW DIAGRAM) 385
V. LIMITATIONS AND SENSITIVITY 392
SUGGESTED READING 395
REFERENCES 395
Chapter 28. Establishment and Maintenance of
396
I. OBJECTIVE 396
II. PRINCIPLE OF THE METHOD 397
III. MATERIALS 398
IV. PROCEDURE 399
V. CRITICAL APPRAISAL 402
SUGGESTED READING 403
REFERENCES 403
Chapter 29. Clonal Growth of Cells in Semisolid or
404
I. INTRODUCTION 404
II. MATERIALS 405
III. PROCEDURE 406
IV. APPLICATIONS 408
SUGGESTED READING 409
REFERENCES 409
Chapter 30. Preparation of Sendai Virus for
412
I. GROWTH OF VIRUS 412
II. TITRATION OF VIRUS 413
III. CONCENTRATION OF VIRUS 414
IV. INACTIVATION OF VIRUS 414
V. ASSAY OF INFECTIVITY 415
REFERENCES 415
Chapter 31. Fusion of Lymphocytes 416
I. OBJECTIVE 416
II. PRINCIPLE OF THE METHOD 416
III. MATERIAL 417
IV. PROCEDURE 417
V. CRITICAL APPRAISAL 419
REFERENCES 420
Chapter 32. Soft Agar Cloning of Lymphoid Tumor Lines: Detection of Hybrid Clones with
422
I. OBJECTIVE AND PRINCIPLE OF THE METHOD 422
II. MATERIAL 422
III. PROCEDURE 423
V. CRITICAL APPRAISAL 423
REFERENCES 426
Chapter 33. Isotope Laboratory 428
I. INTRODUCTION 428
II. MATERIALS 429
III. SPECIAL PROCEDURES 431
IV. RADIATION AND CONTAMINATION SURVEILLANCE 437
ACKNOWLEDGMENTS 442
SUGGESTED READING 442
REFERENCES 442
Chapter 34. Analysis of Immunological Data 444
I. INTRODUCTION 445
II. WORKED EXAMPLES 445
ACKNOWLEDGMENTS 480
REFERENCES 480
Subject Index 482
The Quality of Antibodies and Cellular Receptors
S. Fazekas de St. Groth
Publisher Summary
This chapter discusses the theory and practice of estimating equilibrium constants in immune systems. The simplest overall measure of the forces holding epitope and paratope together is the equilibrium constant. For practical measurement, at least one of the three components making up the equilibrium mixture (free epitopes, free paratopes, or epitope–paratope complexes) must be separated from the other two. If there are reasonable differences in size, the separation presents no problems. The simple treatment of equilibria assumes independence of epitopes and paratopes. The immunoglobulin molecules found in sera are multivalent and so are most natural antigenic molecules and particles. The most common interaction between antibodies landing on neighboring sites is electrical. If such interactions are present, an additional term for electrostatic free energy change arises and the observed K will not remain constant but appear as a function of the density of epitope–paratope complexes.
I INTRODUCTION
Conventional titrations define an end point which depends on both the quantity and quality of reactants. The reaction can be made of practically zero order by working in great excess of one component, and the quantity of the other may then be estimated independently. Quality cannot be assayed in such systems because either it appears confounded with quantity or its effects are eliminated altogether. This holds equally for binary reactions (such as precipitin tests in solution or in gels, active or passive agglutination, complement fixation, etc.) and for ternary systems where an acceptor and an indicator compete for the third component (such as inhibition of any binary reaction, neutralization of toxins or pathogens, etc.).
The simplest overall measure of the forces holding epitope and paratope together, that is, of quality in the immunological sense, is the equilibrium constant. When not only the extent but also the nature of these forces is to be investigated, thermodynamic parameters (such as changes in enthalpy, entropy, heat capacity, etc.) must be determined. All this, however, amounts to no more than measuring equilibrium constants under different environmental conditions (commonly at different temperatures, less frequently by varying pressure or volume experimentally). This chapter deals, therefore, with the theory and practice of estimating equilibrium constants in immune systems.
II SIMPLE EQUILIBRIA
A Theory
1 Monovalent Reactants
Consider the reaction between epitopes at concentration [E] and paratopes at concentration [P]. Both reagents are in thermal motion and have a calculable chance of colliding with each other. Epitope-paratope complexes, x, will be formed at a rate proportional to the concentration of free reagents ([E − x] and [P − x] and to the rate of effective collisions, k1. Formally, the concentration of complexes will increase with time as
[x]dt=k1[E−x][P−x] (1)
The formed complexes dissociate at a rate of k2, and hence their concentration decreases with time as
d[x]dt=k2[x] (2)
The overall rate is obtained by combining Eqs. (1) and (2):
[x]dt=k1[E−x][P−x]−k2[x] (3)
Equilibrium is defined as the state where d[x]/dt = 0, that is, where association and dissociation exactly balance each other (Note 1). Thus, at equilibrium we have
1[E−x][P−x]−k2[x]=0 (4)
NOTE 1.
The approach to equilibrium is obtained by integrating Eq. (3). Thus, the concentration of epitope-paratope complexes at time t is
t]=x1(x0−x2)−x2(x0−x1)exp{(x1−x2)k1(t−t0)}(x0−x2)−(x0−x1)exp{(x1−x2)k1(t−t0)}
or, conversely, the time required to reach a particular concentration of complexes, xt, is
=1k1(x1−x2)ln(xt−x1)(x0−x2)(xt−x2)(x0−x1)
where x0 is the concentration of complexes at zero time and x1,x2 are the roots of Eq. (5),X1, X2 = 1/2{E + P + K + [(E + P + K)2− 4EP]1/2}. The equilibrium concentration, x, reached at is obviouslyX2, the smaller root of Eq. (5).
The equilibrium constant, K, is simply the ratio of k2 and k1, as seen by rearranging Eq. (4):
2k1=[E−x][P−x][x]=k=[free epitopes][free paratopes][complexers] (5)
The equilibrium constant has the dimensions of concentration and is conventionally expressed in terms of molarity (moles per liter). With biological material, such as cells, it is more meaningful to use cgs units, that is, to speak of topes per cubic centimeter (cf. Jerne et al., 1974). Since there are 6.023 × 1023 molecules per mole, and thus 6.023 × 1020 in 1 cm3 of a molar solution, the two notations are readily interconvertible: Kcgs = 6.023 X 1020Kmol.
(Some immunochemists define K as
1k2=[x][E−x][P−x]
a formally unobjectionable alternative which, however, imparts to K the quaint dimension of liters per mole. This may stun those accustomed to thinking in concentrations and has to be inverted in any case for conventional thermodynamic calculations.)
2 Multivalent Reactants
Equation (5) holds for monovalent reagents, such as small haptens and Fab fragments. Considering the valency of whole antibody molecules, that is, a number n of paratopes/antibody (making P = nA), the equilibrium equation becomes
E−x][nA−x][x]=k (5a)
For large multivalent antigens (such as cells, bacteria, viruses) of concentration [C], each carrying n epitopes, the corresponding equation is
nC−x][P−x][x]=k (5b)
(The valency of antibodies can usually be ignored in this situation. To check the correctness of this practice, the behavior of whole Ig molecules may be compared with Fab fragments derived from them.)
3 Microscopic Equilibria
Equation (5b) does not take into account the fact that epitopes are found on a large particle and thus occur in parcels of n. It can be shown, however, that in the absence of interaction between epitopes their behavior is the same, irrespective of whether they occur singly or as part of the surface of large carriers (Note 2).
NOTE 2.
Consider the microscopic equilibrium between the (i -1)th and the ith epitope on, say, a red cell:
i=xt[P−x]xi−1 (6)
Here xi denotes the sum of all classes in which i out of n sites can be occupied. Note that
i=xi−1Ki[P−x]=(c−∑1nxt)∏1iKi[P−x]i (7)
If all sites are identical and equivalent, a single equilibrium constant, K, characterizes all associations and Ki = K[n - (i − 1)]/i, since there are n - (i − 1) ways of adding an antibody molecule to make xi out of xi−1 and i ways in which a molecule can dissociate from xi to make xi−1. Equation (7) may be rewritten accordingly as
i=(ni)(C−∑1nxi)(KP−x)i (7a)
| Erscheint lt. Verlag | 28.6.2014 |
|---|---|
| Sprache | englisch |
| Themenwelt | Sachbuch/Ratgeber ► Natur / Technik ► Naturführer |
| Studium ► Querschnittsbereiche ► Infektiologie / Immunologie | |
| Naturwissenschaften ► Biologie ► Zoologie | |
| ISBN-10 | 1-4832-6999-X / 148326999X |
| ISBN-13 | 978-1-4832-6999-3 / 9781483269993 |
| Haben Sie eine Frage zum Produkt? |
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