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Tables of Contents for Immunochemistry 2
Chapter/Section Title
Page #
Page Count
Contributors
xxi
4
Abbreviations
xxv
1. Antibody affinity measurements
1
30
Helene Saunal
Robert Karlsson
Marc H. V. van Regenmortel
1. Introduction
1
1
2. General theory
2
1
3. Equilibrium constant K measurement
2
9
Equilibrium dialysis
3
1
Ammonium sulfate precipitation
4
1
Measurement of free Ab at equilibrium by ELISA titration
4
5
Measurement of free Ab at equilibrium using BIAcore
9
2
4. Measurement of the kinetic rate constants K(a) and K(d)
11
18
Principles and technology of real time biosensor measurements
11
1
Principle of the method
12
2
Mass transfer during BIAcore experiments
14
3
More complex interactions
17
6
Range of kinetic rate constants measurable with the BIAcore system
23
1
Experimental aspects of the determination of rate and equilibrium constants
24
1
Example of kinetic measurements in BIAcore analyses and interpretation of experimental data
25
4
References
29
2
2. Simple solid phase assays of avidity
31
22
David Goldblatt
1. Introduction
31
2
The measurement of avidity
31
2
2. Competition inhibition assays
33
2
3. Elution assays
35
11
Thiocyanate elution assays
37
5
Urea elution assays
42
2
Diethylamine elution assays
44
2
4. Comparison of elution techniques
46
3
References
49
4
3. Liposomes
53
18
Eric Claassen
Freek van Iwaarden
1. Introduction
53
1
2. Lipids
54
1
3. Multilamellar vesicles
55
3
4. Unilamellar liposomes
58
1
5. Size
59
1
6. Content
59
1
7. Giant liposomes
60
1
8. Cytotoxic T cells
61
1
9. Stealth (PEG; SL) liposomes
62
1
10. Enhanced entrapment
63
1
11. Carbocyanin labelling of liposomes
64
4
12. Conclusions
68
1
References
68
3
4. Immunofluorescence and immunoenzyme histochemistry
71
60
P. Brandtzaeg
T. S. Halstensen
H. S. Huitfeldt
K. N. Valnes
1. Introduction
71
1
2. Immunostaining for fluorescence or light microscopy
72
14
Reagents
72
8
Immunohistochemical staining methods
80
6
3. Multicolour immunostaining and its evaluation
86
24
Primary antibodies from different species
87
1
Primary antibodies from the same species
87
14
Computerized image analysis of multicolour fluorescence
101
9
Confocal microscopy
110
1
4. Methodological considerations
110
7
Tissue preparation methods
110
5
Choice of immunostaining method
115
2
5. Immunohistochemical reliability criteria
117
6
Specificity criteria
117
3
Detection accuracy
120
1
Detection precision
121
1
Detection sensitivity
121
1
Detection efficiency
122
1
6. General conclusions
123
1
Acknowledgements
123
1
References
123
8
5. Confocal laser scanning microscopy
131
18
G. D. Johnson
1. Introduction
131
2
Principle
132
1
2. Practical considerations relating to the equipment
133
3
Microscope specification
133
1
Supplementary equipment
133
1
Operating adjustments
134
2
3. Application of CLSM in immunological studies
136
9
Improved definition
136
1
Image intensity enhancement
137
1
Optical sectioning and 3D reconstruction
137
2
Multichannel analysis
139
3
Quantitative image analysis
142
3
4. Summary
145
3
Future prospects for CLSM in immunology
146
2
References
148
1
6. Flow cytofluorimetry
149
32
G. Damgaard
C. H. Nielsen
R. G. Q. Leslie
1. Introduction
149
1
2. Applications of flow cytofluorimetry
150
2
3. Investigation of membrane glycoprotein expression and molecular interactions at the cell surface
152
7
Quenching and pH dependence
152
1
Calibration
153
1
Measurement of a membrane glycoprotein on blood leucocytes
154
5
Investigation of molecular interactions at the cell surface
159
1
4. Measurement of intracellular proteins and mRNA
159
5
Detection of intracellular proteins
160
1
Measurement of intracellular enzymatic activity
161
1
Measurement of mRNA
162
1
General remarks regarding the investigation of intracellular components
162
2
5. Investigation of cellular activation
164
1
Measurement of intracellular calcium ions
164
1
Measurement of surface activation markers
164
1
6. Investigation of cellular replication
165
3
PCNA and BrdU approaches
166
1
The macromolecular dilution approach
166
2
7. Measurement of cellular uptake and ingestion of soluble and particulate materials
168
4
Binding of opsonized immune complexes to whole blood cells
169
2
Measurement of binding and ingestion by phagocytes
171
1
8. Measurement of phagocytic oxidative processes
172
1
9. Measurement of cell-mediated cytotoxicity and apoptosis
173
5
Cell-mediated cytotoxicity
173
4
Measurement of apoptosis
177
1
References
178
3
7. Analysis of soluble adhesion molecules
181
16
M. G. Bouma
M. P. Laan
M. A. Dentener
W. A. Buurman
1. Introduction
181
1
2. E-selectin
182
1
3. ICAM-1
183
1
4. VCAM-1
184
1
5. Potential roles of soluble adhesion molecules in health and disease
185
1
6. ELISA for sICAM-1
185
8
7. Potential pitfalls
193
1
Acknowledgements
193
1
References
193
4
8. Immunochemical assays for complement components
197
28
R. Wurzner
T. E. Mollnes
B. P. Morgan
1. Introduction
197
4
The complement system
197
1
Clinical importance of the complement system
197
3
Assays for the assessment of complement activation
200
1
2. Collection and preservation of samples
201
2
3. EIAs for the assessment of complement activation
203
4
4. Native-restricted and neoepitope-specific mAbs
207
8
The classical pathway
207
2
C3 and the alternative pathway
209
2
The terminal pathway
211
4
5. mAbs directed against complement control proteins
215
3
6. Allospecific mAbs
218
2
References
220
5
9. Assays for complement receptors
225
32
Istvan Bartok
Mark J. Walport
1. Introduction
225
2
2. Identification of complement receptors using fluorescence-labelled antibodies
227
3
Isolation of human neutrophils
228
1
Assay of cell surface receptors
229
1
3. Radioligand binding assay using C3b dimers to enumerate CR1
230
7
Binding of radiolabelled ligand to CR1
231
2
Calculation of data
233
1
Scatchard plot analysis
234
3
4. Complement receptor production and surface expression
237
5
Immunoprecipitation of surface CR1
237
2
Biosynthetic labelling of CR1
239
3
5. Rosette formation assays
242
11
Generation of fixed fragments on sheep erythrocytes
242
3
Enumeration of complement fragments on sheep erythrocytes
245
3
Rosette formation
248
5
6. Complement receptor-dependent phagocytosis
253
2
Preparing C3bi-coated particles
253
1
Phagocytosis of C3bi-coated fluorescent microspheres
254
1
7. Conclusion
255
1
References
255
2
A1 List of suppliers
257
8
Index
265