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Bioconjugate
Techniques

2nd Edition
This page intentionally left blank
 Bioconjugate
Techniques

2nd Edition
Greg T. Hermanson
Pierce Biotechnology,
Thermo Fisher Scientific,
Rockford, Illinois, USA

AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD

PARIS • SAN DIEGO • SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO
Academic Press is an imprint of Elsevier
The molecular model on the cover is a nitrite reductase enzyme obtained from the RCSB Protein
Data Bank (2afn), as determined by Murphy, M.E., Turley, S., Kukimoto, M., Nishiyama, M.,
Horinouchi, S., Sasaki, H., Tanokura, M., and Adman, E.T. (1995) Structure of Alcaligenes
faecalis nitrite reductase and a copper site mutant, M150E, that contains zinc. Biochemistry 34,
12107–12117. The space-filling model was created from the coordinate file using PovChem and
the final image ray-traced using POV-Ray

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Second edition 2008

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ISBN: 978-0-12-370501-3

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Printed and bound in the United States of America

08 09 10 11 10 9 8 7 6 5 4 3 2
For Amy and Meghan, who, since the first edition was published,
have now become interested in pursuing careers in
microbiology and medicine.
 Contents Overview

PART I
Bioconjugate Chemistry
1. Functional Targets 3
2. The Chemistry of Reactive Groups 169

PART II
Bioconjugate Reagents
3. Zero-Length Crosslinkers 215
4. Homobifunctional Crosslinkers 234
5. Heterobifunctional Crosslinkers 276
6. Trifunctional Crosslinkers 336
7. Dendrimers and Dendrons 346
8. Cleavable Reagent Systems 391
9. Fluorescent Probes 396
10. Bifunctional Chelating Agents and Radioimmunoconjugates 498
11. Biotinylation Reagents 506
12. Iodination Reagents 546
13. Silane Coupling Agents 562
14. Microparticles and Nanoparticles 582
15. Buckyballs, Fullerenes, and Carbon Nanotubes 627
16. Mass Tags and Isotope Tags 649
17. Chemoselective Ligation: Bioorthogonal Reagents 666
18. Discrete PEG Reagents 707

PART III
Bioconjugate Applications
19. Preparation of Hapten–Carrier Immunogen Conjugates 745
20. Antibody Modification and Conjugation 783
21. Immunotoxin Conjugation Techniques 824
22. Preparation of Liposome Conjugates and Derivatives 858
23. Avidin–Biotin Systems 900
24. Preparation of Colloidal Gold-Labeled Proteins 924
25. Modification with Synthetic Polymers 936
26. Enzyme Modification and Conjugation 961
27. Nucleic Acid and Oligonucleotide Modification and Conjugation 969
28. Bioconjugation in the Study of Protein Interactions 1003

vi
 Detailed Contents

Preface to the Second Edition xxiii

Preface to the First Edition xxvi
Acknowledgments xxviii
Health and Safety xxix
Intellectual Property xxx

PART I
Bioconjugate Chemistry
1. Functional Targets
1. Modification of Amino acids, Peptides, and Proteins 3
1.1. Protein Structure and Reactivity 4
Amino Acids 4
Nucleophilic Reactions and the pI of Amino Acid Side Chains 13
Secondary, Tertiary, and Quaternary Structure 15
Prosthetic Groups, Cofactors, and Post-Translational Modifications 19
Protecting the Native Conformation and Activity of Proteins 21
Oxidation of Amino Acids in Proteins and Peptides 23
Solvent Accessibility of Functional Targets in Proteins 29
1.2. Protein Crosslinking Methods 32
2. Modification of Sugars, Polysaccharides, and Glycoconjugates 35
2.1. Carbohydrate Structure and Functionality 36
Basic Sugar Structure 37
Sugar Functional Groups 39
Polysaccharide and Glycoconjugate Structure 44
2.2. Carbohydrate and Glycan Conjugation Methods 49
3. Modification of Nucleic Acids and Oligonucleotides 50
3.1. Polynucleotide Structure and Functionality 51
Nucleotide Functional Groups 53
RNA and DNA Structure 62
3.2. Polynucleotide Crosslinking Methods 66
4. Creating Specific Functionalities 66
4.1. Introduction of Sulfhydryl Residues (Thiolation) 67
Modification of Amines with 2-Iminothiolane (Traut’s Reagent) 67
Modification of Amines with SATA 71
Modification of Amines with SATP 74

vii
viii Detailed Contents

Modification of Amines with SPDP 76

Modification of Amines with SMPT 77
Modification of Amines with N-Acetyl Homocysteine Thiolactone 80
Modification of Amines with SAMSA 81
Modification of Aldehydes or Ketones with AMBH 83
Modification of Carboxylates or Phosphates with Cystamine 84
Modification of Proteins with Cystamine 87
Modification of Nucleic Acids and Oligonucleotides with Cystamine 87
Use of Disulfide Reductants 87
Complete Reduction of Disulfides in Protein Molecules Using DTT 90
Use of DTT to Cleave Disulfide-Containing Crosslinking Agents 91
Ellman’s Assay for the Determination of Sulfhydryls 100
4.2. Introduction of Carboxylate Groups 101
Modification of Amines with Anhydrides 102
Modification of Sulfhydryls with Iodoacetate 109
Modification of Sulfhydryls with BMPA 111
Modification of Hydroxyls with Chloroacetic Acid 113
4.3. Introduction of Primary Amine Groups 114
Modification of Carboxylates with Diamines 114
Modification of Sulfhydryls with N-(-Iodoethyl)trifluoroacetamide [Aminoethyl-8] 118
Modification of Sulfhydryls with Ethylenimine 119
Modification of Sulfhydryls with 2-Bromoethylamine 120
Modification of Sulfhydryls with 2-Aminoethyl-2-aminoethanethiolsulfonate 121
Modification of Carbohydrates with Diamines 122
Modification of Alkylphosphates with Diamines 124
Modification of Aldehydes with Ammonia or Diamines 124
Introduction of Arylamines on Phenolic Compounds 125
Amine Detection Reagents 127
4.4. Introduction of Aldehyde Residues 129
Periodate Oxidation of Glycols and Carbohydrates 130
Oxidase Modification of Sugar Residues 131
Modification of Amines with NHS-Aldehydes (SFB and SFPA) 132
Modification of Amines with Glutaraldehyde 134
Periodate Oxidation of N-Terminal Serine or Threonine Residues 136
4.5. Introduction of Hydrazine or Hydrazide Functionalities 139
Modification of Aldehydes with Bis-Hydrazide Compounds 140
Modification of Carboxylates with Bis-Hydrazide Compounds 142
Modification of Amines with SANH, SHNH, or SHTH 143
Modification of Alkylphosphates with Bis-Hydrazide Compounds 146
4.6. Introduction of Saccharide or Glycan Groups 147
Modification of Amines with Mono(lactosylamido) mono(succinimidyl)suberate 149
Modification of Amine or Hydrazide Molecules by Carbohydrates and Glycans 150
Labeling Glycans with Fluorescent 2-Aminopyridine, 2-Amino Benzamide, or
Anthranilic Acid 153
Synthesis of Glycosylamines for Conjugating Glycans 155
5. Blocking Specific Functional Groups 156
5.1. Blocking Amine Groups 157
Detailed Contents ix
Sulfo-NHS Acetate 157
Acetic Anhydride 158
Citraconic Anhydride 159
Maleic Anhydride 159
5.2. Blocking Sulfhydryl Groups 160
N-Ethyl Maleimide 160
Iodoacetate Derivatives 161
Sodium Tetrathionate 161
Methyl Methanethiosulfonate 163
Ellman’s Reagent 164
Dipyridyl Disulfide Reagents 165
5.3. Blocking Aldehyde Groups 166
Reductive Amination with Tris or Ethanolamine 166
5.4. Blocking Carboxylate Groups 167
Tris or Ethanolamine plus EDC 167

2. The Chemistry of Reactive Groups

1. Amine Reactions 169
1.1. Isothiocyanates 170
1.2. Isocyanates 170
1.3. Acyl Azides 171
1.4. NHS Esters 171
1.5. Sulfonyl Chlorides 173
1.6. Aldehydes and Glyoxals 173
1.7. Epoxides and Oxiranes 174
1.8. Carbonates 175
1.9. Arylating Agents 175
1.10. Imidoesters 176
1.11. Carbodiimides 176
1.12. Anhydrides 178
1.13. Fluorophenyl Esters 179
1.14. Hydroxymethyl Phosphine Derivatives 180
1.15. Guanidination of Amines 181
2. Thiol Reactions 182
2.1. Haloacetyl and Alkyl Halide Derivatives 182
2.2. Maleimides 183
2.3. Aziridines 184
2.4. Acryloyl Derivatives 184
2.5. Arylating Agents 185
2.6. Thiol-Disulfide Exchange Reagents 185
Pyridyl Disulfides 186
TNB-Thiol 187
x Detailed Contents

Disulfide Reductants 187

2.7. Vinylsulfone Derivatives 188
2.8. Metal-Thiol Dative Bonds 188
2.9. Native Chemical Ligation 191
2.10. Cisplatin Modification of Methionine and Cysteine 192
3. Carboxylate Reactions 192
3.1. Diazoalkanes and Diazoacetyl Compounds 193
3.2. Carbonyldiimidazole 194
3.3. Carbodiimides 195
4. Hydroxyl Reactions 195
4.1. Epoxides and Oxiranes 195
4.2. Carbonyldiimidazole 196
4.3. N,N-Disuccinimidyl Carbonate or N-Hydroxysuccinimidyl
Chloroformate 196
4.4. Oxidation with Periodate 197
4.5. Enzymatic Oxidation 198
4.6. Alkyl Halogens 198
4.7. Isocyanates 199
5. Aldehyde and Ketone Reactions 200
5.1. Hydrazine Derivatives 200
5.2. Schiff Base Formation 200
5.3. Reductive Amination 201
5.4. Mannich Condensation 201
6. Active Hydrogen Reactions 202
6.1. Diazonium Derivatives 202
6.2. Mannich Condensation 203
6.3. Iodination Reactions 203
7. Photo-Chemical Reactions 204
7.1. Aryl Azides and Halogenated Aryl Azides 204
7.2. Benzophenones 205
7.3. Anthraquinones 205
7.4. Certain Diazo Compounds 207
7.5. Diazirine Derivatives 208
7.6. Psoralen Compounds 208
8. Cycloaddition Reactions 210
8.1. Diels–Alder Reaction 210
8.2. Complex Formation with Boronic Acid Derivatives 210
8.3. Click Chemistry: Cu1-promoted Azide—Alkyne [32]
Cycloaddition 211
Detailed Contents xi

PART II
Bioconjugate Reagents
3. Zero-Length Crosslinkers
1. Carbodiimides 215
1.1. EDC 216
1.2. EDC plus Sulfo-NHS 219
1.3. CMC 223
1.4. DCC 224
1.5. DIC 226
2. Woodward’s Reagent K 228
3. N,N-Carbonyldiimidazole 228
4. Schiff Base Formation and Reductive Amination 231

4. Homobifunctional Crosslinkers
1. Homobifunctional NHS Esters 235
1.1. DSP and DTSSP 238
1.2. DSS and BS3 241
1.3. DST and Sulfo-DST 243
1.4. BSOCOES and Sulfo-BSOCOES 244
1.5. EGS and Sulfo-EGS 246
1.6. DSG 248
1.7. DSC 249
2. Homobifunctional Imidoesters 250
2.1. DMA 251
2.2. DMP 252
2.3. DMS 253
2.4. DTBP 254
3. Homobifunctional Sulfhydryl-Reactive Crosslinkers 256
3.1. DPDPB 257
3.2. BMH 258
4. Difluorobenzene Derivatives 259
4.1. DFDNB 259
4.2. DFDNPS 260
5. Homobifunctional Photoreactive Crosslinkers 261
5.1. BASED 262
6. Homobifunctional Aldehydes 262
6.1. Formaldehyde 263
6.2. Glutaraldehyde 265
xii Detailed Contents

7. Bis-epoxides 268
7.1. 1,4-Butanediol Diglycidyl Ether 269
8. Homobifunctional Hydrazides 269
8.1. Adipic Acid Dihydrazide 270
8.2. Carbohydrazide 271
9. Bis-diazonium Derivatives 271
9.1. o-Tolidine, Diazotized 272
9.2. Bis-diazotized Benzidine 273
10. Bis-alkyl Halides 274

5. Heterobifunctional Crosslinkers
1. Amine-Reactive and Sulfhydryl-Reactive Crosslinkers 277
1.1. SPDP, LC-SPDP, and Sulfo-LC-SPDP 278
1.2. SMPT and Sulfo-LC-SMPT 281
1.3. SMCC and Sulfo-SMCC 283
1.4. MBS and Sulfo-MBS 286
1.5. SIAB and Sulfo-SIAB 288
1.6. SMPB and Sulfo-SMPB 291
1.7. GMBS and Sulfo-GMBS 292
1.8. SIAX and SIAXX 293
1.9. SIAC and SIACX 295
1.10. NPIA 296
2. Carbonyl-Reactive and Sulfhydryl-Reactive Crosslinkers 297
2.1. MPBH 298
2.2. M2C2H 299
2.3. PDPH 300
3. Amine-Reactive and Photoreactive Crosslinkers 302
3.1. NHS-ASA, Sulfo-NHS-ASA, and Sulfo-NHS-LC-ASA 305
3.2. SASD 306
3.3. HSAB and Sulfo-HSAB 308
3.4. SANPAH and Sulfo-SANPAH 310
3.5. ANB-NOS 312
3.6. SAND 312
3.7. SADP and Sulfo-SADP 314
3.8. Sulfo-SAPB 316
3.9. SAED 316
3.10. Sulfo-SAMCA 319
3.11. p-Nitrophenyl Diazopyruvate 322
3.12. PNP-DTP 323
Detailed Contents xiii

4. Sulfhydryl-Reactive and Photoreactive Crosslinkers 324

4.1. ASIB 325
4.2. APDP 326
4.3. Benzophenone-4-iodoacetamide 328
4.4. Benzophenone-4-maleimide 330
5. Carbonyl-Reactive and Photoreactive Crosslinkers 330
5.1. ABH 331
6. Carboxylate-Reactive and Photoreactive Crosslinkers 332
6.1. ASBA 333
7. Arginine-Reactive and Photoreactive Crosslinkers 333
7.1. APG 334

6. Trifunctional Crosslinkers
1. 4-Azido-2-nitrophenylbiocytin-4-nitrophenyl ester 336
2. Sulfo-SBED 337
3. MTS-ATF-Biotin and MTS-ATF-LC-Biotin 341
4. Hydroxymethyl Phosphine Derivatives 342

7. Dendrimers and Dendrons

1. Dendrimer Construction 346
2. Conjugation to Dendrimers 353
2.1. Coupling to Amine-Dendrimers 356
Modification of Amine-Dendrimers with Sulfo-NHS-LC-SPDP 356
NHS-PEG-Maleimide Coupling to Amine-Dendrimers 359
Coupling Glycoproteins to Amine-Dendrimers by Reductive Amination 361
Blocking of Amines on PAMAM Dendrimers 363
Preparation of Sugar-Dendrimer Derivatives 366
Conjugation of Carboxylate Organic Molecules to Amine-Dendrimers 371
Epoxy Activation of Amine-Dendrimers 373
Biotinylation of Amine-Dendrimers 376
Fluorescent Labeling of Amine Dendrimers 380
3. Dendrimer-Chelate Derivatives for Imaging Applications 383
4. Dendrimer Derivatives as Surface Modification Agents 385
5. Dendrimer Fluorescent Quantum Dots 389

8. Cleavable Reagent Systems

1. Cleavage of Disulfides by Reduction 392
2. Periodate-Cleavable Glycols 393
3. Dithionite-Cleavable Bonds 394
4. Hydroxylamine Cleavable Esters 394
5. Base Labile Sulfones 395
xiv Detailed Contents

9. Fluorescent Probes
1. Fluorescein Derivatives 400
Amine-Reactive Fluorescein Derivatives 401
Sulfhydryl-Reactive Fluorescein Derivatives 406
Aldehyde/Ketone and Cytosine-Reactive Fluorescein Derivatives 412
2. Rhodamine Derivatives 415
Amine-Reactive Rhodamine Derivatives 416
Sulfhydryl-Reactive Rhodamine Derivatives 425
Aldehyde/Ketone and Cytosine-Reactive Rhodamine Derivatives 427
3. Coumarin Derivatives 430
Amine-Reactive Coumarin Derivatives 431
Sulfhydryl-Reactive Coumarin Derivatives 434
Aldehyde- and Ketone-Reactive Coumarin Derivatives 438
4. BODIPY Derivatives 440
Amine-Reactive BODIPY Derivatives 441
Aldehyde/Ketone-Reactive BODIPY Derivatives 444
Sulfhydryl-Reactive BODIPY Derivatives 449
5. Cascade Blue Derivatives 453
Amine-Reactive: Cascade Blue Acetyl Azide 453
Carboxylate-Reactive: Cascade Blue Cadaverine and Cascade
Blue Ethylenediamine 455
Aldehyde/Ketone-Reactive: Cascade Blue Hydrazide 456
6. Lucifer Yellow Derivatives 457
Sulfhydryl-Reactive: Lucifer Yellow Iodoacetamide 458
Aldehyde/Ketone-Reactive: Lucifer Yellow CH 459
7. Phycobiliprotein Derivatives 461
8. Cyanine Dye Derivatives 464
Amine-reactive Cyanine Dyes 467
Thiol-reactive Cyanine Dyes 470
Carbonyl-reactive Cyanine Dyes 472
9. Lanthanide Chelates for Time-resolved Fluorescence 474
10. Quantum Dot Nanocrystals 485
Properties of Quantum Dots 485
Conjugation to QDs 493
Conjugation of Proteins to QDs Using EDC 494
Conjugation to QDs Using sulfo-SMCC 496

10. Bifunctional Chelating Agents and Radioimmunoconjugates

1. DTPA 499
2. DOTA, NOTA, and TETA 500
3. DTTA 501
4. DFA 502
5. Use of Thiolation Reagents for Direct Labeling to Sulfhydryl Groups 503
6. FeBABE 505
Detailed Contents xv

11. Biotinylation Reagents

1. Amine-Reactive Biotinylation Agents 507
1.1. D-Biotin and Biocytin 508
1.2. NHS-Biotin and Sulfo-NHS-Biotin 510
1.3. NHS-LC-Biotin and Sulfo-NHS-LC-Biotin 512
1.4. NHS-Iminobiotin 515
1.5. Sulfo-NHS-SS-Biotin 517
2. Sulfhydryl-Reactive Biotinylation Agents 520
2.1. Biotin-BMCC 520
2.2. Biotin-HPDP 522
2.3. Iodoacetyl-LC-Biotin 524
3. Carbonyl- or Carboxyl-Reactive Biotinylation Agents 525
3.1. Biotin-Hydrazide and Biotin-LC-Hydrazide 526
3.2. Biocytin Hydrazide 528
3.3. 5-(Biotinamido)pentylamine 529
4. Photoreactive Biotinylation Agents 530
4.1. Photobiotin 531
4.2. Psoralen-PEO3-Biotin 533
5. Active Hydrogen-Reactive: p-Aminobenzoyl Biocytin, Diazotized 534
6. Glycan Biotinylation Reagents 537
6.1. Biotinylated Aminopyridine 538
6.2. Biotinyl-L-3-(2-naphthyl)-alanine hydrazide 541
6.3. Biotin-PEG-Phosphine 543

12. Iodination Reagents

1. Chloramine-T 548
2. Iodobeads 550
3. Iodogen 553
4. Lactoperoxidase-Catalyzed Iodination 555
5. Iodinatable Modification and Crosslinking Agents 556
5.1. Bolton–Hunter Reagent 557
5.2. Iodinatable Bifunctional Crosslinking Agents 560

13. Silane Coupling Agents

1. Silane Reaction Strategies 565
1.1. Aqueous/Organic Solvent Deposition 566
1.2. Aqueous Deposition 566
1.3. Organic Solvent Deposition 567
1.4. Vapor Phase Deposition 567
xvi Detailed Contents

2. Functional Silane Compounds 568

2.1. 3-Aminopropyltriethoxysilane and 3-Aminopropyltrimethoxysilane 568
2.2. Carboxyethylsilanetriol 573
2.3. N-(Trimethoxysilylpropyl)ethylenediamine triacetic acid 575
2.4. 3-Glycidoxypropyltrimethoxysilane and
3-Glycidoxypropyltriethoxysilane 577
2.5. Isocyanatopropyltriethoxysilane 579

14. Microparticles and Nanoparticles

1. Particle Types 582
2. Particle Characteristics and Stability 584
3. Particle Concentration 588
4. Polymeric Microspheres and Nanospheres 588
4.1. Passive Adsorption 590
4.2. Covalent Coupling to Polymeric Particles 594
4.3. Coupling to Carboxylate Particles 595
4.4. Coupling to Amine Particles 599
4.5. Coupling to Amine Particles Using Crosslinking Agents 599
4.6. Glutaraldehyde 601
4.7. SPDP Coupling to Amine Particles 602
4.8. NHS-PEGn-Maleimide Coupling to Amine Particles 604
4.9. Coupling to Hydroxyl Particles 606
4.10. Coupling to Hydrazide Particles 613
4.11. Coupling to Epoxy Particles 615
4.12. Coupling to Aldehyde Particles 617
5. Silica Particles 618
5.1. Fluorescent Silica Particles 620
5.2. Silane Functionalization of Silica Particles 625

15. Buckyballs, Fullerenes, and Carbon Nanotubes

1. Buckyballs and Fullerenes 627
1.1. Properties of Fullerenes 627
1.2. Modification of Fullerenes 629
2. Carbon Nanotubes 638
2.1. Nanotube Properties 638
2.2. Nanotube Functionalization 640
2.3. Detergent or Lipid Modification of Carbon Nanotubes 640
2.4. Pyrene Modification of Carbon Nanotubes 644
2.5. Modification of Carbon Nanotubes by Cycloaddition 645
Detailed Contents xvii

16. Mass Tags and Isotope Tags

1. ICAT Reagents 651
2. ECAT Reagents 657
3. Isobaric Tags 659

17. Chemoselective Ligation: Bioorthogonal Reagents

1. Diels–Alder Reagent Pairs 667
2. Hydrazine–Aldehyde Reagent Pairs 669
Protocol for Modification of Amine-Oligo with SANH or SFB 674
Protocol for Modification of Protein or Antibody with SANH or SFB 675
Conjugation Using the Aldehyde/Hydrazine Reaction 675
3. Boronic acid–salicylhydroxamate Reagent Pairs 676
4. Click Chemistry: Cu(1)-promoted Azide–Alkyne [3  2] Cycloaddition 680
5. Staudinger Ligation 690
6. Native Chemical Ligation 697
6.1. Expressed Protein Ligation and Inteins 701

18. Discrete PEG Reagents

1. Homobifunctional PEG Crosslinkers 711
1.1. Bis-NHS Ester PEG Compounds 711
1.2. Bis-Maleimide–PEG Compounds 714
BM(PEG)2, BM(PEG)3, and Bis-MAL–dPEG3 714
2. Heterobifunctional PEG Reagents 718
2.1. Maleimide–PEGn–NHS Ester Compounds 718
2.2. NHS-PEGn-Azide/Alkyne Compounds for Chemoselective Ligation 722
3. Biotinylation Reagents Containing Discrete PEG Linkers 726
3.1. NHS–PEGn–Biotin Compounds 727
3.2. NHS-Chromogenic-PEG3-Biotin 730
3.3. Maleimide-PEGn-Biotin Compounds 732
3.4. Hydrazide-PEG4-Biotin 733
3.5. Biotin-PEGn-Amine Compounds 736
3.6. Biotin–PEG3–Benzophenone 739
4. Discrete PEG Modification Reagents 739

PART III
Bioconjugate Applications
19. Preparation of Hapten–Carrier Immunogen Conjugates
1. The Basis of Immunity 745
2. Types of Immunogen Carriers 747
xviii Detailed Contents

2.1. Protein Carriers 748

KLH 748
BSA and cBSA 749
Thyroglobulin and OVA 751
Tetanus and Diphtheria Toxoids 753
2.2. Liposome Carriers 753
2.3. Synthetic Carriers 754
3. Carbodiimide-Mediated Hapten–Carrier Conjugation 755
4. NHS Ester-Mediated Hapten–Carrier Conjugation 763
5. NHS Ester-Maleimide Heterobifunctional Crosslinker-Mediated
Hapten–Carrier Conjugation 766
6. Active-Hydrogen-Mediated Hapten–Carrier Conjugation 773
6.1. Diazonium Conjugation 773
6.2. Mannich Condensation 776
7. Glutaraldehyde-Mediated Hapten–Carrier Conjugation 779
8. Reductive Amination-Mediated Hapten–Carrier Conjugation 781

20. Antibody Modification and Conjugation

1. Preparation of Antibody–Enzyme Conjugates 787
1.1. NHS Ester–Maleimide-Mediated Conjugation 788
Activation of Enzymes with NHS Ester–Maleimide Crosslinkers 789
Conjugation with Reduced Antibodies 790
Conjugation with 2-Iminothiolane-Modified Antibodies 793
Conjugation with SATA-Modified Antibodies 795
1.2. Glutaraldehyde-Mediated Conjugation 797
One-Step Glutaraldehyde Protocol 798
Two-Step Glutaraldehyde Protocol 800
1.3. Reductive-Amination-Mediated Conjugation 800
Activation of Enzymes with Sodium Periodate 802
Activation of Antibodies with Sodium Periodate 803
Conjugation of Periodate-Oxidized HRP to Antibodies by Reductive
Amination 804
Conjugation of Periodate-Oxidized Antibodies with Amine or
Hydrazide Derivatives 805
1.4. Conjugation Using Antibody Fragments 807
Preparation of F(ab)2 Fragments Using Pepsin 807
Preparation of Fab Fragments Using Papain 808
1.5. Removal of Unconjugated Enzyme from Antibody–Enzyme Conjugates 812
Immunoaffinity Chromatography 813
Nickel-Chelate Affinity Chromatography 814
2. Preparation of Labeled Antibodies 816
2.1. Fluorescently Labeled Antibodies 817
2.2. Radiolabeled Antibodies 819
2.3. Biotinylated Antibodies 821
Detailed Contents xix

21. Immunotoxin Conjugation Techniques

1. Properties and Use of Immunotoxin Conjugates 827
2. Preparation of Immunotoxin Conjugates 829
2.1. Preparation of Immunotoxin Conjugates via Disulfide Exchange
Reactions 833
Pyridyl Disulfide Reagents 834
SPDP 834
SMPT 841
3-(2-Pyridyldithio) Propionate 843
Use of Cystamine, Ellman’s Reagent, or S-Sulfonates 844
2.2. Preparation of Immunotoxin Conjugates via Amine- and
Sulfhydryl-Reactive Heterobifunctional Crosslinkers 847
SIAB 847
SMCC 850
MBS 852
SMPB 854
2.3. Preparation of Immunotoxin Conjugates via Reductive Amination 855
Periodate Oxidation of Glycoproteins Followed by Reductive Conjugation 855
Periodate-Oxidized Dextran as Crosslinking Agent 857

22. Preparation of Liposome Conjugates and Derivatives

1. Properties and Use of Liposomes 858
1.1. Liposome Morphology 858
1.2. Preparation of Liposomes 861
1.3. Chemical Constituents of Liposomes 863
1.4. Functional Groups of Phospholipids 869
2. Derivatization and Activation of Lipid Components 869
2.1. Periodate Oxidation of Liposome Components 870
2.2. Activation of PE Residues with Heterobifunctional Crosslinkers 871
3. Use of Glycolipids and Lectins to Effect Specific Conjugations 877
4. Antigen or Hapten Conjugation to Liposomes 879
5. Preparation of Antibody–Liposome Conjugates 881
6. Preparation of Biotinylated or Avidin-Conjugated Liposomes 883
7. Conjugation of Proteins to Liposomes 885
7.1. Coupling via the NHS Ester of Palmitic Acid 886
7.2. Coupling via Biotinylated PE Lipid Derivatives 888
7.3. Conjugation via Carbodiimide Coupling to PE Lipid Derivatives 888
7.4. Conjugation via Glutaraldehyde Coupling to PE Lipid Derivatives 890
7.5. Conjugation via DMS Crosslinking to PE Lipid Derivatives 892
7.6. Coupling via Periodate Oxidation Followed by Reductive Amination 893
7.7. Conjugation via SPDP-Modified PE Lipid Derivatives 894
7.8. Conjugation via SMPB-Modified PE Lipid Derivatives 895
xx Detailed Contents

7.9. Conjugation via SMCC-Modified PE Lipid Derivatives 896

7.10. Conjugation via Iodoacetate-Modified PE Lipid Derivatives 897

23. Avidin–Biotin Systems

1. The Avidin–Biotin Interaction 900
2. Use of (Strept)avidin–Biotin Interactions in Assay Systems 902
3. Preparation of (Strept)avidin Conjugates 905
3.1. NHS Ester–Maleimide-Mediated Conjugation Protocols 906
3.2. Conjugation using Periodate Oxidation/Reductive Amination 910
3.3. Glutaraldehyde Conjugation Protocol 913
4. Preparation of Fluorescently Labeled (Strept)avidin 914
4.1. Modification with FITC 915
4.2. Modification with Lissamine Rhodamine B Sulfonyl Chloride 916
4.3. Modification with AMCA–NHS 917
4.4. Conjugation with Phycobiliproteins 918
5. Preparation of Hydrazide-Activated (Strept)avidin 919
6. Biotinylation Techniques 920
7. Determination of the Level of Biotinylation 921

24. Preparation of Colloidal Gold-Labeled Proteins

1. Properties and Use of Gold Conjugates 924
2. Preparation of Mono-Disperse Gold Suspensions for Protein Labeling 928
2.1. Preparation of 2 nm Gold Particle Sols 928
2.2. Preparation of 5 nm Gold Particle Sols 928
2.3. Preparation of 12 nm Gold Particle Sols 929
2.4. Preparation of 30 nm Gold Particle Sols 929
3. Preparation of Protein A–Gold Complexes 930
4. Preparation of Antibody–Gold Complexes 931
5. Preparation of Lectin–Gold Complexes 932
6. Preparation of (Strept)avidin–Gold Complexes 934

25. Modification with Synthetic Polymers

1. Protein Modification with Activated Polyethylene Glycols 937
1.1. Trichloro-s-triazine Activation and Coupling 938
1.2. NHS Ester and NHS Carbonate Activation and Coupling 940
1.3. Carbodiimide Coupling of Carboxylate–PEG Derivatives 945
1.4. CDI Activation and Coupling 946
1.5. Miscellaneous Coupling Reactions 948
2. Protein Modification with Activated Dextrans 951
2.1. Polyaldehyde Activation and Coupling 952
Detailed Contents xxi

2.2. Carboxyl, Amine, and Hydrazide Derivatives 954

2.3. Epoxy Activation and Coupling 956
2.4. Sulfhydryl-Reactive Derivatives 960

26. Enzyme Modification and Conjugation

1. Properties of Common Enzymes 961
1.1. Horseradish Peroxidase 961
1.2. Alkaline Phosphatase 963
1.3. -Galactosidase 964
1.4. Glucose Oxidase 965
2. Preparation of Activated Enzymes for Conjugation 966
2.1. Glutaraldehyde-Activated Enzymes 966
2.2. Periodate Oxidation Techniques 966
2.3. SMCC-Activated Enzymes 967
2.4. Hydrazide-Activated Enzymes 967
2.5. SPDP-Activated Enzymes 968
3. Preparation of Biotinylated Enzymes 968

27. Nucleic Acid and Oligonucleotide Modification and Conjugation

1. Enzymatic Labeling of DNA 970
2. Chemical Modification of Nucleic Acids and Oligonucleotides 973
2.1. Diamine or Bis-Hydrazide Modification of DNA 974
Conjugation via Bisulfite Activation of Cytosine 974
Conjugation via Bromine Activation of Thymine, Guanine, and Cytosine 976
Conjugation via Carbodiimide Reaction with 5 Phosphate of DNA
(Phosphoramidate Formation) 978
2.2. Sulfhydryl Modification of DNA 980
Cystamine Modification of 5 Phosphate Groups Using EDC 981
SPDP Modification of Amines on Nucleotides 982
SATA Modification of Amines on Nucleotides 984
2.3. Biotin Labeling of DNA 985
Biotin-LC-dUTP 985
Photobiotin Modification of DNA 987
Reaction of NHS-LC-Biotin with Diamine-Modified DNA Probes 987
Biotin–Diazonium Modification of DNA 989
Reaction of Biotin–BMCC with Sulfhydryl-Modified DNA 990
Biotin–Hydrazide Modification of Bisulfite-Activated Cytosine Groups 990
2.4. Enzyme Conjugation to DNA 992
Alkaline Phosphatase Conjugation to Cystamine-Modified DNA Using
Amine- and Sulfhydryl-Reactive Heterobifunctional Crosslinkers 993
Alkaline Phosphatase Conjugation to Diamine-Modified DNA Using DSS 994
Enzyme Conjugation to Diamine-Modified DNA Using PDITC 996
Conjugation of SFB-Modified Alkaline Phosphatase to
Bis-hydrazide-Modified Oligonucleotides 998
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