Monoclonal Antibodies A Practical Approach

by Shepherd, Philip S.; Dean, Christopher

• ISBN13: 9780199637232
• ISBN10: 0199637237
• Format: Hardcover
• Copyright: 2000-07-13
• Publisher: Oxford University Press

Summary

Monoclonal Antibodies: A Practical Approach covers the preparation, testing, derivation, and applications of monoclonal antibodies. New immunological techniques incorporating tried and tested methodologies are described, making the book of interest to established and inexperienced immunologists. Both the standard somatic hybridization technique and recombinant techniques, including the use of phage libraries, for the preparation of rodent and human monoclonal antibodies are described. Protocols for both the small and large scale production are detailed, as well as purification and labelling (with both radioisotopes and non-radioisotopes) methods. The applications of monoclonal antibodies in immunoblotting, enzyme linked immunoassays, immunofluorescence, and FACS analysis are all covered in detail. Finally protocols are given for the use of monoclonal antibodies in rheumatoid arthritis, tissue typing, detecting DNA modified during chemotherapy, and in the clinical analysis of transplantation samples for malignancy. This book will therefore be an invaluable laboratory companion to anyone using monoclonal antibodies in their research.

Table of Contents

List of protocols	xix
Abbreviations	xxvii
Preparation of rodent monoclonal antibodies by In vitro somatic hybridization	1	(24)
Christopher Dean
Philip Shepherd
Introduction and strategy	1	(2)
Choice of host for immunization and myeloma for cell fusion	3	(1)
Choice of immunogen	4	(1)
Preparation of antigen for immunization	5	(2)
Soluble antigens	5	(1)
Antigens expressed on live cells	5	(1)
Plasmid DNA	6	(1)
Peptides	7	(1)
Route of immunization	7	(2)
Generation of immune spleen cells	7	(1)
Immunization via the Peyer's patches of rats	7	(2)
Growth of myeloma cell lines	9	(1)
Preparation of cells for fusion	10	(1)
Cell fusion	11	(1)
Screening hybridoma culture supernatants for specific antibody	12	(6)
Antigen coated multiwell plates	12	(4)
Live or fixed cells	16	(1)
Ligand binding assays	17	(1)
Cloning of hybridomas	18	(1)
Characterization and use of the antibodies obtained	19	(6)
Isotyping of antibodies	20	(1)
Epitope reactivity	20	(1)
Specific immunoprecipitation	21	(2)
References	23	(2)
Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting	25	(33)
Olivier J. P. Leger
Jose W. Saldanha
Introduction	25	(1)
Preparation of mouse spleen	26	(2)
Isolation of total RNA from spleen	28	(2)
Poly(A+) mRNA isolation	30	(1)
Reverse transcriptase reaction	31	(2)
Primary PCR of antibody genes	33	(4)
Preparation of linker	37	(2)
Assembly of VH and VK gene fragments with linker DNA	39	(2)
Assembly of single chain Fv antibody fragments	41	(2)
Reamplification of assembled scFv DNA	43	(1)
Restriction enzyme digestion of assembled scFv	44	(2)
Purification of pHEN-1 vector by equilibrium centrifugation in CsCI ethidium bromide gradients	46	(3)
Restriction digestion of the phage display vector, pHEN-1	49	(1)
Ligation of pHEN-1 and insert antibody scFv	50	(2)
Preparation of electroporation competent E. coli TG1 strain cells	52	(1)
Electroporation	53	(2)
Analysis of recombinant clones from the library	55	(3)
References	57	(1)
Appendix	58	(405)
The design of the humanized antibody	58	(9)
Issues to consider	59	(5)
References	64	(3)
Selection of antibodies from phage libraries of immunoglobulin genes	67	(24)
Jane K. Osbourn
Introduction	67	(1)
Preparation and storage of phage library stocks	68	(1)
Phagemid libraries	68	(1)
Phage libraries	69	(1)
Maintenance of bacterial stocks and titration of phage preparations	69	(2)
Selection of phage libraries on purified, immobilized antigen	71	(3)
Immobilization of antigen on immunotubes	71	(1)
Elution conditions	72	(1)
Storage and rescue of the phagemid population after selection	72	(1)
Choice of number of selection rounds	73	(1)
Selection of phage libraries on biotinylated antigen	74	(2)
Biotinylation of antigen	74	(1)
Selections using biotinylated antigen	75	(1)
Cell surface selections	76	(2)
Adherent cell selections	77	(1)
Cells in suspension	78	(1)
Screening the output of cell surface selections	78	(1)
Proximity selections	78	(5)
Proximity selection using an existing antibody	80	(1)
Proximity selection using natural ligands	81	(2)
Step back selections	83	(1)
Screening of selected phage	83	(4)
Basic screening assays	83	(4)
Affinity screening	87	(1)
Soluble scFv production and purification	87	(4)
References	89	(2)
ARM complexes for in vitro display and evolution of antibody combining sites	91	(20)
Michael J. Taussig
Maria A. T. Groves
Margit Menges
Hong Liu
Mingyue He
Introduction	91	(2)
Ribosome display methodology	93	(12)
Outline of procedure	93	(1)
Primer design and single chain antibody (VH/K) construction for ARM display	93	(3)
Generation of ARM complexes by coupled transcription/translation in vitro	96	(2)
Antigen selection of ARM complexes	98	(2)
Recovery and amplification of DNA from antigen-selected ARM complexes	100	(3)
Further ARM cycles and cloning	103	(1)
Analysis of clones encoding antibodies by ARM display	104	(1)
Examples of ARM display	105	(2)
ARM specificity	105	(1)
Selection of DB3R VH/K from libraries	106	(1)
Troubleshooting	107	(1)
Background	107	(1)
No DNA recovery	107	(1)
Summary	107	(4)
References	109	(2)
Human monoclonal antibodies to blood group antigens	111	(14)
Belinda M. Kumpel
Introduction	111	(1)
General equipment and reagents required	112	(1)
Selection of donor	113	(1)
Preparation of lymphocytes	113	(1)
EBV transformation of B cells	114	(3)
Preparation of EBV	115	(1)
EBV transformation of B cells and growth of B-LCL	116	(1)
Selection of antigen-specific B cells by rosetting	117	(1)
Fusion of B-LCL with murine myeloma cells (P3X63Ag8.653)	118	(2)
Screening techniques	120	(1)
Cloning	121	(1)
Cryopreservation of cells	122	(3)
References	123	(2)
Laboratory based methods for small scale production of monoclonal antibodies	125	(24)
Bryan Griffiths
Introduction	125	(1)
General principles	125	(4)
Culture parameters	125	(2)
Medium and serum	127	(2)
Stationary cultures	129	(2)
Tissue culture plates and flasks	129	(1)
Specialized (scale-up) culture systems	130	(1)
Stirred cultures	131	(4)
Spinner flasks	131	(2)
SuperSpinner	133	(1)
Stirred bioreactors	134	(1)
Dynamic (non-stirred) culture systems	135	(2)
Roller bottle culture	135	(1)
Airlift fermenters	136	(1)
Perfusion (high cell density) systems	137	(6)
Hollow fibre (Acusyst) culture	138	(1)
Tecnomouse	138	(1)
Membrane culture systems (miniPERM)	139	(1)
Packed bed systems	140	(2)
Fluidized bed bioreactors	142	(1)
Harvesting and concentration	143	(2)
Harvesting and clarification	143	(1)
Concentration	144	(1)
Summary	145	(4)
References	146	(3)
Isolation and purification of monoclonal antibodies from tissue culture supernatant	149	(32)
Geoff Hale
Objectives of antibody purification	149	(1)
Essential information about the antibody	150	(7)
Problems with purifying antibodies from culture supernatant	157
Low concentration of antibody in culture supernatant compared with serum	151	(1)
Potential contamination by bovine IgG	152	(1)
Equipment for antibody purification	153	(3)
Equipment for chromatography	154	(2)
Precipitation methods	156	(2)
Affinity chromatography	158	(4)
Reuse of affinity columns	159	(1)
Choice of affinity ligand	160	(2)
Immunoaffinity purification	162	(1)
Ion exchange chromatography	162	(4)
Cation exchange chromatography	163	(2)
Anion exchange chromatography	165	(1)
Immobilized metal affinity chromatography (IMAC)	166	(2)
Size exclusion chromatography (SEC)	168	(1)
Hydrophobic interaction chromatography	169	(3)
Other chromatographic methods	172	(1)
Choice of method	173	(1)
Purification of antibody fragments	173	(1)
Storage of antibodies	173	(1)
Analysis of purity and activity	174	(5)
Antibody concentration and purity	174	(3)
Endotoxin contamination	177	(2)
Conclusion	179	(2)
Acknowledgements	179	(1)
References	180	(1)
Antibody production in plants	181	(26)
Pascal Drake
Eva Stoger
Liz Nicholson
Paul Christou
Julian K.-C. Ma
Introduction	181	(1)
Expressing recombinant proteins in plants	181	(5)
Plant hosts	182	(1)
Antibodies in plants	182	(3)
Modified viruses for transient expression in plants	185	(1)
Glycosylation of recombinant proteins in transgenic plants	185	(1)
Plant transformation	186	(10)
Gene constructs	187	(1)
Agrobacterium tumefaciens-mediated transformation	188	(4)
Principles of particle bombardment	192	(4)
Plant transformation techniques	196	(5)
Agrobaderium-mediated transformation of tobacco	196	(3)
Transformation of wheat by micro-projectile bombardment	199	(2)
Screening regenerated plantlets for immunoglobulin chain production	201	(1)
Self and cross-fertilization of transgenic plants	202	(1)
The overall advantages in expressing antibodies in plants	202	(5)
References	203	(4)
Radiolabelling of monoclonal antibodies	207	(30)
Stephen J. Mather
Introduction	207	(1)
The choice of radionuclide	207	(1)
In vitro applications of radiolabelled antibodies	207	(10)
Iodine-125	208	(9)
In vivo applications of radiolabelled antibodies	217	(17)
Iodine-123	218	(1)
Indium-111	219	(4)
Technetium-99m	223	(8)
Iodine-131	231	(1)
Yttrium-90	2	(233)
Other therapeutic radionuclides	233	(1)
Antibody fragments and genetically engineered constructs	234	(1)
Quality control of radiolabelled antibodies	234	(3)
References	235	(2)
Non-radioactive antibody probes	237	(10)
G. Brian Wisdom
Introduction	237	(1)
Choice of label	238	(1)
General aspects of labelling	238	(2)
The labelling reactions	238	(1)
The monoclonal antibody	239	(1)
Scale and ratios	239	(1)
Purification and storage of the labelled antibody	239	(1)
Labeiling with an enzyme	240	(2)
Labelling with fluorescein	242	(1)
Labelling with biotin	243	(1)
Labelling with digoxigenin (DIG)	244	(1)
Evaluation of labelled monoclonal antibodies	244	(3)
References	246	(1)
Immunogold probes for light and electron microscopy	247	(18)
Paul Monaghan
David Robertson
Introduction	247	(2)
Pre-embedding labelling for SEM and TEM	249	(2)
Thawed cryosections	251	(4)
Progressive lowering of temperature embedding (PLT)	255	(1)
Rapid freezing	256	(2)
Immunocytochemistry of resin sections	258	(3)
Multiple labelling	261	(1)
Conclusion	262	(3)
References	262	(3)
Characterization of cellular antigens using monoclonal antibodies	265	(32)
Gillian Hynes
Introduction	265	(1)
Initial characterization of monoclonal antibodies	266	(1)
Preparation of cell lysates	266	(4)
Detection of cellular antigens by immunoblotting	270	(13)
Separation of proteins by SDS-PAGE	270	(3)
Electrotransfer of proteins from gels to membranes	273	(4)
Blocking non-specific binding sites on the membrane	277	(1)
Incubation with the primary monoclonal antibody	277	(1)
Detection of secondary antibodies and quantitation of signals	277	(5)
Troubleshooting problems encountered during immunoblotting	282	(1)
Analysis of the electrophoretic properties of polypeptides by 2D PAGE	283	(4)
Analysis of subcellular localization of proteins	287	(1)
Immunofluorescence staining	287	(1)
Subcellular fractionation	287	(1)
Resolution of oligomeric complexes by non-denaturing PAGE	287	(2)
Detection of cellular antigens by immunoprecipitation	289	(6)
Analysis of physiological interactions by immunoprecipitation	292	(1)
Immunoprecipitation under disruptive conditions	292	(1)
Troubleshooting high background signals	292	(3)
Epitope mapping	295	(1)
Further applications of monoclonal antibodies in protein characterization	296	(1)
Acknowledgements	296	(1)
References	296	(1)
Immunoassays	297	(22)
Jane V. Peppard
Introduction	297	(1)
General considerations	297	(4)
Selecting an antibody	297	(1)
Selecting an assay standard	297	(2)
Sample matrix	299	(1)
Sample preparation and dilution	299	(1)
Assay turnaround time	300	(1)
Solid support and separation options	301	(2)
Microtitre plates	301	(1)
Precipitation from solution	302	(1)
Scintillation proximity	303	(1)
Labelling and detection of antibody or antigen	303	(6)
Enzyme labelling	303	(2)
Biotin labelling	305	(1)
Labelling with lanthanide fluorophores	306	(1)
Radioactive labelling	307	(1)
Mass spectrometry	308	(1)
Setting up an assay	309	(10)
Competitive binding immunoassay	309	(2)
'Sandwich' immunoassay	311	(4)
Buffers	315	(1)
Setting up an ELISA	316	(2)
References	318	(1)
Immunoaffinity chromatography of macromolecules	319	(22)
Steve Hobbs
Introduction	319	(1)
Choice of matrix	320	(10)
Physical structure	320	(1)
Coupling methods	320	(1)
Coupling chemistry	321	(8)
Monitoring of coupling efficiency	329	(1)
Crosslink stability	329	(1)
Column storage	330	(1)
Sample preparation	330	(1)
Binding of antigen	331	(3)
Elution	334	(1)
Extremes of pH	334	(1)
Chaotropic ions	334	(1)
Ionic strength	335	(1)
Electroelution	335	(1)
Antigen reconstitution	335	(1)
Analysis of purified antigen	336	(1)
Future developments	337	(4)
References	338	(3)
Immunochemical detection of BrdUrd labelled nuclei for monitoring cell kinetics	341	(14)
George D. Wilson
Introduction	341	(1)
Basic concepts in cell kinetics	341	(2)
Background to the BrdUrd flow cytometry technique	343	(2)
In vitro BrdUrd incorporation	345	(1)
Fixation procedures	345	(1)
Staining procedures	346	(3)
DNA denaturation	346	(2)
Antibody staining	348	(1)
Flow cytometry	349	(1)
Examples of data and analysis	350	(2)
Conclusions	352	(3)
References	352	(3)
Immunofluorescence microscopy	355	(16)
Rainer Pepperkok
Andreas Girod
Jeremy Simpson
Jens Rietdorf
Introduction	355	(1)
Immunofluorescence	355	(8)
Fluorescent labelling of antibodies	356	(2)
Fixation, permeabilization, and staining of cells	358	(5)
Microscopy and imaging equipment	363	(5)
Microscopes and associated hardware	363	(2)
Imaging detectors for 3D sectioning microscopy	365	(2)
Data analysis, image processing, and data presentatio	367	(1)
Immunolabelling and visualization in living cells	368	(2)
Labelling by microinjection of fluorescent antibodies	368	(1)
Microscope set-up for live cell IFM	369	(1)
Examples of IFM in fixed cells	370	(1)
References	370	(1)
FACS analysis of clinical haematological samples in transplantation for cancer	371	(20)
Barbara C. Millar
Introduction	371	(1)
Fluorescence activated cell analysis	371	(6)
General considerations	371	(2)
Choice of antibodies, reagents, and general maintenance of the FACS analyser	373	(1)
Dual fluorescence	374	(2)
Preparation of clinical samples	376	(1)
Quality control	376	(1)
Calibration of the analyser	377	(1)
Assessment of the progenitor cell content in bone marrow (BM) and peripheral blood stem cell (PCSC) harvests	377	(6)
Rationale for measuring the progenitor cell content of BM and PBSC harvests for transplantation after intensive therapy for cancer	377	(1)
The CD34 determinant	378	(1)
Collecting peripheral blood stem cell (PBSC) harvests for CD34+ quantitation	378	(1)
Collecting bone marrow (BM) harvests for CD34+ quantitation	379	(3)
Quantitation of purified CD34+ cells from PBSC harvests from patients with chronic lymphocytic leukaemia	382	(1)
Analysis of lymphocyte subsets in peripheral blood and bone marrow harvests from unrelated donors	383	(8)
T cell depletion of paediatric matched (MUD) and unmatched (U-UD) bone marrow (BM) harvests from unrelated donors	383	(1)
Analysis of lymphocyte subsets after transplantation or autologous rescue	384	(1)
Measurement of intracellular cytokines in mononuclear cells (MNC) and T cells after transplantation or autologous rescue	384	(5)
References	389	(2)
Immunocytochemical staining of cells and tissues for diagnostic applications	391	(20)
Andrew R. Dodson
John P. Sloane
Introduction	391	(1)
Tissue and cell substrates	391	(1)
Tissue sections	391	(1)
Cytological specimens	391	(1)
Fixation and processing for paraffin wax embedded tissues	392	(1)
Fixation	392	(1)
Decalcification	392	(1)
Processing	393	(1)
Section preparation	393	(2)
Section adhesives	393	(1)
Section storage	394	(1)
Primary antibodies	395	(1)
Immunocytochemical methodology	395	(7)
Antigen retrieval	396	(2)
Detection methodology	398	(4)
Reporter molecules	402	(3)
Horseradish peroxidase	402	(2)
Alkaline phosphatase	404	(1)
Enhancement	405	(2)
Biotinylated tyramine	406	(1)
Controls	407	(4)
Positive control	407	(1)
Negative control	407	(1)
External quality assurance scheme (EQA)	407	(1)
Acknowledgements	407	(1)
Suggested Further Reading	407	(1)
References	408	(3)
Detection of chemically modified DNA in lymphocytes of patients undergoing chemotherapy	411	(20)
Michael J. Tilby
Introduction	411	(3)
Types of modifications to which antibodies can be produced	412	(1)
Applications of antibodies against modified DNA	412	(1)
Merits of raising antibodies against modified mono/dinucleotides versus modified polymeric DNA	412	(2)
Requirements for detection of low frequencies of DNA modifications	414	(1)
Production of appropriate antibodies	414	(1)
Immunization	414	(1)
Screening hybridomas	415	(1)
ELISA techniques for quantification of DNA modifications	415	(11)
Coating wells with DNA	416	(2)
Direct binding assays	418	(1)
Competitive assays	419	(7)
Staining for adducts in individual cells	426	(5)
Staining techniques: conventional versus agarose embedded DNA	426	(1)
Staining agarose embedded DNA	427	(2)
References	429	(2)
Monoclonal antibody therapy in organ transplantation	431	(18)
Matt Wise
Diana Zelenika
Introduction	431	(1)
The challenge of antibody therapy in humans	432	(3)
Immunological tolerance versus immunosuppression	432	(1)
Depletion versus non-depletion	433	(1)
Avoiding the antiglobulin response	434	(1)
Can monoclonal antibodies ever be used to induce tolerance in humans?	434	(1)
Animal models of transplantation and mAb therapy: defining the problem	435	(1)
Which mAbs for organ transplantation	436	(13)
Anti-CD3 mAb treatment	437	(3)
Monoclonal antibodies to CD4 and CD8	440	(2)
Monoclonal antibodies to CD2S (IL-2 receptor)	442	(1)
Blockade of co-stimulation through CD4O and CD28 pathways	443	(1)
T cell depletion	444	(1)
Summary	445	(1)
References	445	(4)
Monoclonal antibody therapy In rheumatoid arthritis	449	(14)
Ernest H. S. Choy
Gabrielle H. Kingsley
Gabriel S Panayi
Introduction	449	(1)
Pathogenesis of rheumatoid arthritis	449	(1)
Treatment strategies in rheumatoid arthritis	450	(1)
Therapeutic monoclonal antibodies	451	(1)
Monoclonal antibodies in rheumatoid arthritis	452	(6)
Anti-cytokine mAbs in rheumatoid arthritis	452	(2)
Anti-adhesion molecule mAbs	454	(1)
Anti-T cell mAbs in RA	455	(3)
Conclusion	458	(5)
Acknowledgements	459	(1)
References	459	(4)
List of suppliers	463	(6)
Index	469