Monoclonal Antibodies

Offers modern approachse to generate high quality monoclonals
Provides step-by-step detail essential for reproducible results
Contains key notes and implementation advice from the experts

Monoclonal Antibodies: Methods and Protocols, Second Edition expands upon the previous edition with current, detailed modern approaches to isolate and characterize monoclonal antibodies against carefully selected epitopes. This edition includes new chapters covering the key steps to generate high quality monoclonals via different methods, from antigen generation to epitope mapping and quality control of the purified IgG. Chapters are divided into four parts corresponding to four distinct objectives. Part I covers monoclonal antibody generation, Part II deals with monoclonal antibody expression and purification, Part III presents methods for monoclonal antibody characterization and modification, and Part IV describes selected applications of monoclonal antibodies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.



Authoritative and practical, Monoclonal Antibodies: Methods and Protocols, Second Edition provides crucial initial steps of monoclonal antibody generation and characterization with state-of-the art protocols.



Table of contents

Part I: Antibody Generation

1. Antigen Production for Monoclonal Antibody Generation

Giovanni Magistrelli, Pauline Malinge

2. Method for the Generation of Antibodies Specific for Site- and Post-translational Modifications
Hidemasa Goto and Masaki Inagaki

3. Immunization, Hybridoma Generation and Selection for Monoclonal Antibody Production

Hyung-Yong Kim, Alexander Stojadinovic, and Mina J. Izadjoo

4. Hybridoma Technology for the Generation of Rodent mAbs via Classical Fusion

Efthalia Chronopoulou, Alejandro Uribe-Benninghoff, Cindi R. Corbett, and Jody D. Berry

5. Generation of Rabbit Monoclonal Antibodies

Pi-ChenYam and Katherine L. Knight

6. Screening Hybridomas for Cell Surface Antigens by High-throughput Homogenous Assay and Flow Cytometry

Alejandro Uribe-Benninghoff, Teresa Cabral, Efthalia Chronopoulou, Jody D. Berry and Cindi R. Corbett

7. Screening and Subcloning of High Producer Transfectomas using Semi-solid Media and Automated Colony Picker

Suba Dharshanan and Cheah Swee Hung

8. Design and Generation of Synthetic Antibody Libraries for Phage Display

Gang Chen and Sachdev S. Sidhu

9. Selection and Screening using Antibody Phage Display Libraries

Patrick Koenig and Germaine Fuh

10. Yeast Surface Display for Antibody Isolation: Library Construction, Library Screening, and Affinity Maturation

James A. Van Deventer and Karl Dane Wittrup

11. Human B Cell Immortalization for Monoclonal Antibody Production

Joyce Hui-Yuen, Siva Koganti,and Sumita Bhaduri-McIntosh

12. Using Next-generation Sequencing for Discovery of High-frequency Monoclonal Antibodies in the Variable Gene Repertoires from Immunized Mice

Ulrike Haessler and Sai T. Reddy

Part II: Antibody Expression and Purification

13. Cloning, Reformatting, and Small Scale Expression of Monoclonal Antibody Isolated from Mouse, Rat, or Hamster Hybridoma

Jeremy Loyau and François Rousseau

14. Cloning of Recombinant Monoclonal Antibodies from Hybridomas in a Single Mammalian Expression Plasmid

Nicole Müller-Sienerth, Cécile Crosnier, Gavin J. Wright and Nicole Staudt

15. Monoclonal Antibody Purification by Ceramic Hydroxyapatite Chromatography

Larry J. Cummings, Russell G. Frost and Mark A. Snyder

16. Rapid Purification of Monoclonal Antibodies using Magnetic Microspheres

Pauline Malinge, Giovanni Magistrelli

17. Generation of Cell Lines for Monoclonal Antibody Production

Krista Alvin and Jianxin Ye

18. Expression and Purification of Recombinant Antibody Formats and Antibody Fusion Proteins

Martin Siegemund, Fabian Richter, Oliver Seifert, Felix Unverdorben and Roland E. Kontermann

19. Purification of Antibodies and Antibody Fragments using CaptureSelect Affinity Resins

Pim Hermans, Hendrik Adams, Frank Detmers

20. Reformatting of scFv antibodies into the scFv—Fc format and their Downstream Purification

Emil Bujak, Mattia Matasci, Dario Neri, and Sarah Wulhfard

Part III: Anitbody Characterization and Modification

21. Antibody V and C Domain Sequence, Structure, and Interaction Analysis with Special Reference to IMGT®

Eltaf Alamyar, Véronique Giudicelli, Patrice Duroux and Marie-Paule Lefranc

22. Measuring Antibody Affinities as well as the Active Concentration of Antigens Present on a Cell Surface

Palaniswami Rathanaswami

23. Determination of Antibody Structures

Robyn L. Stanfield

24. Affinity Maturation of Monoclonal Antibodies by Multi Site-Directed Mutagenesis

Hyung-Yong Kim, Alexander Stojadinovic, and Mina J. Izadjoo

25. Epitope Mapping with Membrane-bound Synthetic Overlapping Peptides

Terumi Midoro-Horiuti and Randall M. Goldblum

26. Epitope Mapping by Epitope Excision, Hydrogen/Deuterium Exchange and Peptide-Panning Techniques Combined with in silico Analysis

Nicola Clementi, Nicasio Mancini, Elena Criscuolo, Francesca Cappelletti, Massimo Clementi and Roberto Burioni

27. Fine Epitope Mapping Based on Phage Display and Extensive Mutagenesis of the Target Antigen

Gertrudis Rojas

28. Epitope Mapping with Random Phage Display Library

Terumi Midoro-Horiuti and Randall M. Goldblum

29. Epitope Mapping of Monoclonal and Polyclonal Antibodies using Bacterial Cell Surface Display

Anna-Luisa Volk, Francis Jingxin Hu, and Johan Rockberg

30. Ion Exchange High Performance Liquid Chromatography (IEX-HPLC)

Marie Corbier, Delphine Schrag and Sylvain Raimondi

31. Size Exclusion High Performance Liquid Chromatography (SEC-HPLC)

Delphine Schrag, Marie Corbier and Sylvain Raimondi

32. N-glycosylation Characterization by Liquid Chromatography with Mass Spectrometry

Song Klapoetke

33. Fc-Engineering of Antibodies and Antibody Derivatives by Primary Sequence Alteration and their Functional Characterization

Stefanie Derer, Christian Kellner, Thies Rösner, Katja Klausz, Pia Glorius, Thomas Valerius and Matthias Peipp

Part IV: Applications of Monoclonal Antibodies

34. Labeling and use of Monoclonal Antibodies in Immunofluorescence: Protocols for Cytoskeletal and Nuclear Antigens

Christoph R. Bauer

35. Generation and Use of Antibody Fragments for Structural Studies of Proteins Refractory to Crystallization

Stephen J. Stahl, Norman R. Watts, and Paul T. Wingfield

36. Antibody Array Generation and Use

Carl A.K. Borrebaeck and Christer Wingren

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