The present new version of this popular laboratory manual is at the same time the first one of this text in the English language - and this makes me even a little proud, as it reminds me of probably the first collection of monoclonal recipes in English, written by myself, which circulated for a couple of years in many laboratories. In the meantime many researchers have put enormous effort into improving methods for monoclonal antibody production. The proce­ dures have become more and more standardized and by this have more and more lost the character of magic secrets. Hinrich Peters and Horst Baumgarten, who had followed this good tradition already in the previous edition, written in German, suc­ ceeded in making laboratory tricks teachable. They had contributed their own experiences in cell culture and immunology, and were able to engage a number of experienced authors to contribute to the work. They were all willing to follow the general concept of this book, which contains a brief theoretical background for the methods described and presents the procedures in a highly organized structure. So the book has retained its shape as a "cook-book", which I especially like.

Inhalt

1 Introduction.- 1.1 Principles of Cell Hybridization.- 1.2 Properties and Significance of Monoclonal Antibodies.- 1.3 Use of Monoclonal Antibodies in Human Beings: Quality Control and Legal Aspects.- 2 Preconditions for Hybridoma Technology.- 2.1 Experimental Work with Animals.- 2.1.1 Legal Aspects.- 2.1.2 Animal Maintenance.- 2.2 Equipment of the Cell Culture Laboratory.- 2.3 Equipment for Immunological and Biochemical Work.- 2.4 Organization of the Course of Work (Time Table) and Estimation of Costs.- 3 Immunization.- 3.1 Principles and Strategies for Immunizing Animals.- 3.2 Choice of the Immunogen.- 3.2.1 Native Antigens.- 3.2.2 Modified or Synthetic Antigens.- 3.3 Immunizing the Larger Experimental Animals for Antisera Production.- 3.4 Immunizing Mice.- 3.4.1 The Basics of Immunizing Mice for Hybridoma Production.- 3.4.2 Methods of Immunizing Mice.- 3.5 Influencing the Immune Response.- 3.5.1 Influencing the Immune Response by Use of Selected Mouse Strains.- 3.5.2 Influencing the Immune Response by Use of Adjuvants.- 3.5.3 Influencing the Immune Response by Inducing Tolerance.- 3.5.4 Modifying the Immune Response by Use of Cytostatica.- 3.5.5 Modulating the Immune Response by Masking Especially Immunogenic Epitopes with Antibodies.- 3.5.6 Modifying the Immune Response to Generate Certain Immunoglobulin Subclasses.- 4 Taking Blood and Isolating Cells.- 4.1 Taking Blood from Experimental Animals.- 4.1.1 Taking Blood from Mice.- 4.1.2 Taking Blood from Rats.- 4.1.3 Taking Blood from Rabbits.- 4.1.4 Taking Blood from Sheep and Goats.- 4.2 Isolating Lymphocytes from Spleen and Lymph Nodes.- 4.3 Isolating Human Lymphocytes from Peripheral Blood, Tonsils, or Spleen.- 4.4 Enriching Antigen-Specific Lymphoblasts for Fusion.- 4.5 Isolating Mouse Peritoneal Macrophages for Use as Feeder Cells.- 5 Cell Culture.- 5.1 Requirements for Cell Culture.- 5.1.1 Cleaning, Disinfecting, and Avoiding Toxicity.- 5.1.2 Plastic Ware, Water, Media, Sera, and Additives.- 5.1.3 Culture Conditions.- 5.2 Additives to Media: Growth Factors, Conditioned Media.- 5.3 Cryopreservation of Cells.- 5.3.1 Freeze Storage of Cells Directly After Fusion.- 5.3.2 Freeze Storage of Hybridomas in Cell Culture Plates.- 5.3.3 Storing Lymphocytes in the Cold.- 5.3.4 Keeping Track of Frozen Cells by Use of Computers.- 5.4 Bacterial and Fungal Infections.- 5.5 Limiting an Infection in Multi-Well Plates.- 5.6 Mycoplasmas.- 5.6.1 Mycoplasma Enrichment Cultures in Cell-Free Media.- 5.6.2 Fluorescence Test to Demonstrate Mycoplasma Infections in Cultures of Adherent or Suspended Cells.- 5.6.3 Immunological and Genetical Tests for Mycoplasmas.- 5.6.4 Cleaning Mycoplasma-Infected Cells.- 5.6.4.1 Use of Antibiotics to Eliminate Mycoplasmas.- 5.6.4.2 Clearing Mycoplasmas from Infected Cells by Co-Culture with Macrophages.- 5.7 Cell Viability Testing Using Fluorescent Dyes.- 6 Production of Hybridomas.- 6.1 Basics.- 6.1.1 Properties and Production of Myeloma and Tumor Cell Lines.- 6.1.2 Principles of Selection.- 6.1.3 Survey of Mouse Myelomas.- 6.2 Fusing Cells to Generate Mouse Monoclonal Antibodies.- 6.3 Human Hybridoma Technique.- 6.3.1 Fusion Partner Cell Lines and Methods for Generating Human Monoclonal Antibodies.- 6.3.2 Basics of in Vitro Immunization.- 6.3.3 Preparation of Human B-Lymphocytes for in Vitro Immunization: Principles.- 6.3.4 Preparing the Cells.- 6.3.4.1 Removal of T-Lymphocytes by Panning.- 6.3.4.2 Harvesting Monocytes by Adherence.- 6.3.4.3 Differentiating Out Accessory Cells from Monocytes.- 6.3.4.4 Removal of Lysosome-Rich Cells.- 6.3.5 In Vitro Immunization: Additives.- 6.3.5.1 T-Cell Rosetting and Preparing a Conditioned Medium.- 6.3.6 Procedures for in Vitro Immunization of Human Lymphocytes.- 6.3.7 Fusion of Human Cells.- 6.3.8 Epstein-Barr Virus (EBV) Transformation and EBV Hybridoma Technique.- 6.3.8.1 EBV Transformation.- 6.3.8.2 Heteromyeloma Technique: Fusion of EBV-Transformed B-Lymphocytes with Mouse Myeloma Cells.- 6.3.8.3 Transfecting the Geneticin-Resistance Gene.- 6.3.9 Fusion with Cytoplasts.- 6.3.10 DNA Transformation.- 6.4 Other Fusion Methods.- 6.4.1 Fusion with Viruses.- 6.4.2 Electrofusion.- 6.5 Calculating the Number of Hybridoma Clones To Be Expected.- 6.6 Culture and Enrichment of Hybridomas.- 6.6.1 Growing Hybridomas, Trial Plating.- 6.6.2 Trouble-Shooting During the Generation and Culturing of Hybridomas.- 6.7 Cloning Cells.- 6.7.1 Limiting-Dilution Cloning.- 6.7.2 Cloning Cells with a Cell Sorter.- 6.8 Identifying Human Genomic Material in Mouse-Human Hybridomas.- 6.9 Fine-Tuning Hybridomas.- 6.9.1 Increasing the Proportion of Hybridomas Specific for the Desired Antigen.- 6.9.2 Class-Switch Variants.- 6.9.3 Strategies for Generating Stable Hybridomas Producing Human Monoclonal Antibodies.- 6.9.4 Bispecific, Chimeric, and Recombinant Antibodies.- 6.10 Nomenclature of Monoclonal Antibodies.- 7 Mass Production of Monoclonal Antibodies.- 7.1 Mass Production of Monoclonal Antibodies in Cell Culture or Ascites.- 7.2 Production of Monoclonal Antibodies in Mice.- 7.2.1 Production of Murine Monoclonal Antibodies in the Peritoneal Cavity of the Mouse.- 7.2.2 Production of Human Monoclonal Antibodies in the Peritoneal Cavity of the Mouse.- 7.3 Production of Monoclonal Antibodies in Bioreactors.- 7.4 Serum-Free Cell Culture.- 7.5 Checking the Antibody Properties.- 7.5.1 Protocoling the Production and Quality Control of Monoclonal Antibodies.- 7.5.2 Genome Stability of Mouse Hybridomas.- 8 Purifying Monoclonal Antibodies and Producing Antibody Fragments.- 8.1 Purification of Monoclonal Antibodies: an Overview.- 8.1.1 Ammonium Sulfate Precipitation of Monoclonal Antibodies from Hybridoma Ascites Fluid.- 8.1.2 Protein A/Protein G Column Chromatography.- 8.1.3 Anion Exchange Chromatography for Purification of Monoclonal IgG Antibodies.- 8.2 Producing Immunoreactive Fragments from Mouse Monoclonal Antibodies.- 8.2.1 Preparing Fab Fragments.- 8.2.2 Preparing F(ab)2 Fragments.- 9 Coupling Monoclonal Antibodies.- 9.1 Basic Principles.- 9.2 Conjugation of Enzymes to Monoclonal Antibodies.- 9.2.1 Conjugating Peroxidase.- 9.2.2 Conjugating ß-Galactosidase.- 9.2.3 Conjugating Alkaline Phosphatase.- 9.3 Biotinylating Monoclonal Antibodies.- 9.4 Conjugating Fluorochromes to Monoclonal Antibodies.- 9.4.1 FITC Conjugation.- 9.4.2 Conjugation of Rhodamine, Phycoerythrin, and Cyanine Dyes.- 9.5 Conjugating Monoclonal Antibodies to Solid Phases (Immune Absorption).- 10 Demonstration of Monoclonal Antibodies.- 10.1 How To Find the Correct Monoclonal Antibody.- 10.2 Immunoassays for Soluble Antigens: a Survey.- 10.3 ELISA for Demonstration of Monoclonal Antibodies Against Soluble Antigens.- 10.4 Quantitative Tests to Demonstrate the Synthetic Capacity of Hybridoma Cells.- 10.4.1 Determining Cellul…