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.- 2.2 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.- 3.3 Immunizing the Larger Experimental Animals for Antisera Production.- 3.4 Immunizing Mice.- 3.5 Influencing the Immune Response.- 4 Taking Blood and Isolating Cells.- 4.1 Taking Blood from Experimental Animals.- 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.- 5.2 Additives to Media: Growth Factors, Conditioned Media.- 5.3 Cryopreservation of Cells.- 5.4 Bacterial and Fungal Infections.- 5.5 Limiting an Infection in Multi-Well Plates.- 5.6 Mycoplasmas.- 5.7 Cell Viability Testing Using Fluorescent Dyes.- 6 Production of Hybridomas.- 6.1 Basics.- 6.2 Fusing Cells to Generate Mouse Monoclonal Antibodies.- 6.3 Human Hybridoma Technique.- 6.4 Other Fusion Methods.- 6.5 Calculating the Number of Hybridoma Clones To Be Expected.- 6.6 Culture and Enrichment of Hybridomas.- 6.7 Cloning Cells.- 6.8 Identifying Human Genomic Material in Mouse-Human Hybridomas.- 6.9 Fine-Tuning Hybridomas.- 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.- 7.3 Production of Monoclonal Antibodies in Bioreactors.- 7.4 Serum-Free Cell Culture.- 7.5 Checking the Antibody Properties.- 8 Purifying Monoclonal Antibodies and Producing Antibody Fragments.- 8.1 Purification of Monoclonal Antibodies: an Overview.- 8.2 Producing Immunoreactive Fragments from Mouse Monoclonal Antibodies.- 9 Coupling Monoclonal Antibodies.- 9.1 Basic Principles.- 9.2 Conjugation of Enzymes to Monoclonal Antibodies.- 9.3 Biotinylating Monoclonal Antibodies.- 9.4 Conjugating Fluorochromes to Monoclonal Antibodies.- 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.- 10.5 Selection of a Test System for Antibodies Against Cellular Antigens.- 10.6 Immunofluorescence Demonstration of Cytoplasmic Ig in Fixed Lymphocytes.- 10.7 Immunofluorescence Demonstration of Membrane Antigens on Living Lymphocytes.- 10.8 Immunocytochemical Staining Techniques.- 10.9 Immunocytochemical Demonstration of Membrane Antigens on Living Cells.- 10.10 ELISA Demonstration of Antigens in Fixed Cells (Cell ELISA).- 10.11 Local Demonstration of Specific Antibody.- 10.12 Dot Immunobinding Test.- 10.13 Molecular Weight Determination of Membrane Antigens by Means of Chemiluminescence-Autography and Sequential Immunoprecipitation.- 10.14 Depletion of Cells in Suspension by Use of Particle-Bound Antibodies (Magnetic Particles).-10.15 Typing Class and Subclass (Isotyping) of Mouse Antibodies by Means of ELISA.- 10.16 Analytical HPLC of Monoclonal Antibodies.- 10.17 Analytical SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE).- 10.18 Analytical Isoelectric Focusing of Monoclonal Antibodies.- 10.19 Silver Staining of Polyacrylamide Gels.- 10.20 Protein Blotting, Immunoblotting (“Western Blot”).- 10.21 Epitope Analysis.- 11 Safety Precautions at Work.- 12 Appendix.- 12.1 Monographs.- 12.2 Reference Works for Obtaining Cells, Reagents, and Laboratory Equipment.- 12.3 Addresses of Firms.
