- Chapter 1: Antibody Production by the Immune System
- Chapter 2: The Antibody Molecule
- Chapter 3: Antibody-Antigen Interactions
- Chapter 4: Antibody Responses
- Chapter 5: Selecting the Antigen
- Protocol 1: Modifying Antigens by Dinitrophenol Coupling
- Protocol 2: Modifying Antigens by Arsynyl Coupling
- Protocol 3: Modifying Protein Antigens by Denaturation
- Protocol 4: Preparing Immune Complexes for Injection
- Protocol 5: Coupling Antigens to Red Blood Cells
- Protocol 6: Coomassie Brilliant Blue Staining
- Protocol 7: Sodium Acetate Staining
- Protocol 8: Copper Chloride Staining
- Protocol 9: Side-Strip Method
- Protocol 10: Fragmenting a Wet Gel Slice
- Protocol 11: Lyophilization of a Gel Slice
- Protocol 12: Electroelution of Protein Antigens from Polyacrylamide Gel Slices
- Protocol 13: Electrophoretic Transfer to Nitrocellulose Membranes
- Protocol 14: Autoradiography
- Protocol 15: Cross-Linking Peptides to KLH with Maleimide
- Protocol 16: Preparing GST-Fusion Proteins from Bacteria
- Protocol 17: Preparing His-Fusion Proteins from Bacteria
- Protocol 18: Preparing MBP-Fusion Proteins from Bacteria
- Protocol 19: Sarkosyl Preparation of Antigens from Bacterial Inclusion Bodies
- Protocol 20: Preparing mFc- and hFc-Fusion Proteins from Mammalian Cells
- Protocol 21: Preparing Live Cells for Immunization
- Protocol 22: Preparing Antigens Using a Baculovirus Expression System
- Chapter 6: Immunizing Animals
- Protocol 1: Administering Anesthesia to Mice, Rats, and Hamsters
- Protocol 2: Administering Anesthesia to Rabbits
- Protocol 3: Preparing Freund's Adjuvant
- Protocol 4: Using Ribi Adjuvant
- Protocol 5: Using Hunter's TiterMax Adjuvant
- Protocol 6: Using Magic Mouse Adjuvant
- Protocol 7: Preparing Aluminum Hydroxide (Alum) Adjuvant
- Protocol 8: Injecting Rabbits Subcutaneously
- Protocol 9: Subcutaneous Injections with Adjuvant into Mice and Rats
- Protocol 10: Subcutaneous Injections without Adjuvant into Mice and Rats
- Protocol 11: Immunizing Mice and Rats with Nitrocellulose-Bound Antigen
- Protocol 12: Injecting Rabbits Intramuscularly
- Protocol 13: Injecting Rabbits Intradermally
- Protocol 14: Injecting Rabbits Intravenously
- Protocol 15: Injecting Mice Intravenously
- Protocol 16: Intraperitoneal Injections with Adjuvant into Mice and Rats
- Protocol 17: Intraperitoneal Injections without Adjuvant into Mice and Rats
- Protocol 18: Immunizing Mice, Rats, and Hamsters in the Footpad or Hock
- Protocol 19: Sampling Rabbit Serum from the Marginal Ear Vein
- Protocol 20: Sampling Mouse Serum from the Tail Vein
- Protocol 21: Sampling Rat Serum from the Tail Vein
- Protocol 22: Sampling Mouse and Rat Serum from the Retro-Orbital Sinus
- Protocol 23: Sampling Mouse and Rat Serum from the Submandibular Vein
- Protocol 24: Sampling Mouse and Rat Serum from the Saphenous Vein
- Protocol 25: Serum Preparation
- Protocol 26: Induction of Ascites Using Freund's Adjuvant
- Protocol 27: Induction of Ascites in BALB/c Mice Using Myeloma Cells
- Protocol 28: Standard Immunization of Mice, Rats, and Hamsters
- Protocol 29: Standard Immunization of Rabbits
- Protocol 30: Repetitive Immunization at Multiple Sites (RIMMS) of Mice, Rats, and Hamsters
- Protocol 31: Subtractive Immunization for Mice, Rats, and Hamsters
- Protocol 32: Decoy Immunization for Mice, Rats, and Hamsters
- Protocol 33: Adoptive Transfer Immunization of Mice
- Protocol 34: cDNA Immunization of Mice, Rats, and Hamsters
- Protocol 35: Euthanizing Mice, Rats, and Hamsters Using CO2 Asphyxiation
- Protocol 36: Harvesting Spleens from Mice, Rats, and Hamsters
- Chapter 7: Generating Monoclonal Antibodies
- Protocol 1: Antibody Capture in Polyvinyl Chloride Wells: Enzyme-Linked Detection (Indirect ELISA)
- Protocol 2: Antibody Capture in Polyvinyl Chloride Wells: Enzyme-Linked Detection When Immunogen Is an Immunoglobulin Fusion Protein (Indirect ELISA to Detect Ig Fusion Proteins)
- Protocol 3: Antibody Capture on Nitrocellulose Membrane: Dot Blot
- Protocol 4: Antibody Capture on Nitrocellulose Membrane: High-Throughput Western Blot Assay for Hybridoma Screening
- Protocol 5: Antibody Capture on Whole Cells: Cell-Surface Binding (Surface Staining by Flow Cytometry/FACS)
- Protocol 6: Antibody Capture on Permeabilized Whole Cells Binding (Intracellular Staining by Flow Cytometry/FACS)
- Protocol 7: Antibody Capture on Whole Cells: Cell-Surface Binding (Surface Staining by Immunofluorescence)
- Protocol 8: Antibody Capture on Permeabilized Whole Cells (Immunofluorescence)
- Protocol 9: Antibody Capture on Tissue Sections (Immunohistochemistry)
- Protocol 10: Antigen Capture in 96-Well Plates (Capture or Sandwich ELISA)
- Protocol 11: Antigen Capture on Nitrocellulose Membrane: Reverse Dot Blot
- Protocol 12: Antigen Capture in Solution: Immunoprecipitation
- Protocol 13: Screening for Good Batches of Fetal Bovine Serum
- Protocol 14: Preparing Peritoneal Macrophage Feeder Plates
- Protocol 15: Preparing Myeloma Cell Feeder Layer Plates
- Protocol 16: Preparing Splenocyte Feeder Cell Cultures
- Protocol 17: Preparing Fibroblast Feeder Cell Cultures
- Protocol 18: Screening for Good Batches of Polyethylene Glycol
- Protocol 19: Polyethylene Glycol Fusion
- Protocol 20: Fusion by Sendai Virus
- Protocol 21: Electro Cell Fusion
- Protocol 22: Single-Cell Cloning by Limiting Dilution
- Protocol 23: Single-Cell Cloning by Growth in Soft Agar
- Protocol 24: Determining the Class and Subclass of a Monoclonal Antibody by Ouchterlony Double-Diffusion Assays
- Protocol 25: Determining the Class and Subclass of Monoclonal Antibodies Using Antibody Capture on Antigen-Coated Plates
- Protocol 26: Determining the Class and Subclass of Monoclonal Antibodies Using Antibody Capture on Anti-Immunoglobulin Antibody-Coated Plates
- Chapter 8: Growing Hybridomas
- Protocol 1: Counting Myeloma or Hybridoma Cells
- Protocol 2: Viability Checks
- Protocol 3: Freezing Cells for Liquid Nitrogen Storage
- Protocol 4: Recovering Cells from Liquid Nitrogen Storage
- Protocol 5: Ridding Cell Lines of Contaminating Microorganisms by Antibiotics
- Protocol 6: Ridding Cell Lines of Contaminating Microorganisms with Peritoneal Macrophages
- Protocol 7: Ridding Cell Lines of Contaminating Microorganisms by Passage through Mice
- Protocol 8: Testing for Mycoplasma Contamination by Growth on Microbial Medium
- Protocol 9: Testing for Mycoplasma Contamination by Hoechst Dye 33258 Staining
- Protocol 10: Testing for Mycoplasma Contamination Using PCR
- Protocol 11: Testing for Mycoplasma Contamination Using Reporter Cells
- Protocol 12: Ridding Cells of Mycoplasma Contamination Using Antibiotics and Single-Cell Cloning
- Protocol 13: Ridding Cells of Mycoplasma by Passage through Mice
- Protocol 14: Inducing and Collecting Ascites
- Protocol 15: Collecting Tissue Culture Supernatants
- Protocol 16: Storing Tissue Culture Supernatants and Ascites
- Protocol 17: Selecting Myeloma Cells for HGPRT Mutants with 8-Azaguanine
- Chapter 9: Characterizing Antibodies
- Chapter 10: Antibody Purification and Storage
- Chapter 11: Engineering Antibodies
- Chapter 12: Labeling Antibodies
- Protocol 1: Labeling Antibodies with NHS-LC-Biotin
- Protocol 2: Biotinylating Antibodies Using Biotin Polyethylene Oxide (PEO) Iodoacetamide
- Protocol 3: Biotinylating Antibodies Using Biotin-LC Hydrazide
- Protocol 4: Labeling Antibodies Using NHS-Fluorescein
- Protocol 5: Labeling Antibodies Using a Maleimido Dye
- Protocol 6: Conjugation of Antibodies to Horseradish Peroxidase
- Protocol 7: Labeling Antibodies with Cy5-Phycoerythrin
- Protocol 8: Labeling Antibodies Using Europium
- Protocol 9: Labeling Antibodies Using Colloidal Gold
- Protocol 10: Iodination of Antibodies with Immobilized Iodogen
- Chapter 13: Immunoblotting
- Protocol 1: Preparing Whole-Cell Lysates for Immunoblotting
- Protocol 2: Preparing Protein Solutions for Immunoblotting
- Protocol 3: Preparing Immunoprecipitations for Immunoblotting
- Protocol 4: Resolving Proteins for Immunoblotting by Gel Electrophoresis
- Protocol 5: Semi-Dry Electrophoretic Transfer
- Protocol 6: Wet Electrophoretic Transfer
- Protocol 7: Staining the Blot for Total Protein with Ponceau S
- Protocol 8: Blocking and Incubation with Antibodies: Immunoblots Prepared with Whole-Cell Lysates and Purified Proteins (Straight Western Blotting)
- Protocol 9: Blocking and Incubation with Antibodies of Immunoblots Prepared with Immunoprecipitated Protein Antigens (Immunoprecipitation/Western Blotting)
- Protocol 10: Detection with Enzyme-Labeled Reagents
- Protocol 11: Detection with Fluorochromes
- Chapter 14: Immunoprecipiation
- Protocol 1: Metabolic Labeling of Antigens with [35S]Methionine
- Protocol 2: Pulse-Chase Labeling of Antigens with [35S]Methionine
- Protocol 3: Metabolic Labeling of Antigens with [32P]Orthophosphate
- Protocol 4: Freezing Cell Pellets for Large-Scale Immunoprecipitation
- Protocol 5: Detergent Lysis of Tissue Culture Cells
- Protocol 6: Detergent Lysis of Animal Tissues
- Protocol 7: Lysis Using Dounce Homogenization
- Protocol 8: Differential Detergent Lysis of Cellular Fractions
- Protocol 9: Lysing Yeast Cells with Glass Beads
- Protocol 10: Lysing Yeast Cells Using a Coffee Grinder
- Protocol 11: Denaturing Lysis
- Protocol 12: Cross-Linking Antibodies to Beads Using Dimethyl Pimelimidate (DMP)
- Protocol 13: Cross-Linking Antibodies to Beads with Disuccinimidyl Suberate (DSS)
- Protocol 14: Immunoprecipitation
- Protocol 15: Tandem Immunoaffinity Purification Using Anti-FLAG and Anti-HA Antibodies
- Protocol 16: Chromatin Immunoprecipitation
- Chapter 15: Immunoassays
- Chapter 16: Cell Staining
- Protocol 1: Growing Adherent Cells on Coverslips or Multiwell Slides
- Protocol 2: Growing Adherent Cells on Tissue Culture Dishes
- Protocol 3: Attaching Suspension Cells to Slides Using the Cytocentrifuge
- Protocol 4: Attaching Suspension Cells to Slides Using Poly-L-Lysine
- Protocol 5: Preparing Cell Smears
- Protocol 6: Attaching Yeast Cells to Slides Using Poly-L-Lysine
- Protocol 7: Preparing Frozen Tissue Sections
- Protocol 8: Preparing Paraffin Tissue Sections
- Protocol 9: Additional Protocol: Heat-Induced Epitope Retrieval
- Protocol 10: Preparing Cell Smears from Tissue Samples or Cell Cultures
- Protocol 11: Embedding Cultured Cells in Matrigel
- Protocol 12: Fixing Attached Cells in Organic Solvents
- Protocol 13: Fixing Attached Cells in Paraformaldehyde or Glutaraldehyde
- Protocol 14: Fixing Suspension Cells with Paraformaldehyde
- Protocol 15: Lysing Yeast
- Protocol 16: Binding Antibodies to Attached Cells or Tissues
- Protocol 17: Binding Antibodies to Cells in Suspension
- Protocol 18: Detecting Horseradish PeroxidaseLabeled Cells Using Diaminobenzidine
- Protocol 19: Detecting Horseradish PeroxidaseLabeled Cells Using Diaminobenzidine and Metal Salts
- Protocol 20: Detecting Horseradish PeroxidaseLabeled Cells Using Chloronaphthol
- Protocol 21: Detecting Horseradish PeroxidaseLabeled Cells Using Aminoethylcarbazole
- Protocol 22: Detecting Alkaline PhosphataseLabeled Cells Using NABP-NF
- Protocol 23: Detecting Alkaline PhosphataseLabeled Cells Using BCIP-NBT
- Protocol 24: Detecting -Galactosidase-Labeled Cells Using X-Gal
- Protocol 25: Detecting Fluorochrome-Labeled Reagents
- Protocol 26: Detecting Gold-Labeled Reagents
- Protocol 27: Detecting Iodine-Labeled Reagents
- Protocol 28: Counterstaining Cells
- Protocol 29: Mounting Cell or Tissue Samples in DPX
- Protocol 30: Mounting Cell or Tissue Samples in Gelvatol or Mowiol
- Chapter 17: Antibody Screening using High Throughput Flow Cytometry
- Chapter 18: Appendix I: Electrophoresis
- Chapter 19: Appendix II: Protein Techniques
- Chapter 20: Appendix III: General Information
- Chapter 21: Appendix IV: Bacterial Expression
- Chapter 22: Appendix V: Cautions
- Chapter 23: Index
Creation of Recombinant Antibodies Using Degenerate Oligonucleotides
(Protocol summary only for purposes of this preview site)In this method, mRNA is obtained from B cells as a source of heavy- and light-chain sequences. Typically, this procedure uses an existing hybridoma or myeloma. Extreme care is required to obtain high-quality mRNA, because mRNA is readily degraded by ubiquitous RNases that are common laboratory contaminants. Our laboratory has had good experience using RNAzol reagent and the method available from Molecular Research Center, Inc. Additional methods for preparing intact RNA and avoiding contamination with RNases are available in Chapter 6 of Green and Sambrook (2012). cDNA is prepared using purified mRNA and either oligo(dT) or a C-region 3 primer. The cDNA negative strand is complementary to the coding sequence of the heavy or light chain. A large number of papers have described the design of degenerate oligonucleotides for amplifying either mouse (LeBoeuf et al. 1989; Orlandi et al. 1989; Coloma et al. 1991; Wang et al. 2000, 2006; Rohatgi et al. 2008; Lim et al. 2010) or human (Larrick et al. 1989; Marks et al. 1991; Lim et al. 2010) heavy- and light-chain genes. However, inasmuch as there are approximately 100 variable gene segments in mouse or human, there may be a substantial advantage to having some knowledge regarding the antibody's subfamily when using this strategy. We have made use of anti-Ig agarosebased immunoprecipitation followed by SDS-PAGE and LC-MS/MS to identify amino acid sequences for some hybridomas (JR Dasch, unpubl.). This approach can aid in the design of a variable region primer or in selection of the appropriate degenerate primer for a specific variable gene subfamily.
