Preface
Contents
Contributors
Part I: Introduction
Chapter 1: Antibody Phage Display
1 Introduction
2 Antibody Structure and Function
2.1 Recombinant Formats
3 Antibody Phage Display Libraries
3.1 Immune Antibody Library
3.2 NaΓ―ve Antibody Library
3.3 Synthetic and Semisynthetic Antibody Library
4 Conclusion
References
Part II: Construction of Antibody Phage Display Libraries
Chapter 2: Construction of Human Immune and Naive scFv Phage Display Libraries
1 Introduction
2 Materials
2.1 Isolation of Lymphocytes
2.2 Sorting of B Lymphocytes/Plasma Cells (Optional)
2.3 cDNA Synthesis
2.4 First and Second Antibody Gene PCR
2.5 First Cloning Step - VL
2.6 Second Cloning Step - VH
2.7 Colony PCR
2.8 Library Packaging and scFv Phage Production
2.9 Phage Titration
3 Methods
3.1 Isolation of Lymphocytes (Peripheral Blood Mononuclear Cells (PBMC))
3.2 Fluorescence-Automated Sorting of B Lymphocytes/Plasma Cells
3.3 cDNA Synthesis
3.4 First Antibody Gene PCR
3.5 Second Antibody Gene PCR
3.6 First Cloning Step - VL
3.7 Second Cloning Step - VH
3.8 Colony PCR
3.9 Library Packaging and scFv Phage Production
3.10 Phage Titration
4 Notes
References
Chapter 3: Construction of NaΓ―ve and Immune Human Fab Phage Display Library
1 Introduction
2 Materials
2.1 Isolation of B Cells
2.2 First-Strand cDNA Synthesis
2.3 Amplification of HC and LC Fab Gene Repertoire
2.4 Two-Step Cloning
2.4.1 First Step Cloning (Fab HC)
2.4.2 Second Step Cloning (Fab LC)
2.5 Colony PCR
2.6 Fab Phage Library Packaging
2.7 Phage Titration
2.8 Fab Library Panning
2.8.1 Fab Selection
2.8.2 Polyclonal and Monoclonal Phage ELISA
3 Methods
3.1 Isolation of B Cells
3.2 First-Strand cDNA Synthesis
3.3 Amplification of HC and LC Fab Gene Repertoire
3.4 Two-Step Cloning
3.4.1 First Step Cloning (Fab HC)
3.4.2 Second Step Cloning (Fab LC)
3.4.3 Library Size Estimation
3.4.4 Preparation of Bacteria Library Stock
3.5 Colony PCR
3.6 Fab Phage Library Packaging
3.7 Phage Titration
3.8 Fab Library Panning
3.8.1 Fab Selection
3.8.2 Polyclonal and Monoclonal Phage ELISA
4 Notes
References
Chapter 4: Construction of Synthetic Antibody Phage Display Libraries
1 Introduction
2 Materials
3 Methods
3.1 Phagemid Design
3.2 Library Construction
3.2.1 Purification of dU-ssDNA Template
3.2.2 In Vitro Synthesis of Heteroduplex CCC-dsDNA: Oligonucleotide Phosphorylation
3.2.3 In Vitro Synthesis of Heteroduplex CCC-dsDNA: Annealing of Phosphorylated Oligonucleotides to the dU-ssDNA Template
3.2.4 In Vitro Synthesis of Heteroduplex CCC-dsDNA: Enzymatic Synthesis of CCC-dsDNA
3.3 Conversion of CCC-dsDNA into a Phage-Displayed Antibody Library
4 Notes
References
Chapter 5: Construction of Chicken and Ostrich Antibody Libraries
1 Introduction
2 Materials (See Note 1)
2.1 RNA Isolation (See Note 2)
2.2 cDNA Synthesis, PCR, and Ligation
2.3 Electroporation and Growth of E. coli and Bacteriophage
3 Methods
3.1 RNA Isolation from Tissue
3.2 RNA Isolation from Blood
3.3 cDNA Synthesis and Amplification by PCR
3.4 Assembly of scFv Genes Based on Natural VH and VL
3.5 Assembly of scFv Genes Based on Natural VL and Synthetic, Randomized VH CDR3s
3.6 Restriction Digestion of the scFv Gene Construct and pHEN1 Vector
3.7 Ligation of the scFv Gene and Vector
3.8 Electroporation
3.9 Phage Rescue
3.10 PEG Precipitation of Phages
3.11 Determining Phage Titer
3.12 Crystal Violet Stained Gels (See Note 14)
4 Notes
References
Chapter 6: Construction of Rabbit Immune Antibody Libraries
1 Introduction
2 Materials
3 Method
3.1 Rabbit Immunization
3.2 Preparation of Total RNA from the Spleens of Immunized Rabbits
3.3 Synthesis of First-Strand cDNA
3.4 Assembly of Rabbit/Human Chimeric Fab Repertoire by PCR
3.4.1 First Round of PCR
3.4.2 Second Round of PCR
3.4.3 Third Round of PCR
3.5 Construction of Fab Library
3.6 Phage Antibody Library Rescue
4 Notes
References
Chapter 7: Isolation and Characterization of Single-Domain Antibodies from Immune Phage Display Libraries
1 Introduction
2 Material
2.1 Antigen Preparation, Llama Immunization, and Immune Response Monitoring
2.2 VHH Library Construction
2.3 Phage Rescue from the Library
2.4 Biopanning of Phage-Displayed Library
2.5 Polyclonal and Monoclonal Phage ELISA Screening (See Note 10)
2.6 Sub-cloning of R3/R4 Phage Pool, In Vitro Translation and Screening of the Soluble VHHs (An Alternative to Monoclonal Phag...
2.7 VHH Sub-cloning, Soluble Expression, and Purification
2.8 Characterization of VHH Binders: Surface Plasmon Resonance (SPR)
2.9 Cell Binding of VHHs by Flow Cytometry
2.10 Immunoprecipitation of the Cell Surface Receptor with VHHs
3 Methods
3.1 Antigen Preparation, Llama Immunization, and Immune Response Monitoring
3.2 VHH Library Construction
3.3 Phage Rescue of the VHH Library
3.4 VHH Phage Library Biopanning
3.5 Polyclonal and Monoclonal Phage ELISA Screening
3.6 Sub-cloning of R3/R4 Phage Pool, In Vitro Translation and Screening of the Soluble VHHs (an Alternative to Monoclonal Phag...
3.6.1 Sub-cloning of R3/R4 Phage Pool
3.6.2 In Vitro Translation and Screening of the Soluble VHHs (an Alternative to Monoclonal Phage ELISA Screening)
3.7 VHH Sub-cloning, Soluble Expression, and Purification
3.7.1 Large-Scale VHH Expression in E. coli
3.7.2 Preparation of the Biotin Ligase BirA Extract
3.8 Characterization of VHH Binders by Surface Plasmon Resonance (SPR)
3.9 Cell Binding of VHHs by Flow Cytometry
3.10 Validating VHH Specificity by Immunoprecipitation of the Target Antigen
4 Notes
References
Chapter 8: Phagekines: Directed Evolution and Characterization of Functional Cytokines Displayed on Phages
1 Introduction
2 Materials
2.1 Displaying Cytokines on Filamentous Phages
2.2 Quantifying Cytokine Display Levels by ELISA
2.3 Screening Receptor Binding Properties of Phage-Displayed Cytokines by ELISA
2.4 Assessing CTLL-2 Proliferation Induced by Phage-Displayed IL-2 and IL-2 Variants
2.5 Measuring Trans-Signaling Induced by Phage-Displayed IL-6
2.6 Producing Single-Stranded DNA Templates for Cytokine Library Construction
2.7 Kunkel Mutagenesis Diversification Reactions
2.8 Preparation of Electrocompetent Cells and Electroporation with Mutagenesis Products to Construct Libraries of Cytokine Var...
2.9 Library Phage Production at a 300 mL Scale
2.10 Phage Panning and Amplification
2.11 Phage Production at 96-Well Scale
2.12 Clonal Screening by ELISA and Sequencing
3 Methods
3.1 Displaying Cytokines on Filamentous Phages
3.2 Quantifying Cytokine Display Levels by ELISA
3.3 Screening Receptor Binding Properties of Phage-Displayed Cytokines by ELISA
3.4 Assessing CTLL-2 Proliferation Induced by Phage-Displayed IL-2 and IL-2 Variants
3.5 Measuring Trans-Signaling Induced by Phage-Displayed IL-6
3.6 Producing Single-Stranded DNA Templates for Cytokine Library Construction
3.7 Kunkel Mutagenesis Diversification Reactions
3.8 Preparation of Electrocompetent Cells and Electroporation with Mutagenesis Products to Construct Libraries of Cytokine Var...
3.9 Library Phage Production at a 300 mL Scale
3.10 Phage Panning and Amplification
3.11 Phage Production at 96-Well Scale
3.12 Clonal Screening by ELISA and Sequencing
4 Notes
References
Chapter 9: Efficient Cloning of Inserts for Phage Display by Golden Gate Assembly
1 Introduction
2 Materials
2.1 Polymerase Chain Reactions (PCR)
2.2 1% Agarose Gel
2.3 Golden Gate Assembly Reactions
2.4 Transformation
2.5 Plasmid Extraction and Sequencing
2.6 Chemical Biotinylation of Proteins
2.7 Phage ELISA
3 Methods
3.1 PCR Amplification
3.2 Golden Gate Assembly
3.3 Transformation
3.4 Plating
3.5 Sequencing
3.6 Chemical Biotinylation of Proteins
3.7 Phage Amplification
3.8 Phage Enzyme-Linked Immunosorbent Assay (ELISA)
4 Notes
References
Chapter 10: Construction of an Ultra-Large Phage Display Library by Kunkel Mutagenesis and Rolling Circle Amplification
1 Introduction
2 Materials
2.1 Preparation of Single-Stranded Circular DNA
2.2 1% Agarose Gel
2.3 Kunkel Mutagenesis
2.4 Rolling Circle Amplification (RCA)
2.5 Restriction Enzyme Digestion and Ligation
2.6 Transformation and Library Production
2.7 Plasmid Extraction and DNA Sequencing
2.8 Affinity Selection
2.9 Phage ELISA
3 Methods
3.1 Preparation of Single-Stranded Circular DNA
3.2 Kunkel Mutagenesis
3.3 Rolling Circle Amplification (RCA)
3.4 Ethanol Precipitation of Ligated DNA
3.5 Bacterial Transformation and Library Production
3.6 Sanger Sequencing of Library Clones
3.7 Next-Generation Sequencing (NGS)
3.8 Affinity Selection of the Library
3.9 Phage ELISA
3.10 Competition ELISA
4 Notes
References
Chapter 11: Construction of Semisynthetic Shark vNAR Yeast Surface Display Antibody Libraries
1 Introduction
2 Materials
2.1 Shark Handling and Blood Isolation
2.2 Preparation of Total RNA from Whole Blood
2.3 cDNA Synthesis and Gene-Specific Amplification of vNAR Regions as Template for Library Construction
2.4 Yeast Surface Display Library Construction
3 Methods
3.1 Blood Collection
3.2 Total RNA Preparation
3.3 cDNA Synthesis
3.4 Amplification of the Natural vNAR Repertoire
3.5 Generation of the CDR3-Randomized PCR Insert for Library Establishment Using Yeast Surface Display as Platform Technology
3.5.1 First PCR
3.5.2 Second PCR
3.5.3 Third PCR
3.6 Shark vNAR Library Generation for Yeast Surface Display
3.6.1 Digestion of pCT Plasmid
3.6.2 Yeast Transformation
3.7 Affinity Maturation by CDR1 Diversification and Sublibrary Establishment of Target-Enriched Binders
3.7.1 First PCR
3.7.2 Second PCR
3.7.3 Third PCR
4 Notes
References
Part III: Selection Strategies for Antibodies
Chapter 12: Antibody Selection via Phage Display in Microtiter Plates
1 Introduction
2 Materials
2.1 Coating of Microtiter Wells
2.2 Panning
2.3 Phage Titration
2.4 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates
2.5 ELISA of Soluble Monoclonal Antibody Fragments
3 Methods
3.1 Coating of Microtiter Plate Wells
3.2 Panning
3.3 Phage Titration
3.4 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates
3.5 ELISA of Soluble Monoclonal Antibody Fragments
3.6 Fast On-Rate Panning
4 Notes
References
Chapter 13: Antibody Selection in Solution Using Magnetic Beads
1 Introduction
2 Materials
2.1 Preparation of the Magnetic Beads
2.2 Panning
2.3 Phage Titration
2.4 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates
2.5 ELISA of Soluble Monoclonal Antibody Fragments
3 Methods
3.1 Preparation of the Magnetic Beads
3.2 Panning
3.3 Phage Titration
3.4 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates
3.5 ELISA of Soluble Monoclonal Antibody Fragments
4 Notes
References
Chapter 14: Streptavidin-Coated Solid-Phase Extraction (SPE) Tips for Antibody Phage Display Biopanning
1 Introduction
2 Materials
2.1 Preparation of Phage Display Antibody Library
2.1.1 Phage Display Antibody Libraries and E. Coli Host Strains
2.1.2 Preparation of Antibody Library
2.2 Phage Display MSD Biopanning
2.3 Phage ELISA
2.4 DNA Sequencing
2.5 Soluble Antibody Fragment Detection
3 Methods
3.1 Preparation of Antibody Library Phage
3.2 Library Phage Packaging
3.3 Preparation of First-Generation Stock
4 Phage Display Biopanning
4.1 MSIA Streptavidin D.a.R.TΒ΄s Loading of Biotinylated Antigen
4.2 MSIA Streptavidin D.a.R.TΒ΄s Antibody Biopanning
5 Phage ELISA
5.1 Polyclonal Phage ELISA
5.2 Monoclonal Phage Propagation
5.3 Monoclonal ELISA
6 DNA Sequencing
7 Generation of Soluble Antibody Fragments
7.1 Expression and Extraction of Soluble Antibody
7.2 Soluble ELISA
8 Analysis
9 Notes
References
Chapter 15: Magnetic Nanoparticle-Based Semi-automated Panning for High-Throughput Antibody Selection
1 Introduction
2 Materials
2.1 Loading of Magnetic Beads
2.2 Semi-automated Panning Using a Magnetic Particle Processor
2.3 Packaging of Phagemids
2.4 Titration of Phage Particles
2.5 Magnetic Particle ELISA of Polyclonal Antibody Phage
2.6 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates
2.7 ELISA of Soluble Monoclonal Antibody Fragments in Microtiter Plates
3 Methods
3.1 Loading of Magnetic Beads
3.2 Semi-automated Panning on Magnetic Particle Processor
3.3 Packaging of Phage Particles
3.4 Titration of Phage Particles
3.5 ELISA of Polyclonal Antibody Phage
3.6 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates
3.7 ELISA of Soluble Monoclonal Antibody Fragments in Microtiter Plates
4 Notes
References
Chapter 16: Antibody Selection on Cells Targeting Membrane Proteins
1 Introduction
2 Materials
2.1 Phage Display on Cells
2.2 Phage Titration
2.3 High-Throughput Screening of Binders by Flow Cytometry
3 Methods
3.1 Phage Display on Cells
3.1.1 Preparation of Cells
3.1.2 Depletion of Phage Display Library on Cells
3.1.3 Selection of scFv Binders on Target Expressing Cells
3.1.4 Amplification of Phage Particles
3.2 Phage Titration
3.3 Screening of scFv on Cells
3.3.1 Production of Soluble Monoclonal Antibody Fragments in Microtiter Plates
3.3.2 Screening of scFv Binders by Flow Cytometry
4 Notes
References
Chapter 17: Targeting Intracellular Antigens with pMHC-Binding Antibodies: A Phage Display Approach
1 Introduction
2 Materials
2.1 Phage Panning via Dynabeads
2.2 Phage Panning via T2 Cells
2.3 Amplification
2.4 Clone Selection for Dynabeads Panning
2.5 Clone Selection for Cell Panning
2.6 Screening of Phage Clones via ELISA
2.7 Screening of Phage Clones via FACS
2.8 Large-Scale Expression
2.9 Fc Fusion Protein Cloning and Expression
3 Methods
3.1 Panning via Dynabeads
3.2 Panning via Cell Panning
3.3 Amplification
3.4 Clone Selection for Dynabeads Panning
3.5 Clone Selection for Cell Panning
3.6 ELISA Screening for pMHC Binding Phage Clones
3.7 FACS Screening for pMHC Binding Phage Clones
3.8 Large-Scale Expression
3.9 Generating Fc Fusion Proteins
4 Notes
References
Chapter 18: Antibody Isolation from Human Synthetic Libraries of Single-Chain Antibodies and Analysis Using NGS
1 Introduction
2 Materials
2.1 Panning
2.2 Phage-Seq
3 Methods
3.1 Affinity Selection of scFv-Displaying Phages on Immobilized Antigen
3.1.1 Growth and Helper Phage Rescue of the Library (See Note 2)
3.1.2 Affinity Selection (Panning) on Immobilized Antigen
3.2 Identification of Antigen Binders
3.3 Phage-Seq
4 Notes
References
Chapter 19: Selection of Affibody Affinity Proteins from Phagemid Libraries
1 Introduction
2 Materials
3 Methods
3.1 Phage Stock Preparation
3.2 Target Antigen Preparation
3.3 Phage Display Selection
3.4 Phage-ELISA Screening
4 Notes
References
Part IV: Complementary Approaches for Antibody Phage Display Selections
Chapter 20: Antibody Affinity and Stability Maturation by Error-Prone PCR
1 Introduction
2 Materials
2.1 Error-Prone PCR
2.2 Library Construction
2.3 Library Validation
2.4 Library Packaging
2.5 Titration
2.6 Selection by Panning
2.7 Production of Soluble, Monoclonal Antibody Fragments
2.8 Monoclonal ELISA and Thermal Stability Screening
3 Methods
3.1 Error-Prone PCR
3.2 Library Construction
3.3 Library Validation
3.4 Library Packaging
3.5 Titration
3.6 Selection by Panning
3.7 Production of Soluble Monoclonal Antibody Fragments
3.8 ELISA of Soluble Monoclonal Antibody Fragments
3.9 Stability Screening of Soluble Monoclonal Antibody Fragments
4 Notes
References
Chapter 21: Antibody Batch Cloning
1 Introduction
2 Materials
2.1 Preparation of scFv-Encoding DNA After Panning
2.2 Ligation
2.3 Heat-Shock Transformation
2.4 Colony Identification and Inoculation
2.5 Plasmid Isolation
3 Methods
3.1 Preparation of scFv-Encoding DNA
3.2 Ligation
3.3 Heat-Shock Transformation
3.4 Colony Identification and Inoculation
3.5 Plasmid Isolation
4 Notes
References
Chapter 22: Deep Mining of Complex Antibody Phage Pools
1 Introduction
2 Materials
3 Methods
3.1 Protein Depletion Followed by Panning on Whole Cells
3.1.1 Coating of Polystyrene Beads
3.1.2 Binding to Streptavidin Beads
3.1.3 Coupling of Tosylactivated M-280 Beads
3.1.4 Depletion of Phage Pool
3.1.5 Panning on Cells
3.1.6 Elution of Phages Using Trypsin
3.2 Panning on Whole Cells with Protein Competition
3.2.1 Calculations of Protein Concentrations
3.2.2 Panning on Cells
3.2.3 Elution of Phages Using Trypsin
3.3 Panning on Whole Cells with Antibody Blocking
3.3.1 Calculations of Antibody Concentrations
3.3.2 Panning on Cells
3.3.3 Elution of Phages Using Trypsin
4 Notes
References
Chapter 23: High-Throughput IgG Reformatting and Expression Using Hybrid Secretion Signals and InTag Positive Selection Techno...
1 Introduction
2 Materials
2.1 General Reagents
2.2 Preparation of Linearized Vector and Adaptor for Cloning
2.3 PCR Amplification of LC and VH Regions
2.4 In-Fusion Cloning
2.5 Isolation of Plasmid DNA and Sequencing Analysis
2.6 Transient Transfection
3 Methods
3.1 Preparation of Linearized Vector and Adaptor
3.2 PCR Amplification of Antibody Light Chain and Variable Heavy Chain
3.3 In-Fusion Cloning (Also see Note 5)
3.4 Transformation
3.5 IgG Reformatting Using the Cut-Paste Method
3.6 Isolation of Plasmid DNA and Sequence Confirmation
3.7 Transient Transfection
4 Notes
References
Chapter 24: Validation and the Determination of Antibody Bioactivity Using MILKSHAKE and Sundae Protocols
1 Introduction
2 Materials and Reagents
3 Methods
3.1 Construction of Sortase-Acceptor Plasmid
3.2 Preparation of MILKSHAKE Protein Inoculant
3.3 Expression of MILKSHAKE Protein
3.4 Purification of MILKSHAKE Protein
3.5 Modified Maltose Binding Protein Conjugation to Peptide
3.5.1 Conjugation Controls
3.5.2 Peptide Design
3.5.3 Modified Maltose Binding Protein Conjugation to Peptides
3.6 Ab Validation Using Western Blot
3.6.1 Protein Electrophoresis and Membrane Transfer
3.6.2 Developing and Exposing the Membrane
3.6.3 Interpretation of Data
3.7 Construction of Genetically Encoded Target Sequence Plasmid (Sundae)
3.8 Sundae Protocol-Preparation of Inoculant
3.9 Expression of Sundae Protein
3.10 Purification of Sundae Protein
3.11 Ab Validation Using Sundae ELISA
4 Notes
References
Chapter 25: Mapping Polyclonal Antibody Responses to Infection Using Next-Generation Phage Display
1 Introduction
2 Materials
2.1 General
2.2 Biopanning Against Polyclonal Immunoglobulin G
2.3 Bioinformatic Analysis to Identify Enriched Peptides Specific for Infection
2.4 Confirmation of Peptide Specificity for Infection
3 Methods
3.1 Biopanning Against Polyclonal Immunoglobulin G
3.2 Bioinformatic Analysis to Identify Enriched Peptides Specific for Infection
3.3 Confirmation of Infection-Specific Diagnostic Peptides
4 Notes
References
Chapter 26: Applications of High-Throughput DNA Sequencing to Single-Domain Antibody Discovery and Engineering
1 Introduction
2 Materials
2.1 Core NGS Workflow
2.1.1 Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
2.1.2 RNA Extraction and cDNA Synthesis
2.1.3 Construction and Panning of Phage-Displayed sdAb Libraries
2.1.4 Illumina MiSeq Sequencing
2.1.5 Data Analysis
2.2 Identification of sdAbs Against Prespecified Epitopes
2.2.1 Isolation of PBMCs
2.2.2 RNA Extraction and cDNA Synthesis
2.2.3 Construction of Phage-Displayed sdAb Libraries
2.2.4 Panning of Phage-Displayed sdAb Libraries with Competitive Elution
2.2.5 Illumina MiSeq Sequencing
2.2.6 Data Analysis
2.3 Direct Selection of Antigen-Specific B Cells from PBMCs and Identification of Antigen-Specific sdAbs
2.3.1 Isolation of PBMCs
2.3.2 Preparation of MBP-Int277-Coupled Magnetic Beads
2.3.3 Positive Selection of MBP-Int277-Reactive B cells
2.3.4 RNA Extraction and cDNA Synthesis
2.3.5 Illumina MiSeq Sequencing
2.3.6 Data Analysis
2.4 Affinity Maturation of sdAbs Using NGS
2.4.1 Construction of Random sdAb Mutagenesis Libraries
2.4.2 Construction of Site-Saturating sdAb Mutagenesis Libraries
2.4.3 Panning of sdAb Mutagenesis Libraries
2.4.4 Illumina MiSeq Sequencing
2.4.5 Data Analysis
3 Methods
3.1 Core NGS Workflow
3.1.1 Isolation of PBMCs
3.1.2 RNA Extraction and cDNA Synthesis
3.1.3 Construction and Panning of Phage-Displayed Single-Domain Antibody Libraries
3.1.4 Illumina MiSeq Sequencing
3.1.5 Data Analysis
3.2 Identification of sdAbs Against Prespecified Epitopes
3.2.1 Isolation of PBMCs
3.2.2 RNA Extraction and cDNA Synthesis
3.2.3 Construction of Phage-Displayed sdAb Libraries
3.2.4 Panning of Phage-Displayed sdAb Libraries with Competitive Elution
3.2.5 Illumina MiSeq Sequencing
3.2.6 Data Analysis
3.3 Direct Selection of Antigen-Specific B Cells from PBMCs and Identification of Antigen-Specific sdAbs
3.3.1 Isolation of PBMCs
3.3.2 Preparation of MBP-Int277-Coupled Magnetic Beads
3.3.3 Positive Selection of MBP-Int277-Reactive B cells
3.3.4 RNA Extraction and cDNA Synthesis
3.3.5 Illumina MiSeq Sequencing
3.3.6 Data Analysis
3.4 Affinity Maturation of sdAbs Using NGS
3.4.1 Construction of Random sdAb Mutagenesis Libraries by Error-Prone PCR
3.4.2 Construction of Site-Saturating sdAb Mutagenesis Libraries
3.4.3 Panning of Random and Site-Saturating sdAb Mutagenesis Libraries
3.4.4 Illumina MiSeq Sequencing
3.4.5 Expression of sdAbs and Affinity Determination by Surface Plasmon Resonance
4 Notes
References
Part V: Epitope Mapping and Biomarker Discovery by Phage Display
Chapter 27: Biomarker Discovery by ORFeome Phage Display
1 Introduction
2 Materials
2.1 Isolation of Genomic DNA
2.2 Amplification of Genomic DNA
2.3 Fragmentation of DNA
2.4 DNA End Repair
2.5 Library Construction
2.6 Antigen Library Packaging
2.7 Colony PCR
2.8 Antigen Panning and Screening-ELISA
3 Methods
3.1 Isolation of Genomic DNA
3.2 Amplification of Genomic DNA (Optional)
3.3 DNA Fragmentation
3.4 Removal of Cohesive Ends
3.5 Phagemid-Fragment Ligation and Library Construction
3.6 Library Quality Control
3.7 Library Packaging and ORF Enrichment
3.8 Colony PCR
3.9 Antigen Panning
3.10 Monoclonal Phage Production and Screening ELISA
4 Notes
References
Chapter 28: Mapping Epitopes by Phage Display
1 Introduction
2 Materials
2.1 Antigen Library Construction
2.2 Antigen Panning and Screening
3 Methods
3.1 Gene Amplification, Fragmentation, and End-Repair
3.2 Phagemid-Fragment Ligation and Library Construction
3.3 Library Quality Control and Packaging
3.4 Antigen Panning
3.5 Monoclonal Phage Production and Screening
3.6 Selection and Sequencing of Positive Hits and Epitope Determination
4 Notes
References
Chapter 29: Epivolve: A Protocol for Site-Directed Antibodies
1 Introduction
2 Materials, Reagents, and Equipment
3 Methods
3.1 Peptide Design
3.2 Phage Display Library
3.3 Isolation of mod1-specific scFvs by Phage Display Biopanning.
3.4 Site-Specific scFv Protein Titration ELISA
3.5 Error-Prone PCR
3.6 Discovery Maturation (DisMat) and Affinity Maturation (AffMat) Biopanning
3.6.1 DisMat Modifications
3.6.2 AffMat Modifications
3.7 Converting scFv Hits into Full-Length IgG Proteins and Final Antibody Validation.
4 Notes
References
Index