Genotype Phenotype Coupling: Methods and Protocols

Description

This detailed new edition broadens the scope of the first edition by moving beyond classical display technologies. This book explores methodologies for the generation of natively paired antibody libraries, single cell technologies, alternative scaffolds, and in silico antibody sequence assessments are described. The application of these methods may allow for a generation of improved therapeutics and diagnostic reagents in a shorter time frame. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.  Authoritative and practical, Genotype Phenotype Coupling: Methods and Protocols, Second Edition serves as an ideal guide for researchers seeking to expand their knowledge of antibody-based therapeutics. Preface Contents Contributors Chapter 1: Quantitative Determination of Staphylococcus aureus Using Aptamer-Based Recognition and DNA Amplification Machinery 1 Introduction 2 Materials 2.1 Strains 2.2 Oligonucleotides 2.3 Media 2.4 Preparation and Modification of Gold Electrode 2.5 Detection of S. aureus 3 Methods 3.1 Cultivation and Preparation of Bacterial Strains 3.2 Preparation of Aptamer/W, AS/RP Duplexes 3.3 Formation of the Circular DNA 3.4 Verification Through Polyacrylamide-Gel Electrophoresis (PAGE) 3.5 Preparation and Modification of Gold Electrode 3.6 Target Incubation and Exo III Enzymatic Hydrolysis 3.7 Rolling Circle Amplification Reaction 3.8 Detection of S. aureus 3.9 Optimization of the Conditions for Electrochemical Detection of S. aureus 3.9.1 Optimization of the Formation of Circular DNA 3.9.2 Optimization of the Complementary Length of Aptamer/W and AS/RP Duplexes 3.9.3 Optimization of the Concentration Ratio of Aptamer/W and AS/RP Duplex 3.9.4 Optimization of the Concentration of Exo III and Reaction Time 3.9.5 Optimization of the Concentration of phi29 DNA Polymerase and Reaction Time 3.10 Field Emission Scanning Electron Microscopy Characterization of DNA Nanoflowers 3.11 Detection of S. aureus Using Aptamer-Based Recognition and DNA Amplification Machinery 3.12 Specificity Studies of the Detection Method 4 Notes References Chapter 2: Construction of Synthetic VHH Libraries in Ribosome Display Format 1 Introduction 2 Materials 2.1 Oligonucleotides 2.1.1 Primers Used for Generation of the 5′-Flanking Region of the Ribosome Display Construct and the Randomized Positions of ... 2.1.2 Primers for the Amplification of tolA Linker Encoded by pFP-RDV3 2.1.3 Primers for the Final Assembly of the 5′-Construct and tolA Linker 2.1.4 Primers for PCR to Prepare NGS Samples 2.2 PCR 3 Methods 3.1 Production of Input Library 3.1.1 Production of the VHH Library Fragments 3.1.2 Production of the tolA Fragment 3.1.3 Production of the Ribosome Display Construct 3.1.4 Next-Generation Sequencing 4 Notes References Chapter 3: Isolation of Adhirons Specific for Plant Protoplast Membrane Biomarkers Is Simplified by Phagemid Design 1 Introduction 2 Materials 2.1 Protoplast Isolation 2.2 Protoplast Assessments 2.3 Biopanning 2.4 Screening 3 Methods 3.1 Protoplast Isolation 3.2 Biopanning 3.2.1 Coating of Beads with the Antigen (See Note 10) 3.2.2 Bead and Library Blocking 3.2.3 Washing 3.2.4 Phage Elution and Amplification 3.2.5 Phage Production 3.2.6 Phage Precipitation 3.3 Second Round of Biopanning 3.3.1 Phage Extraction for Screening 3.3.2 Bacteria Transformation 3.4 Screening 3.4.1 Colony Amplification of Transformed Bacteria 3.4.2 In-Plate Phage Production 3.4.3 Protoplast Preparation 3.4.4 Blocking and Phage-Antigen Binding 3.4.5 Washing 3.4.6 Analysis by Flow Cytometer 3.4.7 Screening in Triplicates 4 Notes References Chapter 4: Facile One-Step Generation of Camelid VHH and Avian scFv Libraries for Phage Display by Golden Gate Cloning 1 Introduction 2 Materials 2.1 cDNA Synthesis and Gene-Specific Amplification of VHH or VH/VL for Library Generation 2.2 Library Construction Phage Display 2.2.1 Plasmids (Table 1) 2.2.2 Buffers, Enzymes, Primers, and Devices for Golden Gate Cloning 3 Methods 3.1 cDNA Synthesis 3.2 Amplification of Natural Variable Antibody Domain Repertoire 3.2.1 Amplification of Camelid Heavy Chain Only Variable Domain Repertoire 3.2.2 Amplification of Chicken Variable Antibody Domain Repertoire 3.3 Golden Gate Cloning for the Generation of Phage Display Libraries 3.3.1 GGC for Camelid VHH Libraries 3.3.2 GGC for Chicken scFv Libraries 4 Notes References Chapter 5: Identification of New Antibodies Targeting Tumor Cell Surface Antigens by Phage Display 1 Introduction 2 Materials 2.1 Immunization of Mice and Analysis of Antibody Titer in Serum 2.2 Generation of Murine scFv Antibody Library from Spleen 2.2.1 Preparation of Total RNA from Spleen 2.2.2 Generation of First Strand cDNA by Reverse Transcription 2.2.3 PCR Amplification of V-Regions and Assembly of scFvs 2.3 Phage Display with Cellular Panning 2.4 Whole Cell ELISA 3 Methods 3.1 Immunization of Mice and Analysis of Antibody Titer in Serum 3.2 Generation of Murine scFv Antibody Library from the Spleen 3.2.1 Preparation of Total RNA from the Spleen 3.2.2 Generation of the First Strand cDNA by Reverse Transcription 3.2.3 Amplification of Mouse V-Regions by PCR 3.2.4 Generation of VH and VL Sub-Libraries 3.2.5 Assembly of VH and VL to scFv 3.3 Phage Display with Cellular Panning 3.3.1 Preparation of Phages 3.3.2 Phage Titration 3.3.3 Cellular Panning 3.3.4 Binding Analysis of Polyclonal Phage Preparations by Flow Cytometry 3.4 Characterization of Monoclonal Phage Antibodies 3.4.1 Production of Monoclonal Phages 3.4.2 Whole Cell ELISA 3.5 Sequencing Analyses 3.5.1 Sanger Sequencing of Monoclonal Phages 3.5.2 Next-Generation Sequencing (NGS) of Libraries (Optional) 4 Notes References Chapter 6: Phage Display of Bovine Ultralong CDRH3 1 Introduction 2 Materials 2.1 Harvesting Bovine Lymphocytes 2.2 Lymphocyte RNA Extraction 2.3 Reverse Transcription PCR of Lymphocyte RNA 2.4 Primary PCR Amplification of CDRH3 2.5 Secondary PCR Amplification of Ultralong CDRH3 2.6 PCR Restriction Digestion 2.7 Plasmid Digestion and Ligation with Amplified Ultralong CDRH3 2.8 Precipitation of Ligation Product 2.9 Electroporation of E. coli and Plating Library 2.10 Phage Rescue 2.11 Biopanning 2.12 Monoclonal Phage Rescue 2.13 Phage ELISA 3 Method 3.1 Harvesting Bovine Lymphocytes 3.2 Lymphocyte RNA Extraction 3.3 Reverse Transcription PCR of Lymphocyte RNA 3.4 Primary PCR Amplification of CDRH3 3.5 Secondary PCR Amplification of Ultralong CDRH3s 3.6 PCR Restriction Digestion 3.7 Plasmid Construction 3.8 Plasmid Digestion and Ligation with Amplified Ultralong CDRH3´s 3.9 Precipitation of Ligation Product 3.10 Electroporation of E. coli and Plating Library 3.10.1 Electroporation 3.10.2 Titer Plates 3.10.3 Library Plate 3.11 Phage Rescue 3.11.1 Collecting Biomass in Liquid Media 3.11.2 Helper Phage Infection 3.11.3 Phage Precipitation 3.12 Biopanning 3.12.1 Phage Preparation 3.12.2 Streptavidin Beads Preparation 3.12.3 Phage Binding, Washing, and Elution 3.12.4 Infection and Titration 3.13 Monoclonal Phage Rescue 3.14 Phage ELISA 4 Notes References Chapter 7: Bacterial Cell Display for Selection of Affibody Molecules 1 Introduction 1.1 Affibody Molecules 1.2 Display of Affibody Molecules on E. coli 1.3 Display of Affibody Molecules on S. carnosus 2 Materials 2.1 MACS of Bacteria-Displayed Affibody Libraries 2.1.1 E. coli Display-Specific Reagents 2.1.2 S. carnosus Display-Specific Reagents 2.1.3 General Reagents 2.1.4 Equipment 2.2 FACS of Bacteria-Displayed Affibody Libraries 2.2.1 Reagents 2.2.2 Equipment 3 Methods 3.1 MACS of Bacteria-Displayed Affibody Libraries 3.2 FACS of Bacteria-Displayed Affibody Libraries 4 Notes References Chapter 8: Isolation of Antigen-Specific Unconventional Bovine Ultra-Long CDR3H Antibodies Using Cattle Immunization in Combin... 1 Introduction 2 Materials 2.1 Strains 2.2 Plasmids 2.3 Media and Buffers 2.4 PCR Amplification of Cow UL-CDR3H Regions 2.5 Digestion of the Destination Plasmid 2.6 Transformation of S. cerevisiae BJ5464 with LC Plasmid 2.7 Library Transformation of S. cerevisiae EBY100 2.8 Labeling and Selection of Yeast Cells with Fluorescence-Activated Cell Sorting (FACS) 2.9 Retransformation of Enriched Yeast Library to E. coli 2.10 General Equipment 3 Methods 3.1 General 3.2 Amplification of Bovine Ultra Long CDR3H Domains 3.3 Transformation of BJ5464 Yeast with the Fixed LC Plasmid 3.4 Digestion of the Destination Plasmid 3.5 Transformation of Yeast for CDR3H Library Generation 3.6 Cryopreservation for Long-Term Storage of Yeast 3.7 Mating of the CDR3H Yeast Library with the Fixed LC Clone 3.8 Induction of Expression for Antibody Yeast Surface Display 3.9 Fluorescence-Activated Cell Sorting for Detection and Selection of Antigen Binding Bovine UL-CDR3H-Based Fab Fragments 3.9.1 Staining of the Library and Controls for FACS Analysis 3.9.2 Cell Handling Following FACS Analysis 3.10 Sequencing the Display Vector from Yeast Libraries 3.11 Yeast Lysis and E. coli Transformation 4 Notes References Chapter 9: Selection of High-Affinity Heterodimeric Antigen-Binding Fc Fragments from a Large Yeast Display Library 1 Introduction 1.1 Bispecific Antibodies and mAb2 Antibody Molecules 1.2 Directed Evolution of Fcab Libraries 1.3 Fcab Fragment-Antigen Interaction 1.4 Design of Heterodimeric Fcab Libraries 1.5 Heterodimeric Fcab Libraries Constructed with Yeast Mating 1.6 Yeast Display Using Combined Genome-Integrated and Episomal Expression Cassette 1.7 Sorting of Heterodimeric Fcab Library and Identification of an Antigen-Specific Clone 1.8 Future Prospects 2 Materials 2.1 Reagents 2.2 Solutions and Buffers 2.3 Media 2.4 Kits 2.5 Equipment 2.6 Plasmids, Bacterial Strains, Yeast Strains, and Cell Lines 3 Methods 3.1 Construction of the Recipient Strain with the Genome-Integrated Fcab Expression Cassette 3.2 PCR Screening of the Yeast Colonies 3.3 Transformation of the Variant Heterodimer Chain Library 3.3.1 Library-Encoding PCR Fragment Preparation 3.3.2 Recipient Vector 3.3.3 Library Transformation 3.4 Quality Control and Sorting of Heterodimer Fc Yeast Display Libraries 3.4.1 Sequencing of Library Clones 3.4.2 Staining of Displayed Fc Heterodimer Mutants 3.4.3 Selection of Heterodimeric Fc Libraries 3.5 Thermal Stability of Yeast-Displayed Heterodimeric Fcab Fragments 3.6 Expression of Selected Fcab Clones in Mammalian Expression System 4 Notes References Chapter 10: A Two-Step Golden Gate Cloning Procedure for the Generation of Natively Paired YSD Fab Libraries 1 Introduction 2 Materials 2.1 Strains 2.2 Plasmids 2.3 Reagents for Nested PCR 2.4 Reagents for Gel Purification of VH-VL Insert 2.5 Reagents for First GGC Step 2.6 Reagents for E. coli Transformation 2.7 Reagents for Plasmid Preparation 2.8 Reagents for Second GGC Step 2.9 Reagents for S. cerevisiae Library Generation 3 Methods 3.1 Introduction of Esp3I Restriction Sites to RT-OE-PCR Product 3.2 Agarose Gel Electrophoresis 3.3 Gel Extraction of the VH-VL Insert 3.4 First GGC Step 3.5 E. coli Transformation 3.6 Plasmid Preparation 3.7 Second GGC Step 3.8 Yeast Transformation for Library Generation 4 Notes References Chapter 11: Single-Cell B-Cell Sequencing to Generate Natively Paired scFab Yeast Surface Display Libraries 1 Introduction 1.1 Background 1.2 Overview of Method 2 Materials 2.1 Mouse Immunization 2.2 Single-Cell Sequencing 2.3 Lead Clone Characterization and Refinement 2.4 Library Generation and YSD Expression 3 Methods 3.1 Mouse Immunization 3.1.1 Mouse Immunization 3.1.2 Harvesting B-Cells from Immune-Adapted Mice 3.1.3 Negative Selection for B-Cell Isolation 3.1.4 Positive Selection for B-Cell Isolation 3.2 Single-Cell Sequencing of BCRs 3.2.1 Prepare GEM Reaction Mix 3.2.2 Load the Chromium Next GEM Chip K 3.2.3 Post-GEM-RT Cleanup Using Dynabeads 3.2.4 cDNA Amplification 3.2.5 SPRIselect cDNA Cleanup 3.2.6 cDNA QC and Quantification 3.2.7 V(D)J Amplification 1 3.2.8 SPRIselect Post-V(D)J Amplification 1 Cleanup 3.2.9 V(D)J Amplification 2 3.2.10 SPRIselect Post-V(D)J Amplification 2 Cleanup 3.2.11 Fragmentation, End Repair, and A-Tailing 3.2.12 Adaptor Ligation 3.2.13 SPRIselect Post-Ligation Cleanup 3.2.14 Sample Index PCR 3.2.15 SPRIselect Post-Sample Index PCR Cleanup 3.2.16 GEX Library Fragmentation, End Repair, and A-Tailing 3.2.17 SPRIselect Post-Fragmentation, End Repair, and A-Tailing Cleanup 3.2.18 GEX Adaptor Ligation 3.2.19 SPRIselect Post-Ligation Cleanup 3.2.20 GEX Sample Index PCR 3.2.21 SPRIselect Post-GEX Sample Index PCR Cleanup 3.3 Lead Clone Characterization and Refinement 3.4 Library Generation and YSD Expression 3.4.1 Prepare VH and VL Inserts 3.4.2 Prepare Yeast Expression Vector 3.4.3 Ligation 3.4.4 Transformation 3.4.5 Prepare Plasmid 3.4.6 Transform Clones Into Yeast: Option A-Electroporation 3.4.7 Transform Clones Into Yeast: Option B-EZ-Yeast Transformation 4 Notes References Chapter 12: One-Pot Droplet RT-OE-PCR for the Generation of Natively Paired Antibody Immune Libraries 1 Introduction 2 Materials 2.1 Reagents for Preparation of Single-Cell Suspensions from Lymphatic Tissues 2.2 Reagents for B Cell Isolation 2.3 Reagents for B Cell Staining for Flow Cytometry 2.4 Oligonucleotides 2.5 Reagents for Droplet RT-OE-PCR Mix (2x) 2.6 Preparation of B Cells for Droplet Encapsulation 2.7 Droplet Generation 2.8 Microscopic Analysis 2.9 Droplet RT-OE-PCR 2.10 Purification of VH-VL PCR Product 2.11 Nested PCR 3 Methods 3.1 Preparation of Single-Cell Suspensions from Lymphatic Tissues 3.2 B Cell Isolation 3.3 B Cell Staining for Flow Cytometry 3.4 Preparation of Droplet RT-OE-PCR Mix (2x) 3.5 B Cell Preparation for Droplet Encapsulation 3.6 Droplet Generation 3.7 Microscopic Analysis of Droplet Generation and Stability 3.8 Droplet RT-OE-PCR 3.9 Purification of VH-VL PCR Product 3.10 Small-Scale Nested PCR Test 3.11 Large-Scale Nested PCR 4 Notes References Chapter 13: Affinity Maturation of the Natural Ligand (B7-H6) for Natural Cytotoxicity Receptor NKp30 by Yeast Surface Display 1 Introduction 2 Materials 2.1 Strains 2.2 Plasmids 2.3 Media 2.4 Reagents for Amplification of Diversified ΔB7-H6 DNA 2.5 Reagents for Destination Vector (pDest) Digestion 2.6 Reagents for ΔB7-H6 Transformation into EBY100 2.7 Reagents for YSD and FACS Analysis 2.8 Equipment 3 Methods 3.1 PCR Amplification of N-Terminal V-Like Domain of B7-H6 (ΔB7-H6) Diversity 3.2 Destination Vector (pDest) Digestion 3.3 Yeast Transformation for Library Generation 3.4 Sequence Analysis of Yeast Cell Display Vector 3.5 Cryopreservation for Long-Term Storage of Yeast Cells 3.6 Induction of ΔB7-H6 Yeast Surface Expression 3.7 Fluorescence-Activated Cell Sorting (FACS) Analysis for the Detection of Yeast Surface Display and NKp30 Binding 3.7.1 Surface Display Control 3.7.2 Library Staining for Affinity Maturation Purposes 3.7.3 Treatment of Sorted Cells After FACS Analysis 4 Notes References Chapter 14: Accessing Transient Binding Pockets by Protein Engineering and Yeast Surface Display Screening 1 Introduction 2 Materials 2.1 Protein Engineering 2.1.1 Random Mutagenesis 2.1.2 Site Saturation Mutagenesis 2.1.3 Primers for Protein Randomization 2.2 Yeast Surface Display (YSD) Library Generation and Sorting 2.2.1 Testing Correct Protein Cell Surface Display and Optimal Ligand Concentration for Library Screening 2.2.2 YSD Library Generation 2.2.3 Library Sorting by FACS 2.2.4 Single Clone Analysis 2.3 Production of Identified Protein Variants 3 Methods 3.1 Protein Engineering 3.1.1 Random Mutagenesis 3.1.2 Site Saturation Mutagenesis 3.2 Yeast Surface Display (YSD) 3.2.1 Testing Correct Protein Cell Surface Display and Optimal Ligand Concentration for Library Screening 3.2.2 Yeast Surface Display (YSD) Library Generation 3.2.3 Library Sorting by FACS 3.2.4 Single Clone Analysis 3.3 Production of Identified Protein Variants 3.4 Evaluation of Identified Protein Variants 4 Notes References Chapter 15: Tyrosine Phosphorylation Screening on the Yeast Surface by Magnetic Bead Selection and FACS 1 Introduction 2 Materials 2.1 Mutant Library Preparation 2.1.1 Media, Buffers, and Reagents 2.1.2 Equipment and Consumables 2.1.3 Cell Lines 2.2 Flow Cytometry Analysis and FACS of Yeast Displaying Mutant Substrate Libraries 2.2.1 Media, Buffers, and Reagents 2.2.2 Equipment and Consumables 2.2.3 Cell Lines 2.3 Sorting of Yeast Cells Displaying Phosphorylated Mutant Libraries Through Magnetic Bead Selection 2.3.1 Media, Buffers, and Reagents 2.3.2 Equipment and Consumables 2.3.3 Cell Lines 3 Methods 3.1 Mutant Library Preparation 3.1.1 Mutagenic PCR Primer Design 3.1.2 Mutant Substrate Generation Through Mutagenic PCR 3.1.3 Insert Preparation Through Assembly and Extension PCR 3.1.4 Insert DNA Concentration Through Ethanol Precipitation 3.1.5 Plasmid Digest for Substrate Incorporation 3.1.6 Plasmid DNA Concentration Through Ethanol Precipitation 3.1.7 Mutated Substrate Incorporation Through Yeast Electroporation Transformation 3.1.8 Cell Growth of Yeast Harboring Mutated Substrate Libraries 3.1.9 Induction of Protein Expression 3.2 Flow Cytometry Analysis and FACS of Yeast Displaying Mutant Substrate Libraries 3.2.1 Biotinylation of 4G10 Anti-Phosphotyrosine Antibody 3.2.2 Cell Labeling for Flow Cytometry Analysis of Phosphorylated Domains 3.2.3 Evaluation of Displayed Domains Through Flow Cytometry Analysis 3.2.4 Cell Labeling for FACS of Yeast Displaying Phosphorylated Domains 3.2.5 Yeast Surface Display Library Sorting Through FACS 3.2.6 Protein Induction of Yeast Harboring Sorted Mutated Substrate Libraries 3.3 Sorting of Yeast Cells Displaying Phosphorylated Mutant Libraries Through Magnetic Bead Selection 3.3.1 Cell Growth of Yeast Harboring Mutated Substrate Libraries for Magnetic Bead Sorting 3.3.2 Protein Induction of Yeast Harboring Mutated Substrate Libraries for Magnetic Bead Sorting 3.3.3 Biotinylation of 4G10 Anti-Phosphotyrosine Antibody 3.3.4 Magnetic Bead Preparation for Sorting of Phosphorylated Domains 3.3.5 Mutant Library Preparation for Magnetic Bead Sorting of Phosphorylated Domains 3.3.6 Magnetic Bead Sorting of Yeast Cells Displaying Phosphorylated Domains 3.3.7 Protein Induction of Yeast Harboring Magnetic Bead Sorted Mutated Substrate Plasmids 3.3.8 Cell Labeling for Flow Cytometry Analysis of Sorted Phosphorylated Domains 3.3.9 Yeast Surface Display Evaluation Through Flow Cytometry Analysis of Sorted Cells 4 Notes References Chapter 16: Bulk Reformatting of Antibody Fragments Displayed on the Surface of Yeast Cells to Final IgG Format for Mammalian ... 1 Introduction 2 Materials 2.1 Yeast Surface Display (YSD) Generation and Sorting 2.1.1 Library Generation for YSD 2.1.2 Library Sorting Using Fluorescence-Activated Cell Sorting (FACS) 2.1.3 Primers for YSD Generation 2.2 Reformatting 2.2.1 Transfer of Entire ORF from YSD to MD Vector 2.2.2 Bidirectional Promoter Exchange for Mammalian Expression 2.3 Production and Characterization 2.3.1 Transient Transfection and Purification of mAbs 3 Methods 3.1 Yeast Surface Display (YSD) Library Generation and Sorting 3.1.1 Library Generation for YSD 3.1.2 Library Sorting by FACS 3.2 Reformatting 3.2.1 Transfer of Both Entire ORFs from YSD to MD Vector 3.2.2 Bidirectional Promoter Exchange for Mammalian Expression 3.3 Production of mAbs 3.3.1 Transient Production and Purification of mAbs 4 Notes References Chapter 17: Antibody-Secreting Cell Isolation from Different Species for Microfluidic Antibody Hit Discovery 1 Introduction 2 Materials 2.1 Isolation from Mouse Tissue via MACS 2.2 Isolation from Human Peripheral Blood via MACS 2.3 Isolation from Rat Tissues via FACS 2.4 Storage and Revival of B Cells 2.5 ASC Staining with Cell Tracker or Anti-CD138 Antibody 2.6 Equipment 3 Methods 3.1 Isolation of Antibody-Secreting Cells 3.1.1 Isolation from Mouse Tissue via MACS 3.1.2 Isolation from Human Peripheral Blood via MACS 3.1.3 Isolation from Rat Tissues via FACS 3.2 Storage and Revival of B Cells 3.3 ASC Staining with Cell Tracker or Anti-CD138 Antibody 3.3.1 Staining with Cell Tracker 3.3.2 Anti-CD138 Detection 4 Notes References Chapter 18: Efficient Microfluidic Downstream Processes for Rapid Antibody Hit Confirmation 1 Introduction 2 Materials 2.1 Droplet-Based Microfluidic Antibody Discovery Using Cyto-Mine 2.2 Single-Cell Antibody Gene PCR Recovery 2.3 High-Throughput Antibody Reformatting 2.4 High-Throughput Transient Antibody Production 2.5 Single-Cell Sub-Cultivation 2.6 Supernatant Binding Confirmation via Biolayer Interferometry 2.7 Equipment 3 Methods 3.1 Droplet-Based Microfluidic Antibody Discovery Using Cyto-Mine 3.2 Single-Cell Antibody Gene PCR Recovery 3.2.1 cDNA Synthesis 3.2.2 First PCR 3.2.3 Nesting PCR 3.2.4 PCR Analysis via 2% Agarose Gel 3.3 High-Throughput Antibody Reformatting 3.4 High-Throughput Transient Antibody Production 3.5 Single-Cell Sub-Cultivation 3.6 Supernatant Binding Confirmation via Biolayer Interferometry 4 Notes References Chapter 19: Cell Line Development Using Targeted Gene Integration into MAR-Rich Landing Pads for Stable Expression of Transgen... 1 Introduction 2 Materials 2.1 Plasmids 2.2 Cell Culture 2.3 Transfection Using Neon Transfection System and Selection 2.4 Clone Isolation 2.5 Assessment of Productivity of Selected Clones and Clone Stability 3 Methods 3.1 Preparation of LP, Donor, and Helper Vectors for Transfection 3.2 Generation of LP Clones 3.3 Isolation of LP Clones 3.4 Evaluation of LP Clone Stability 3.5 Transfection with Donor and Helper Vectors for the Generation of mAb-Expressing Clones 3.6 Isolation and Evaluation of mAb-Expressing Clones 3.7 Evaluation of Product Stability 4 Notes References Chapter 20: Generation of Human 293-F Suspension NGFR Knockout Cells Using CRISPR/Cas9 Coupled to Fluorescent Protein Expressi... 1 Introduction 2 Materials 2.1 CRISPR/Cas9-sgRNA Expression Plasmids 2.2 Transient Expression 2.3 Tissue Culture 2.4 Flow Cytometry and Cell Sorting 3 Methods 3.1 Transient Transfection of 293-F Cells 3.2 Determination of Transfection Efficiency 3.3 Analysis of CRISPR/Cas9-Mediated NGFR Knockout 3.4 Depletion of NGFR- and FP-Positive Cells 4 Notes References Chapter 21: Antibody Display Technology (ADbody) to Present Challenging and Unstable Target Proteins on Antibodies 1 Introduction 2 Materials 2.1 Software for Construct Design 2.2 Expression Vectors 2.3 Tissue Culture 2.4 IMAC and In-Gel Fluorescent Imaging 3 Methods 3.1 Search Protein Model and Structure-Based Construct Design 3.2 Validate the Designed Model Computationally 3.3 Molecular Cloning 3.4 Small-Scale Protein Expression and Purification 3.4.1 Day 1 3.4.2 Day 2 3.5 IMAC and In-Gel Fluorescent Imaging 4 Notes References Chapter 22: SUMO: In Silico Sequence Assessment Using Multiple Optimization Parameters 1 Introduction 2 Materials 3 Methods 3.1 Sequence Annotation and Calculation of In Silico Properties 3.2 MHC-II Binding Predictions 3.3 Sequence Clustering 3.4 Generation of an Overview Table on the In Silico Developability Assessment of Multiple Antibody/VHH Sequences 3.5 Generation of a Detailed View on Specific Relevant Features of the Variable Antibody Sequence 3.6 Generation of PyMOL Session Files 4 Notes References Chapter 23: Streamlined Data Analysis Pipeline for Deep Sequence-Coupled Biopanning Identification of Pathogen-Specific Antibo... 1 Introduction 2 Materials 3 Methods 4 Notes References Index