Genotype Phenotype Coupling Methods and Protocols

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. 

Quantitative Determination of Staphylococcus aureus Using Aptamer-Based Recognition and DNA Amplification Machinery.- Construction of Synthetic VHH Libraries in Ribosome Display Format.- Isolation of Adhirons Specific for Plant Protoplast Membrane Biomarkers is Simplified by Phagemid Design.- Facile One-Step Generation of Camelid VHH and Avian scFv Libraries for Phage Display by Golden Gate Cloning.- Identification of New Antibodies Targeting Tumor Cell Surface Antigens by Phage Display.- Phage Display of Bovine Ultralong CDRH3.- Bacterial Cell Display for Selection of Affibody Molecules.- Isolation of Antigen-Specific Unconventional Bovine Ultra-Long CDR3H Antibodies Using Cattle Immunization in Combination with Yeast Surface Display.- Selection of High-Affinity Heterodimeric Antigen-Binding Fc Fragments from a Large Yeast Display Library.- A Two-Step Golden Gate Cloning Procedure for the Generation of Natively Paired YSD Fab Libraries.- Single-Cell B-Cell Sequencing to Generate Natively-Paired scFab Yeast Surface Display Libraries.- One-Pot Droplet RT-OE-PCR for the Generation of Natively Paired Antibody Immune Libraries.- Affinity Maturation of the Natural Ligand (B7-H6) for Natural Cytotoxicity Receptor NKp30 by Yeast Surface Display.- Accessing Transient Binding Pockets by Protein Engineering and Yeast Surface Display Screening.- Tyrosine Phosphorylation Screening on the Yeast Surface by Magnetic Bead Selection and FACS.- Bulk Reformatting of Antibody Fragments Displayed on the Surface of Yeast Cells to Final IgG Format for Mammalian Production.- Antibody Secreting Cell Isolation from Different Species for Microfluidic Antibody Hit Discovery.- Efficient Microfluidics Downstream Processes for Rapid Antibody Hit Confirmation.- Cell Line Development Using Targeted Gene Integration into MAR-Rich Landing Pads for Stable Expression of Transgenes.- Generation of Human 293-F Suspension NGFR Knockout Cells Using CRISPR/Cas9 Coupled to Fluorescent Protein Expression.- Antibody Display Technology (ADbody) to Present Challenging and Unstable Target Proteins on Antibodies.- SUMO: In Silico Sequence Assessment Using Multiple Optimization Parameters.- Streamlined Data Analysis Pipeline for Deep Sequence-Coupled Biopanning Identification of Pathogen-Specific Antibody Responses in Serum.