The specific goal of this proposal is to develop a high-throughput method to identify scFv antibodies that recognize post-translationally modified proteins. We propose to complete a proof-of-concept for a new type of affinity screening process based on emulsion, secretion, and capture, which we term "ESCape". In the proposed phage ESCape method, E. coli transformed with a phagemid encoding an scFv antibody-gpIII fusion library is infected with M13K07-H9 (His6::gpIX) helper phage. The infected bacterium is then compartmentalized along with a cobalt-chelate "capture-bead" in a water-in-oil (W/O) emulsion. Consequently, in each isolated compartment during overnight incubation, thousands of copies of the recombinant phage displaying an scFv protein at one end (fused to the gpIII protein) and a his6 peptide at the opposite end (fused to the gpIX protein) are produced (Fig. 1). The his6-end of the display phage will bind to the cobalt-chelate capture-bead. The emulsion will be broken, and these beads, along with any phage bound to them, will be isolated. The phage-bound beads will be incubated with an Alexa 488-labeled antigen and then washed to remove any unbound ligand. The labeled beads will then be sorted (together with the phage attached to them) from the population of beads by flow cytometry. Enriched beads can be plated onto a lawn of E. coli and the phagemid-encoded scFv further tested for specificity and affinity. We will compare this phage ESCape method with traditional methods for phage biopanning to identify scFv antibodies that recognize the glycosylated extracellular domain of the c-ErbB2/Her2 protein. The number of specific scFvs identified, their binding affinities, the time and cost associated with the screening method, and the ability to automate the assay will be evaluated.
Public Health Relevance: The goal of this proposal is to develop a high-throughput method to identify scFv antibodies that recognize post-translationally modified proteins. A novel method is described for amplifying single bacteriophage within an emulsion droplet and subsequently capturing and displaying the phage on the surface of a 3 micron bead. We will apply this phage-based surface display method toward identification of single chain variable fragment (scFv) antibodies that specifically recognize a glycosylated protein. The number of specific scFvs identified, their binding affinities, the time and cost associated with the screening method, and the ability to automate the assay will be evaluated and compared with traditional phage biopanning.
Thesaurus Terms: Atgn; Affinity; Antibodies; Antibody Specificity; Antigens; Assay; Bacteria; Bacteriophages; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological Assay; Biological Function; Biological Models; Biological Process; Blood Serum; Cells; Co Element; Cobalt; Cytofluorometry, Flow; Dose; E Coli; Erbb2; Erbb2 Gene; Emulsions; Escherichia Coli; External Domain; Extracellular Domain; Figs; Figs - Dietary; Flow Cytofluorometries; Flow Cytometry; Flow Microfluorimetry; Genes, Her-2; Genes, Her2; Genes, Erbb-2; Genes, Neu; Goals; Her -2; Her-2; Her2; Her2/Neu; High Throughput Assay; Human; Human Egf Receptor 2 Gene; Human, General; Hydrogen Oxide; Image; Incubated; Individual; Investigators; Label; Libraries; Ligand Binding; Ligands; Man (Taxonomy); Man, Modern; Methods; Microfluorometry, Flow; Model System; Models, Biologic; Molecular; Molecular Interaction; Nci; Nci Organization; National Cancer Institute; Oils; Peptides; Phage Display; Phages; Population; Process; Production; Proteins; Proteome; Proteomics; Reagent; Recombinants; Research Personnel; Researchers; Screening Procedure; Serum; Sorting - Cell Movement; Specificity; Surface; System; System, Loinc Axis 4; Tkr1; Technology; Testing; Time; Water; Bacterial Virus; Base; C-Erbb-2; C-Erbb-2 Genes; C-Erbb-2 Proto-Oncogenes; Cost; Flow Cytophotometry; Gene Product; High Throughput Screening; Imaging; Immunogen; Novel; Public Health Relevance; Response; Screening; Screenings; Sorting; Tool
