title: Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.
abstract: The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv) in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.
authors.affiliation: Seattle Biomedical Research Institute, Seattle, Washington, United States of America.
authors.forename: Sean A
authors.initials: SA
authors.lastname: Gray
authors.forename: Kris M
authors.initials: KM
authors.lastname: Weigel
authors.forename: Ibne K M
authors.initials: IK
authors.lastname: Ali
authors.forename: Annie A
authors.initials: AA
authors.lastname: Lakey
authors.forename: Jeremy
authors.initials: J
authors.lastname: Capalungan
authors.forename: Gonzalo J
authors.initials: GJ
authors.lastname: Domingo
authors.forename: Gerard A
authors.initials: GA
authors.lastname: Cangelosi
mesh_terms: D000911:Antibodies, Monoclonal / Q000191:economics* / Q000276:immunology; D000953:Antigens, Protozoan / Q000191:economics / Q000276:immunology*; D003365:Costs and Cost Analysis; D004748:Entamoeba histolytica / Q000276:immunology*; D005455:Fluorescent Antibody Technique; D005612:Freeze Drying / Q000191:economics* / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents / Q000191:economics; D057230:Limit of Detection; D008856:Microscopy, Fluorescence; D015335:Molecular Probes / Q000191:economics / Q000378:metabolism; D015800:Protozoan Proteins / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000191:economics / Q000276:immunology*; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D007202:Indicators and Reagents; D015335:Molecular Probes; D015800:Protozoan Proteins; D057127:Single-Chain Antibodies