title: High throughput identification of monoclonal antibodies to membrane bound and secreted proteins using yeast and phage display.
abstract: Antibodies are ubiquitous and essential reagents for biomedical research. Uses of antibodies include quantifying proteins, identifying the temporal and spatial pattern of expression in cells and tissue, and determining how proteins function under normal or pathological conditions. Specific antibodies are only available for a small portion of the proteome, limiting study of those proteins for which antibodies do not exist. The technologies to generate target-specific antibodies need to be improved to obtain high quality antibodies to the proteome at reasonable cost. Here we show that renewable, validated, and standardized monoclonal antibodies can be generated at high throughput, without the need for antigen production or animal immunizations. In this study, 60 protein domains from 24 selected secreted proteins were expressed on the surface of yeast and used for selection of phage antibodies, over 400 monoclonal antibodies were identified within 3 weeks. A subset of these antibodies was validated for binding to cancer cells that overexpress the target protein by flow cytometry or immunohistochemistry. This approach will be applicable to many of the membrane-bound and the secreted proteins, 20-40% of the proteome, accelerating the timeline for Ab generation while reducing the cost.
authors.affiliation: Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco General Hospital, San Francisco, California, United States of America.
authors.forename: Lequn
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco General Hospital, San Francisco, California, United States of America.
authors.forename: Liang
authors.initials: L
authors.lastname: Qu
authors.affiliation: Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco General Hospital, San Francisco, California, United States of America.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco General Hospital, San Francisco, California, United States of America.
authors.forename: Zhengda
authors.initials: Z
authors.lastname: Sun
authors.affiliation: Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco General Hospital, San Francisco, California, United States of America.
authors.forename: Hao
authors.initials: H
authors.lastname: Zou
authors.affiliation: Departments of Pathology & Laboratory Medicine, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Yunn-Yi
authors.initials: YY
authors.lastname: Chen
authors.affiliation: Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco General Hospital, San Francisco, California, United States of America.
authors.forename: James D
authors.initials: JD
authors.lastname: Marks
authors.affiliation: Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco General Hospital, San Francisco, California, United States of America.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhou
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D000914:Antibodies, Viral / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D001435:Bacteriophages / Q000276:immunology*; D045744:Cell Line, Tumor; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D008565:Membrane Proteins