title: Isolation of cell surface-specific human monoclonal antibodies using phage display and magnetically-activated cell sorting: applications in immunohematology.
abstract: A method is described for the isolation of filamentous phage-displayed human monoclonal antibodies directed at unpurifiable cell surface-expressed molecules. To optimize the capture of antigen-specific phage and minimize the binding of irrelevant phage antibodies, a simultaneous positive and negative selection strategy is employed. Cells bearing the antigen of interest are pre-coated with magnetic beads and diluted into an excess of unmodified antigen-negative cells. Following incubation of the cell admixture with a Fab/phage library, the antigen-positive cell population is retrieved using magnetically-activated cell sorting and antigen-specific Fab/phage are eluted and propagated in bacterial culture. Utilizing this protocol with magnetically-labeled Rh(D)-positive and excess unlabeled Rh(D)-negative human red blood cells and a Fab/phage library constructed from human peripheral blood lymphocytes, dozens of unique clinically-useful gamma 1 kappa and gamma 1 lambda anti-Rh(D) antibodies were isolated from a single alloimmunized individual. This cell-surface selection method is readily adaptable for use in other systems, such as for the identification of putative tumor-specific antigens and provides a rapid (< 1 month), high-yield approach for isolating self-replicative antibody reagents directed at novel or conformationally-dependent cell-surface epitopes.
authors.affiliation: Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA. siegeld@mail.med.upenn.edu
authors.forename: D L
authors.initials: DL
authors.lastname: Siegel
authors.forename: T Y
authors.initials: TY
authors.lastname: Chang
authors.forename: S L
authors.initials: SL
authors.lastname: Russell
authors.forename: V Y
authors.initials: VY
authors.lastname: Bunya
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity; D000954:Antigens, Surface / Q000276:immunology* / Q000378:metabolism; D010582:Bacteriophage lambda / Q000276:immunology*; D001710:Biotin / Q000378:metabolism; D001788:Blood Grouping and Crossmatching / Q000379:methods*; D003001:Cloning, Molecular; D004912:Erythrocytes / Q000276:immunology; D015723:Gene Library; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000032:analysis; D018189:Immunomagnetic Separation / Q000379:methods*; D007202:Indicators and Reagents; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism; D012204:Rh-Hr Blood-Group System / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D007140:Immunoglobulin Fab Fragments; D007202:Indicators and Reagents; D012204:Rh-Hr Blood-Group System; D001710:Biotin