Isolation of cell surface-specific human monoclonal antibodies using phage display and magnetically-activated cell sorting: applications in immunohematology

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 negativ...

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Bibliographic Details
Published in:Journal of immunological methods 1997-08, Vol.206 (1), p.73-85
Main Authors: Siegel, Don L, Chang, Tylis Y, Russell, Shari L, Bunya, Vatinee Y
Format: Article
Language:English
Subjects:
Antibodies, Monoclonal - chemistry
Antibody Specificity
Antigens, Surface - immunology
Antigens, Surface - metabolism
Bacteriophage lambda - immunology
Biotin - metabolism
Blood Grouping and Crossmatching - methods
Cloning, Molecular
Erythrocytes - immunology
Gene Library
Humans
Immunoglobulin Fab Fragments - analysis
Immunomagnetic Separation - methods
Indicators and Reagents
Lymphocytes - immunology
Lymphocytes - metabolism
Monoclonal antibody
Phage display
Red blood cell
Repertoire cloning
Rh antigen
Rh-Hr Blood-Group System - immunology
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Summary: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 γ 1 κ and γ 1 λ 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 (
ISSN:0022-1759
1872-7905
DOI:10.1016/S0022-1759(97)00087-2