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-exp ressed molecules. To optimize the capture of antigen-specific phage an d minimize the binding of irrelevant phage antibodies, a simultaneous positive and negative selection strategy is employed. Cells bearing th e antigen of interest are pre-coated with magnetic beads and diluted i nto an excess of unmodified antigen-negative cells. Following incubati on of the cell admixture with a Fab/phage library, the antigen-positiv e cell population is retrieved using magnetically-activated cell sorti ng and antigen-specific Fab/phage are eluted and propagated in bacteri al culture. Utilizing this protocol with magnetically-labeled Rh(D)-po sitive 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) lambd a anti-Rh(D) antibodies were isolated from a single alloimmunized indi vidual. This cell-surface selection method is readily adaptable for us e in other systems, such as for the identification of putative tumor-s pecific 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. (C) 1997 Elsevier Sc ience B.V.