Discovery of human antibodies to cell surface antigens by capture lift screening of phage-expressed antibody libraries.

摘要: An assay for the rapid identification and cloning of antibody fragments (Fabs) reactive with cell surface antigens was established used to identify Fabs selectively tumor antigens. The were produced by a phage expression system screened modified plaque lift approach in which nitrocellulose filters coated an anti-immunoglobulin reagent blocked bovine serum albumin prior application phage-infected bacterial lawn. Subsequently, capture lifts incubated biotinylated antigen identified streptavidin conjugates. This screening method, termed lift, results immobilization greater quantities Fab decreases binding unrelated host proteins, resulting more sensitive assay. permits simultaneous analysis thousands clones and, importantly, can be crude detergent-solubilized extracts, permitting discovery bind integral membrane proteins present heterogeneous mixtures Optimal conditions utilizing phage-expressed BR96 horseradish peroxidase conjugate Lewis Y, soluble cross-reactive antigen. it demonstrated that functional postnuclear detergent extracts isolated from surface-biotinylated cells expressing Purification target not required. To demonstrate novel human library constructed tumor-infiltrating B lymphocytes screened. Multiple reacted identified. Upon further characterization portion these displayed selectivity cells, as fixed live but normal fibroblasts.