Filter screening of antibody Fab fragments secreted from individual bacterial colonies: Specific detection of antigen binding with a two-membrane system

Abstract

Recently antibody fragments have been expressed in a functional form from bacteria. We have devised a simple method to detect the binding of antigen to antibody Fab fragments secreted by bacterial colonies. Bacteria harboring plasmid vectors that direct the secretion of Fab fragments into the bacterial periplasm are grown on one membrane. The secreted fragments are allowed to diffuse to a second “capture” membrane coated with anti-globulin, and are probed with antigen. Using enzyme or colloidal gold conjugates, the binding of antigen is detected on the second membrane as a colored spot. The colonies can be regrown on the first membrane, and the antigen binding signal on the second membrane is free of noise contributed by bacterial debris.