Synthetic peptide immunogens elicit polyclonal and monoclonal antibodies specific for linear epitopes in the D motifs of Staphylococcus aureus fibronectin-binding protein, which are composed of amino acids that are essentialfor fibronectin binding

A fibronectin (Fn)-binding adhesin of Staphylococcus aureus contains three tandem 37- or 38-amino-acid motifs (D1, D2, and D3), which function to bind Fn, Plasma from patients with S. aureus infections contain antibodies that preferentially recognize ligand induced binding sites in the D motifs and do not inhibit Fn binding (F, Casolini, L, Visai, D, Joh, P, G. Conaldi, A. Toniolo, M. Hook, and P. Speziale, Infect. Immun, 66:5433-5442, 1998). To eliminate the influence of Fn binding on antibody development, we used synt hetic peptide immunogens D1(21-34) and D3(20-33), which each contain a cons erved pattern of amino acids that is essential for Fn binding but which can not bind Fn without N- or C-terminal extensions, The D3(20-33) immunogen pr omoted the production of polyclonal antibodies that were 10-fold more effec tive as inhibitors of Fn-binding to the D3 motif than antibodies obtained b y immunizing with an extended peptide D3(16-36), which exhibits functional Fn binding. The D3(20-33) immunogen also facilitated the production of a mo noclonal antibody, 9C3, which was highly specific for the epitope SVDFEED, and abolished Fn binding by the D3 motif, When mixed with polyclonal anti-D 1(21-34) immunoglobulin G, 70% inhibition of Fn binding to the three tandem D motifs was achieved compared to no more than 30% inhibition with either antibody preparation alone. Therefore, by immunizing with short synthetic p eptides that are unable to bind Fn, we have effectively stimulated the prod uction of antibodies specific for epitopes comprised of amino acids that ar e essential for Fn binding. Although these epitopes occur within a conserve d pattern of amino acids that is required for Fn binding, the antibodies re cognized specific linear epitope sequences and not a conserved structure co mmon to all repeated motifs.