Binding and orientation of fibronectin on polystyrene surfaces using immobilized bacterial adhesin-related peptides U. Klueh, 1–3 J. D. Bryers, 1,4 D. L. Kreutzer 1–3,5 1 The Center for Biomaterials, University of Connecticut, School of Medicine, School of Dental Medicine, Farmington, Connecticut 06030 2 Department of Pathology, University of Connecticut, School of Medicine, School of Dental Medicine, Farmington, Connecticut 06030 3 Department of Biomedical Engineering, University of Connecticut, School of Medicine, School of Dental Medicine, Farmington, Connecticut 06030 4 Department of Biostructure and Function, University of Connecticut, School of Medicine, School of Dental Medicine, Farmington, Connecticut 06030 5 Department of Surgery, University of Connecticut, School of Medicine, School of Dental Medicine, Farmington, Connecticut 06030 Received 3 August 2002; Revised 4 October 2002; Accepted 4 October 2002 Abstract: Fibronectin (FN) is known to bind to bacteria via high affinity receptors on bacterial surfaces known as ad- hesins. The binding of bacteria to FN is thought to have a key role in foreign device associated infections. For example, previous studies have indicated that Staphylococcus aureus adhesins bind to the 29 kDa NH 3 terminus end of FN, and thereby promote bacteria adherence to surfaces. Recently, the peptide sequences within the S. aureus adhesin molecule that are responsible for FN binding have been identified. Based on these observations, we hypothesize that functional FN can be bound and specifically oriented on polystyrene surfaces using bacterial adhesin-related (BRP-A) peptide. We further hypothesize that monoclonal antibodies that re- act with specific epitopes on the FN can be used to quantify both FN binding and orientation on these surfaces. Based on this hypothesis, we initiated a systematic investigation of the binding and orientation of FN on polystyrene surfaces using BRP-A peptide. To test this hypothesis, the binding and orientation of the FN to immobilized BRP-A was quantified using 125 I-FN, and monoclonal antibodies. 125 I-FN was used to quantitate FN binding to peptide-coated polystyrene sur- faces. The orientation of bound FN was demonstrated by the use of monoclonal antibodies, which are reactive with the amine (N) or carboxyl (C) termini of the FN. The results of our studies demonstrated that when the BRP-A peptide was used to bind FN to surfaces that: 1. functional FN was bound to the peptide; 2. anti-C terminus antibodies bound to the peptide FN; and 3. only limited binding of anti-N terminus antibodies to peptide-bound FN occurred. We believe that the data that indicate an enhanced binding of anti-C anti- bodies reactive to anti-N antibodies are a result of the FN binding in an oriented manner with the N termini of FN bound tightly to the BRP-A on the polystyrene surface. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 36 – 43, 2003 Key words: fibronectin; orientation; bacterial-related pep- tides; anti-fibronectin antibodies INTRODUCTION Recently, we have demonstrated the ability of de- fined peptides, such as the collagen-related peptides I and II (CRP-I and CRP-II), to bind fibronectin (FN) on polystyrene surfaces. 1 These studies also demon- strated that monoclonal antibodies to specific FN epitopes can be used to quantify not only FN binding, but also the orientation of the FN molecules immobi- lized on surfaces. Based on these studies, we hypoth- esize that other FN-binding proteins or peptides may also be useful in immobilizing and orienting FN on various surfaces. Previously, it was demonstrated that microorganisms, such as bacteria, contain cell-surface molecules, which are capable of specifically binding to FN. 2,3 These molecules, referred to as FN-binding pro- Correspondence to: D. L. Kreutzer; e-mail: kreutzer@ sun.uchc.edu Contract grant sponsor: National Institutes of Health; con- tract grant number: R14171 Contract grant sponsor: National Science Foundation; contract grant number: DEB-9817719 © 2003 Wiley Periodicals, Inc.