Research paper Comparison of random and oriented immobilisation of antibody fragments on mixed self-assembled monolayers Kristien Bonroy b, ⁎ , Filip Frederix a , Gunter Reekmans a , Ellen Dewolf a , Randy De Palma a , Gustaaf Borghs a , Paul Declerck b , Bruno Goddeeris c,d a IMEC, MCP-ART, Kapeldreef 75, B-3001 Leuven, Belgium b K.U.Leuven, Laboratory for Pharmaceutical Biology and Phytopharmacology, Van Evenstraat 4, B-3000 Leuven, Belgium c K.U.Leuven, Laboratory for Physiology and Immunology of Domestic Animals, Kasteelpark Arenberg 30, B-3001 Leuven, Belgium d U.Gent, Laboratory for Immunology, Salisburylaan 133, B-9820 Merelbeke, Belgium Received 29 June 2005; received in revised form 1 February 2006; accepted 16 March 2006 Available online 25 April 2006 Abstract The sensitivity of immunosensors is strongly dependent on the amount of immobilised antibodies and their remaining antigen binding properties. The use of smaller and well-oriented antibody fragments as bioreceptor molecules influences the final immunosensor signal. The aim of this study was to compare the immunosensor responses of different immobilised antibody fragments, such as F(ab′) 2 and Fab′, with their parental IgG. In addition, we evaluated the oriented versus the random covalent immobilisation method of the Fab′ fragments. First, an optimisation of cleavage protocol to generate these F(ab′) 2 and Fab′ fragments was performed. Subsequently, we pursued a study with limited denaturation effects during immobilisation of the bioreceptor molecules and with reduced steric hindrance during antigen binding using mixed self-assembled monolayers (SAM) of thiols as the chemical linking layer. The Surface Plasmon Resonance technique was used to evaluate the degree of immobilisation of the antibody fragments and their parental IgGs on the mixed SAMs and the binding signals of their specific antigens. In this study, we demonstrate that for a particular antibody/antigen system (anti-hIgG/hIgG), the optimised fragmentation protocol in combination with an oriented immobilisation of Fab′ fragments on mixed SAMs leads to a > 2-fold increase of the antigen binding signals compared to randomly covalent immobilised full-length antibodies. © 2006 Elsevier B.V. All rights reserved. Keywords: Antibody fragments; Mixed self-assembled monolayers; SPR biosensor; Oriented coupling 1. Introduction In recent years, there has been an increasing need for the detection of low concentrations of (bio)chemical substances. Biosensors could provide a rapid and convenient alternative to conventional analytical meth- ods for monitoring these substances in various applica- tion fields (Pearson et al., 2000; Rogers, 2000; Nakamura and Karube, 2003). In general, a biosensor consists of two parts, i.e. a transducer and a biological interface, which lead to the variation of a physical quantity when the analyte of interest binds to the sensor system. The biological interface of an immunosensor Journal of Immunological Methods 312 (2006) 167 – 181 www.elsevier.com/locate/jim ⁎ Corresponding author. Tel.: +32 16 281050; fax: +32 16 281097. E-mail address: kristien.bonroy@imec.be (K. Bonroy). 0022-1759/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2006.03.007