Investigation of surface plasmon resonance biosensor for skin sensitizers studies Christine Achilleos a , Magalie Tailhardat b , Pascal Courtellemont b , Béatrice Le Varlet b,1 , Didier Dupont c, * a INRA, UR342 Technologie et Analyses Laitières, BP 20089, F-39800 Poligny, France b LVMH Recherche, 185 Avenue de Verdun, F-45804 Saint Jean de Braye, France c INRA, UMR 1253 Science et Technologie du Lait et de l’Oeuf, 65 rue de St. Brieuc, F-35000 Rennes, France article info Article history: Received 16 June 2008 Accepted 23 November 2008 Available online 3 December 2008 Keywords: Allergens Biosensor Skin sensitization Small molecule Surface plasmon resonance abstract Non-animal testing methods are a current challenge in terms of the assessment of skin sensitization potential for new chemicals. Our objective was to investigate a surface plasmon resonance (SPR) biosen- sor to screen allergens against nucleophilic amino acids (cysteine, lysine and histidine) in a direct binding assay. Amino acids were immobilized on the sensor surface and exposed to different skin allergens (chemicals and fragrances) with varying sensitizing potential. Cysteine was found to be more reactive than lysine while histidine showed the lowest reactivity. The interactions observed were different depending on the allergen/amino acids involved. It appeared that weak allergens could quickly dissociate from the ligand, whereas strong and extreme allergens remained bound to the amino acids. The SPR report points allowed a good discrimination of the tested allergens. With this technology, we can observe low energy bindings and get information on the stability of the hapten/amino acid complex which seem relevant for the determination of skin sensitization potential. This prospective experiment showed the potential of real-time SPR to generate specific report points to refine the skin sensitization allergen assessment. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Skin exposure to allergens can result in allergic contact derma- titis which is an important health issue. Currently, the accepted ap- proaches for sensitization hazard assessment are in vivo animal tests such as the local lymph node assay (LLNA) described by Kimber et al. (1994). It is the method of choice for the determina- tion of skin sensitization potential for the purposes of categoriza- tion (Basketter et al., 2001; Kimber et al., 2003). However, animal testing of cosmetic ingredients will be banned within the EU in 2009 for several tests, and in 2013 for repeated-dose toxicity including skin sensitization studies in agreement with the seventh amendment of the European Council Directive 76/768/EEC. As a result of this directive, it has become necessary to develop alternative strategies that are based solely on in vitro methods to generate useful data for prediction of skin sensitization potential. Some of the published in vitro methods are based on keratinocytes and skin dendritic cell lines (Ashikaga et al., 2006; Hirota and Moro, 2006; Kimber et al., 2001; Ryan et al., 2007; Vandebriel et al., 2005; Van Och et al., 2005). Schoeters et al. (2007) investi- gated whether the differential gene expression patterns in den- dritic cells are relevant for the prediction of the sensitizing potential of chemicals using microarray technology. Alternative approaches to predict skin sensitization are reviewed elsewhere (Casati et al., 2005). Other strategies are based on electrophilic assays. The electro- philic properties of allergens may enable reaction with skin nucle- ophiles to form macromolecular immunogens (Aptula et al., 2005). The reactivity with nucleophiles might then be used as a skin sen- sitizers screening tool. The strongest potential nucleophiles in pro- teins are the sulfhydryl group of cysteine, methionine, the e-amino group of lysine and the imidazole group of histidine (Divkovic et al., 2003). Recently published methods are based on human ser- um albumin (HSA) reactivity to identify the types of amino acids modified and the nature of haptenation by accurate mass shift determinations and sequencing (Aleksic et al., 2007), non-enzy- matic thiol reactivity using glutathione in combination with in vitro toxicity measurement (Aptula et al., 2006), and synthetic peptides reactivity using HPLC (Gerberick et al., 2004). Surface plasmon resonance (SPR) biosensor technology has been applied to the study of numerous protein–protein interac- tions. It involves label-free binding interactions between an ana- lyte in solution and an immobilized ligand being monitored directly by changes in refractive index at the biosensor surface. The response is directly proportional to the mass of the bound ana- lyte. Recently published studies demonstrate the applicability of SPR in characterizing small molecule interactions: drug–protein interactions (Touil et al., 2005; Wear et al., 2005), antigen–anti- body interactions (Aizawa et al., 2007; Kawazumi et al., 2005; 0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.11.007 * Corresponding author. Tel.: +33 2 23 48 57 44; fax: +33 2 23 48 53 50. E-mail address: didier.dupont@rennes.inra.fr (D. Dupont). 1 Present address: Links Ingénierie, Paris, France. Toxicology in Vitro 23 (2009) 308–318 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit