497 Surface Plasmon Resonance Imaging: New Tool for Immunodiagnostics of Food Allergy

BACKGROUND: Food allergy affects as many as 5 to 8% of children and 2 to 3% of adults. The milk is an important source of food allergens. The Surface Plasmon Resonance imaging (SPRi) is an optical technique used for measuring simultaneously several hundreds of biomolecular interactions (about 400 interactions) in real-time and in a label free environment. The aim of our study was to measure avidity of milk allergens with their antibodies by the SPRi. METHODS: The biochip gold surface was functionalized by mixed layers of acid and alcohol. Milk allergens (β-lactoglobulin and α-lactalbumin) and ovalbumin (used as negative control) were then immobilized by covalent bonds on the biochip surface. After saturation step, some solutions with different concentrations of polyclonal antibodies - whole serum and purified antibodies - were injected in the SPRi-plex™ system (Horiba Scientific, Genoptics). The surface coverage and the detection limits of the target antibody were measured. The avidity of the couple antigen/antibody was then calculated. RESULTS: For the couple β-lactoglobulin and whole serum from rabbit sensitized with this allergen, the surface coverage of antibody increased from 0.7 to 1160 pg/mm(2), when we injected 4 and 340 nM of antibody, respectively. The avidity of this couple is 0.7 nM. The detection limits are 2 and 0.8 nM by SPRi and ELISA, respectively. For the couple α-lactalbumin/purified anti-α-lactalbumin antibody, the surface coverage of antibody increased from 20 to 1000 pg/mm2, when we injected 10 nM and 100 μM of antibody, respectively. The avidity of this couple is about 5.2 nM and 60 nM by SPRi and capilary electrophoresis, respectively. The rate of antibody detection limit obtained by SPRi compared to what is obtained by ELISA is 35%. CONCLUSIONS: A Good proportionality of SPRi and ELISA signals was observed according to antibody concentrations. A high specificity binding and an excellent avidity between allergens and their antibodies was also shown. Limits of detection obtained with SPRi were comparable to those obtained with ELISA.