16 May 2010
DEVELOPING DETECTION METHODS FOR COCCIDIOSTATS

Coccidiostats are the only anti-bacterial substances still authorised as feed additives and constitute the main choice to fight against coccidiosis, one of the major parasitic disease occurring in poultry. The conditions of use of coccidiostats are regulated but to protect consumers, the correct use of these feed additives must be monitored and maximum levels (ML’s) have been set by the European Union (regulation 124/2009). Within the work package WP2a (coccidiostats) of the CONffIDENCE project, one of the major objectives is the development and validation of a flow cytometry-based multiplex immunoassay (FCIA) using the MultiAnalyte Profiling (xMAP) technology of Luminex (Austin, Texas, USA) that would allow the simultaneous detection of six target coccidiostats at residue or cross-contamination levels of concentration in eggs and in non-target feed. The antigens (drug-protein conjugates) are covalently coupled onto carboxylated polystyrene microspheres which are internally dyed with a red and orange fluorophore. By using different ratios of these two fluorophores, it is in principle possible to create up to 100 different microsphere sets. After incubation with coccidiostat-specific antibodies and green fluorescent labelled second antibodies, the microspheres are analysed in a dual laser reader containing a red laser for identification of the microsphere sets and a green laser for the quantification of the amount of antibodies bound to the beads. This combination makes it possible to simultaneously measure up to 100 different biomolecular reactions in a single well.

During the second year of the CONffIDENCE project, the set of necessary immunochemical reagents was successfully completed by the two partners CER (Belgium) and QUB (North Ireland); selective and sensitive antibodies against monensin, salinomycin, diclazuril, lasalocid and nicarbazin were produced and characterised both by ELISA whenever possible (CER) and using the FCIA coupled to the xMAP technology (RIKILT, The Netherlands). Individual (single-plex) FCIAs could therefore be developed by RIKILT. These assays involved the coupling of the beads with ovalbumin and horse radish peroxidase conjugates or directly with the target coccidiostat. The performance achieved in buffer was satisfactory enough to trigger the combination of the single-plex assays into two multiplex FCIAs  that allow the simultaneous determination on the one-hand of narasin, salinomycin, nicarbazin and diclazuril and on the other hand of lasalocid and monensin. The sensitivity of these two multiplex assays showed to be within the range of the maximum target levels; the two multiplex assays are therefore ready to be tested on the feed (MasterLab, The Netherlands) and egg (CER, Belgium, FERA, UK) sample materials containing coccidiostats also produced within the project.