Development and validation of Biacore methods for the detection of streptomycin/dihydrostreptomycin, gentamicin and neom

1. Development and validation of Biacore methods for the detection of streptomycin/dihydrostreptomycin, gentamicin and neomycin in milk VIIth International Conference on Agri-Food Antibodies, Uppsala (Sweden), September 10-13, 2003 Valérie GAUDIN, Céline HEDOU, Pascal SANDERS Community Reference Laboratory for Veterinary Drug Residues, AFSSA, BP 90203, 35302 Fougères, France e-mail : v.gaudin@fougeres.afssa.fr Streptomycin (STRP), dihydrostreptomycin (DHS), gentamicin (GTM) and neomycin (NEO) are four antibacterial (AB) substances used in veterinary medicine belonging to the family of aminoglycosides. Residues of antibiotics could be present in food of animal origin after treatment. Therefore some sensitive analytical methods are needed to detect these residues. Among immunological methods, the biosensor technology has already been applied to the development of methods for the screening or post-screening of antibiotic residues in animal matrices [1-6]. During this study different antibodies have been tested for their binding capacity to the concerned antibiotics. Some parameters have been optimised like flow rate, antibody/sample ratio, regeneration… 3 different protocols have been developed for GTM, NEO and STRP. 2 were validated according to the European Decision 2002/657/EC: 1 for the detection of streptomycin/dihydrostreptomycin, and 1 for neomycin in milk. Biacore X MRLs in milk (µg/l): streptomycin 200 dihydrostreptomycin 200 gentamicin 100 neomycin 1500 PROTOCOLS Flow rate = 20 µl/min for the 3 protocols ANTIBODIES The monoclonal antibody against dihydrostreptomycin and streptomycin (codified 504 10.2) was kindly supplied by A. van Amerongen (ATO, Wageningen, The Netherlands. The monoclonal antibodies against neomycin and against gentamicin were respectively purchased from Interchim (produced by Biodesign) and Eurodignostica (Abcys France). VALIDATION SELECTIVITY / SPECIFICITY Cross-reactivities were tested at this time only for streptomycin antibody with spiked milk samples at 10000 µg/l for various AB: streptomycin (100 %), DHS (80 %), mixing penicillin G/DHS (400/200 µg/l) (34 %), mixing penicillin G/DHS (100/200 µg/l) (24 %) and 0 % for other aminoglycosides and other ABs. Streptomycin: 1/30 of the blank milk samples was detected upper than CC. 2/30 of the blank milk samples were detected upper than LOD. Neomycin and gentamicin : 0/30 of the blank milk samples was detected upper than CC or LOD. This table summarises the combination of the experiments to determine performance parameters with minimum workload: CALIBRATION CURVES These graphs represents the mean of 11, 14, 16 calibration curves performed on 11, 14, 16 different days of analysis for GTM, NEO and STRP respectively. PRECISION: REPEATABILITY/WITHIN-LABORATORY REPRODUCIBILITY CONCLUSION : Repeatability CV were very satisfactory for the 3 protocols and all days (except day 4 for neomycin).Overall CV were high for streptomycin but it is noticed that the sensor chip was changed between day 1 and day 2. The experiment with other technician was also very satisfactory. In all cases we were able to discriminate between blank and spiked samples at the MRL and between samples spiked at 0.5, 1 and 1.5 times the MRL. The analysis of 6 replicates of milk samples spiked at 0.5, 1 and 1.5 times the MRL was performed during 6 days for repeatability study. Within-lab reproducibility was tested when the analysis was performed by other technician. Mean, SD (standard deviation) and CV (%) were calculated with the calculated concentration for a set of spiked samples at 0.5, 1 and 1.5 times the MRL of each aminoglycoside. CC / CC At or above CC, a sample is declared non-compliant (probability error ).CC is the smallest content that may be detected (probability error 1-).This way of calculating CC and CC was not representative and adapted to these BIACORE methods. CC and CC should probably be determined each day with the calibration curves . The LOD was more adapted to declare compliant or non-compliant samples. The LODs were lower than the MRLs for the 3 protocols. All NEO and STRP samples spiked at 0.5 MRL were calculated lower than the LOD (except day 1-STRP) and all samples spiked at 1 MRL were upper than the LOD. STABILITY C/C0 (%) : concentration at day x / concentration at day 0 * 100 Stability of stock solutions (1 mg/ml) at +4 °C and -20°C (1, 2, 4 and 8 weeks) and stability of antibiotic at the MRL in milk at -20°C (1, 2, 4 and 20 weeks). Decision limit: CC=concentration at the MRL + 1.64*SD (=5%) Detection capability: CC= CC + 1.64*SD (=5%) LOD (limit of detection): LOD= the concentration corresponding to a relative response of 100% - 3*SD LOQ (limit of quantification): LOQ= the concentration corresponding to a relative response of 100% - 10*SD). CONCLUSION For gentamicin, the first results were satisfactory but the reproducibility of the immobilisation was very bad. It was not possible to obtain the same results on the second chip. The immobilisation protocol of neomycin should also be improved because of the decrease of the signal on the surface little by little. Other antibodies against streptomycin and gentamicin (including polyclonal antibody S27 against STRP from C. Elliott (Belfast, Northern Ireland) will be tested in the next future. References: Baxter JA et al.Journal of Agriculture and Food Chemistry 2001, 49, 3204-3207 Ferguson JP et al. The Analyst 2002, 127, 951-956 Gaudin V. et al.JAOAC 1999, 82 (6), 1316-1320 Gaudin V. et al. Analytica Chimica Acta 2001, 436, 191-198 Gaudin V. et al.Food and Agricultural Immunology 2001, 13 (2), 77-86 Haasnoot W et al. Food and Agricultural Immunology 2002, 14, 15-27 Finally the tested antibodies were very satisfactory but the immobilisation was the limiting factor and needs to be improved for gentamicin and neomycin.