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Results of a European proficiency test for the detection of streptomycin/dihydrostreptomycin, gentamicin and neomycin in milk by ELISA and Biacore methods EURORESIDUE V,Noordwijkerhout (The Netherlands), 10-12 May, 2004 Valrie GAUDIN, N. CADIEU, Pascal SANDERS Community Reference Laboratory for Veterinary Drug Residues, AFSSA, BP 90203, 35302 Fougres, France e-mail : v.gaudin@fougeres.afssa.fr Aminoglycoside antibiotics are commonly used in veterinary medicine for treatment of bacterial infections. Neomycin (NEO), gentamicin (GTM), streptomycin (STR) and dihydrostreptomycin (DHS) are included in Annex I of Council Regulation (EEC) 2377/90 [1] and their respective MRLs in milk are : 1500, 100, 200 and 200 g/kg. In 2003, an interlaboratory study was proposed to the European Community and Third countries National Reference Laboratories (NRL) for the analysis of aminoglycoside residues in bovine milk by ELISA. This test was intended to allow the participants to control their aminoglycoside ELISA methods when used routinely and also to compare the performance of various ELISA kits for the detection of neomycin, gentamicin, streptomycin and DHS in bovine milk. RESULTS AND DISCUSSION ORGANISATION OF THE PROFICIENCY TEST CONCLUSION Test materials : 12 random coded frozen samples were sent in dried ice (including 2 blank samples and 10 spiked milk samples) : STR at 100 ng/ml (0.5*MRL) and at 300 ng/ml (1.5*MRL) in blind duplicate, DHS at 100 ng/ml (0.5*MRL) and at 300 ng/ml (1.5*MRL), GTM at 50 ng/ml (0.5*MRL) and at 150 ng/ml (1.5*MRL), NEO at 75 ng/ml (0. 5*MRL) and at 225 ng/ml (0.15*MRL). It is important to underline first that the NEO samples were 10 times less concentrated than it was planned. So all the samples were well below the MRL. Instructions to the participants : Each sample had to be analysed in triplicate (that means 3 different extractions for each sample) with the ELISA kits of their choice (commercial or in-house), if possible looking for STR/DHS, GTM and NEO. If not, it was recommended to test only STR/DHS twice, with two different batches of the same kit. It was underlined that each sample may contain or not aminoglycosides in the following list: STR, DHS, GTM, NEO. Homogeneity and stability studies : The 10 spiked samples were tested according to the reference publications [2,4]. It was concluded that all materials were sufficiently homogenous and were stable until the analyses deadline. Qualitative evaluation Two steps should be considered in a screening test : 1.the performance of the kit and its capacity to detect one analyte at concentrations upper than a threshold value 2.the decision of the lab concerning the presence or absence of the analyte (non- compliant or compliant) in the sample. Then depending on the laboratory strategy a non-compliant decision is followed or not by a confirmation by physico-chemical methods. Definitions: A false compliant sample : a sample declared compliant while it contains the aminoglycoside (s) detected by the concerned ELISA kit (whichever was the real concentration). A false non-compliant sample : a sample declared non-compliant whereas it does not contain any aminoglycoside nor the aminoglycoside (s) detected by the concerned ELISA kit (whichever was the real concentration). Then according to the Mac Clure publication [3] for the validation of screening qualitative methods, four parameters were calculated: False non-compliant and false compliant rates, sensitivity and specificity. This inter-laboratory testing study on ELISA kits for the screening of aminoglycosides in milk was globally satisfactory. The global false compliant rates (0.0 to 1.0 %) were lower than 5 % at the screening step whichever was the kit used. The problem is only at the decision step because some laboratories took a wrong decision. The global false non-compliant rates were included between 14 and 45 %. So a confirmation step is needed to take the right decision. Finally these kits could be considered as satisfactory tests for screening purposes (with qualitative results). Acknowledgements Many thanks to all the participants to this inter-laboratory study: G. Suhren, M. Brandtner, I. Shwaiger, Y. Govaert, W. Rybroeck, U. Pertilla, N. Cadieu, A.M. Ferrini, C. Arts, S. Stead, L. Lynas, A. Honzlova and V. Titajevs. Quantitative evaluation A quantitative exploitation was also performed by calculating the assigned value for each material [2]. Then the evaluation of laboratorys performance was carried out by calculating accuracy and repeatability z-scores [4]. Combined accuracy and repeatability z-scores STR 300 g/kg 6.2 4.8 Accuracy z-scores GTM 150 g/kg The impact of batch kits and laboratories The different ELISA kits used by the 14 participants for their analyses: In grey colour the labs which used the same batch of kits. Examples of the graphical representation of the individual lab results: Performing the analyses of STR/DHS with different batches of the same kit in one lab did not produce significantly different quantitative results in term of concentration. Moreover the decision (compliant or non-compliant result) was the same in all labs. Furthermore the decision taken by different labs was the same for all aminoglycosides and all kits suppliers, whichever was the batch (except in lab Z). A statistical evaluation was performed to evaluate the quantitative effect of different parameters: batches, laboratories and various kits. The mean calculated concentrations were compared (t-test (Student test) or one-way analysis of variance). YES: effect of the parameter NO: No effect of the parameter References: 1.Council Regulation n2377/90 of 26 June 1990, L251, 1-8 2.2. ISO /DIS 13528 working document: Statistical methods for proficiency testing by interlaboratory comparisons. 3. McClure, F.D. (1990) Design and analysis of qualitative collaborative studies : minimum collaborative program. JAOAC Int., 73 (6), 953-960 4. Thompson, M. & WOOD, R. (1993) International harmonised protocol for proficiency testing of analytical laboratories. JAOAC Int., 76 (4), 926-940 -Whichever was the manufacturer of the ELISA kit or the Biacore kit, questionable or unsatisfactory z-scores were found. -High variability of the quantitative results (16 labs) for STR/DHS. -The assigned values for the STR/DHS materials were very near to the real concentration of the spiked samples. -Regarding the individual laboratory results, the STR/DHS tests can not be considered as quantitative tests. Some laboratories always give the lowest concentrations (for example F and Z) and other laboratories the highest results (laboratory H). However accuracy z-scores for most of the labs (except labs H and Z mainly) were satisfactory. -The repeatability of the results for STR/DHS was very satisfactory whichever was the kit. -With the GTM kits, concentrations far from the assigned values were only obtained by lab H (Eurodiagnostica kit as other labs). The assigned values were very satisfactory. -Considering NEO kits, the variability was very low. All the 6 laboratories found concentrations lower than the MRL (1500 g/l) Satisfactory CRL-AFSSA Fougres Satisfactory z-scores are included between 2 and +2 ().