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 :
[email protected] 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
().