C.R .L.CNEVA - Fo u gèr es

Development and validation of Biacore Development and validation of Biacore methods for the detection of methods for the detection of

streptomycin/dihydrostreptomycin, streptomycin/dihydrostreptomycin, gentamicin and neomycin in milkgentamicin and neomycin in milk

VIIth International Conference on Agri-Food Antibodies, Uppsala (Sweden), September 10-13, 2003Valérie GAUDIN, Céline HEDOU, Pascal SANDERSCommunity 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 aftertreatment. 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.

References:Baxter JA et al. Journal of Agriculture and Food Chemistry 2001, 49, 3204-3207Ferguson JP et al. The Analyst 2002, 127, 951-956Gaudin V. et al. JAOAC 1999, 82 (6), 1316-1320Gaudin V. et al. Analytica Chimica Acta 2001, 436, 191-198Gaudin V. et al. Food and Agricultural Immunology 2001, 13 (2), 77-86 Haasnoot W et al. Food and Agricultural Immunology 2002, 14, 15-27

ANTIBODIESANTIBODIESThe 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).

VALIDATIONVALIDATION

PROTOCOLSPROTOCOLS Flow rate = 20 µl/min for the 3 protocols

CONCLUSIONCONCLUSIONFor 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.

CALIBRATION CURVESCALIBRATION CURVESThese 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.

CCCC / 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.

SELECTIVITY / SPECIFICITYSELECTIVITY / SPECIFICITYCross-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.

STABILITYSTABILITYC/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).

MRLs in milk (µg/l):

streptomycin 200

dihydrostreptomycin 200

gentamicin 100

neomycin 1500

Mean calibration curve for neomycin:

y = -0,0148x + 94,632

R2 = 0,9929

0

20

40

60

80

100

0 500 1000 1500 2000 2500Neomycin concentration (µg/l)

Rela

tive r

esp

on

se (

%)

PRECISION: REPEATABILITY/WITHIN-LABORATORY REPRODUCIBILITYPRECISION: REPEATABILITY/WITHIN-LABORATORY REPRODUCIBILITY

CONCLUSION :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 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 samples at the MRL and between samples spiked at 0.5, 1 and 1.5 times the MRL.the MRL.

Mean calibration curves for streptomycin and gentamicin:

y = -0,0947x + 92,48

R2 = 0,9931

y = -0,7548x + 101,52

R2 = 0,9798

0

20

40

60

80

100

0 50 100 150 200 250 300 350 400 450

Antibiotic concentration (µg/l)

Re

lati

ve

re

sp

on

se

(%

)

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.

Streptomycin Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Overall

Other

technician

0.5 LMR Mean 330.5 121.8 96.3 57.2 166.0 109.5 146.9 176.7

SD 15.9 16.8 7.9 8.5 42.4 17.3 91.6 12.7

CV 4.8 13.8 8.2 14.9 25.5 15.8 62.4 7.2

LMR Mean 423.9 261.2 201.6 197.2 293.3 204.4 263.6 327.1

SD 28.8 55.1 11.1 10.0 19.8 57.7 87.7 10.3

CV 6.8 21.1 5.5 5.1 6.8 28.2 33.3 3.2

1.5 LMR Mean 473.3 349.7 313.8 301.9 392.5 330.5 360.3 344.9

SD 17.9 8.1 11.1 12.7 16.7 5.6 60.3 14.6

CV 3.8 2.3 3.5 4.2 4.3 1.7 16.7 4.2

Gentamicin Day 1 Day 2 Day 3 Day 4 Day 5 Overall

0.5 LMR Mean 48.5 43.1 48.5 48.3 53.5 48.4

SD 4.8 2.8 10.6 5.6 5.4 6.8

CV 9.8 6.4 21.8 11.7 10.2 14.0

LMR Mean 62.8 53.1 51.2 64.5 67.0 59.7

SD 4.4 3.8 3.8 11.4 4.5 8.7

CV 7.1 7.2 7.4 17.7 6.7 14.6

1.5 LMR Mean 78.2 69.1 69.8 74.1 83.1 74.9

SD 2.8 2.7 8.3 5.7 4.7 7.2

CV 3.5 4.0 11.9 7.7 5.7 9.7

Neomycin Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Overall

Other

technician

0.5 LMR Mean 801.6 574.8 891.9 269.9 820.0 645.4 667.3 1159.9

SD 65.3 80.9 128.1 168.8 130.1 87.2 236.3 157.0

CV 8.1 14.1 14.4 62.5 15.9 13.5 35.4 13.5

LMR Mean 1608.3 1477.3 1782.7 1010.2 1211.6 1749.8 1473.3 1799.8

SD 44.4 92.5 70.9 39.7 69.8 150.6 295.9 189.2

CV 2.8 6.3 4.0 3.9 5.8 8.6 20.1 10.5

1.5 LMR Mean 2234.9 2131.5 2509.2 2780.0 1811.2 1817.7 2214.1 2273.0

SD 47.4 112.1 57.9 140.2 86.5 114.2 366.3 97.4

CV 2.1 5.3 2.3 5.0 4.8 6.3 16.5 4.3

Stability of neomycin during 2 weeks:

0

20

40

60

80

100

120

140

0 7 14Time (in days)

C/C

0 (%

)

in milk stock solution at +4°Cstock solution at -20°C

Stability of streptomycin during 2 months:

0

20

40

60

80

100

120

1 10 14 29 57Time (in days)

C/C

0 (

%)

in milk stock solution at +4°Cstock solution at -20°C

CC CC LOD LOQ

Streptomycin 347 437 140 590

Neomycin 2070 2580 1230 4600

Gentamicin 55 63 48 150

Finally the tested antibodies were very satisfactory but the immobilisation was the limiting factor and needs to be improved for gentamicin and neomycin.

MOLECULE

(ANTI BODY) I MMOBI LI SATI ON MI LK PREPARATI ON ASSAY CONDI TI ONS REGENERATI ON

External

STRP 20 mg/ ml in

borate buff er 50 mM

pH 7.5

External

NEO 20 mg/ ml in

borate buff er 10 mM

pH 8.5

I nternal

GTM 2 mg/ ml in

borate buff er 10 mM

pH 8.5

10 µl of HCl 100 mM + 20%

acetonitrile + 10 µl NaOH

100 mM

4 µl of NaOH 100 mM

3 µl of NaOH 100 mM

Mix 40 µl Ab 1/ 80 with

40 µl sample

I nject 40 µl of mixing

Mix 30 µl Ab 1/ 75 with

30 µl sample

I nject 30 µl of mixing

Mix 30 µl Ab 1/ 250

with 30 µl sample

I nject 30 µl of mixing

Pasteurisation (5 min at

100°C), 10 min centrifugation,

3000 rpm. Dilute 1/ 100 in HBS

buff er

10 min centrifugation, 2600

rpm. Dilute 1/ 10 in HBS

buff er

Streptomycin/DHS

(504 10.2)

Neomycin

(Biodesign)

Gentamicin

(Eurodiagnostica)

10 min centrifugation, 2600

rpm. Dilute 1/ 100 in HBS

buff er

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).

This table summarises the combination of the experiments to determine performance parameters with minimum workload:

Samples toanalyse

30 blank samplesf rom diff erent

origin

Spiked samples at ½MRL (30), MRL (60),1.5 MRL (30) f romdiff erent origin

20 spikedsamples at CC

Samples spiked withantibiotic diff erent

f rom the AB of interestat 10000 µg/ l

Validationparameters

Selectivity/specificity

Precision, specificity,CC, stability

CC Selectivity / specificity

Biacore X