Anaiytza Chumca Acta, 275 (1993) 313-316 Elsewer Science Pubbshers B V , Amsterdam

313

Detection of streptomycins in raw milk by an antibody-capture immunoassay

P Hammer, H Knchhoff and G. Hahn Federal Dauy Research Centre, Instuute for Hywne, Hermann Wegmann-Stmsse 1, W-2300 fiel (Germany)

(Recewed 21st September 1992, revised manuscnpt recensed 2nd November 1992)

Abstract

An mdlrect competltlve enzyme-hnked nnmunosorbent assay camed out as an antdnxly-capture test was developed for the determmation of streptomycms III mdk The antIserum was raised m rabbits usmg streptomycm hnked to a bactenal protem as lmmunogen The antkxxlres were concentrated by threefold ammomum sulphate preclpltation and reacted equally Hnth streptomycm and dlhydrostreptomycm No other cross-reactlons were ob- served To perform the test muxotltre plates were coated overmght with streptomycm (1 mg ml-‘) The mdk to be exammed was slummed and treated mth oxabc acid Antierum and mdk samples were nuxed and mcubated for 1 h Dependmg on the amount of strept&ycm or dlhydrostreptomycm m the sample, more orjess ant&x$ molecules remam free to be bound to the streptomycm coat The sensltmty of the test IS better than 16 Kg kg-’ Quant&ication IS possible up to 100 bg kg-’ wthout sample ddutlon

Keynxx& Enzymatic methods, Immunoassay, Antlblotax, An&x&capture test, Enzyme-lmked mmmnosorbent assay, MI& Streptomycms

Streptomycms are routmely apphed m the m- tramammary treatment of bovme mastrtrs The wrthholdmg perrod after treatment IS based on a concentratron range of resrdues m mrlk between 100 and 200 pg kg- ’ (AD1 concept) Wrdely used mrcrobrologrcal mhrbrtor tests with BuczZlus stearothemophdus var caluzkkzctrs or Streptococ- cus thermophdus have detection hmrts between 500 and 10000 pg kg-’ and can only meet the reqwrements for the technologrcal safety of mrlk [ll

The Codex Cornrmttee on Residues of Veterr- nary Drugs m Food (CCRVDF) has suggested maxrmum residue lnnrts (MRLs) for a number of antrbrotrcs, whrch are based on toxrcologuxl eval- uations Marls for streptomycm and drhydro-

Correspondence to P Hammer, Federal Dauy Research Cen- tre, Institute for Hymene, Hermann Wemann Strasse 1, W-2300 fiel (Germany)

streptomycm wdl be drscussed m the future [Jomt FAO/WHO Expert Commrttee on Food Addr- trves and Contammants (JECFAC)] 121 Results are expected in 1993 at the level of 100 pg kg-’ (FDA safe level = 125 pg kg-‘) [3]

The am of this study was to develop a rapid and sensrtrve screenmg method for the detection of streptomycms m nnlk to be used m routine laboratones for the exammatron of large numbers of samples

EXPERIMENTAL

Antzbodzes Antisera were raised m Zrka-hybrid rabbits

(3-month-old females) usmg a streptomycm- bactenal protem comugate (undrsclosed composr- non, Charm Scrences, Malden, MA) as vaccme, apphcated m ascendmg concentrattons from 100

0003~2670/93/$06 00 Q 1993 - Elsevler Science Publishers B V All nghts reserved

314 P Hammer et ai /Anal. Chtm Acta 275 &J93) 313-316

to 400 pg per anunal AdditIonally the vaccme contained Alu Gel S (Serva, Heidelberg, Ger- many), complete Freund’s adluvant (Dlfco, De- troit, MI) for the first inJectloon and mcomplete Freund’s aauvant (Dfico) for further mnjetions Intracutaneous mJectlons (multiple pomt at the back) were made at weekly mtervals for 4 weeks Booster mJectlons were performed monthly wtth a vacxme contammg 300 pg of nnmunogen Sera were collected 10 days after the last nnmumza- tron AntIbodies were concentrated by threefold ammomum sulphate preclpltatlon [4]

Sample preparation Raw milk samples of 10 ml each were skunmed

by centrrfugatlon 00 mm at 2200 g) To 5 ml of skunmed rrulk 556 ~1 of 4% (w/v) oxahc acid (Merck) were added with subsequent gentle shak- mg at room temperature for 10 mm After cen- Wugatron (20 mm at 2200 g), to 2 ml of the clear supernatant 60 ~1 of 1 mol 1-l NaOH (Merck) and 50 ~1 of Tween 20 (2 1 mm01 1-l) m dlstllled water (Merck-Schuchardt, Hohenbrunn, Ger- many) were added Standards were prepared by addmg dlhydrostreptomycm sesqulsulphate 3/2 H,SO, (Sigma, St Louis, MO) to antlblotlc-free bulk milk (research farm) at concentrations of 16, 3 1, 6 3, 12 5, 25, 50 and 100 pg kg-’ Stan- dards were prepared as mdlcated above The prepared samples could be stored at 4°C for several (4-5) days and at - 18°C for up to 6 months

Enzyme-lmked unmunosorbent assay Mlcrotltre plates (Nunc Maxi Sorp) (Nunc,

Roskdde, Denmark) were coated overnight at 37°C vvlth 100 ~1 per well of dlhydrostreptomycm (1 mg ml-’ m 0 1 M carbonate buffer, pH 9.6) Blockmg was performed with 0 5% (w/v> chicken albumin (Sigma, grade III) m 0 01 M phosphate- buffered salme (PBS)-0 5 M NaCl-0 05 mM Tween 20 (pH 7 4) (300 ~1 per well) at 37°C for 45 mm Prepared samples (300 ~1) and antlbodles diluted m PBS (as above, but Hrlthout chicken albummj (50 ~1) were mured to give a final con- centration of antibodies of 1 3000 and were mcu- bated at room temperature for 1 h on a rotary shaker (250 rpm) Neutrahzed samples (100 ~1

per well) were transferred to the mlcrotltre wells and incubated at 37°C for 90 mm to allow free antibodies to be nnmoblllzed on the dlhydro- streptomycin coat A pig antl-rabbit-antibody en- zyme cowugate (alkalme phosphatase, Dakopatts, Glostrup, Denmark) diluted 1 1000 (v/v) m PBS (100 ~1 per well) was added and mcubated at 37°C for 90 mm As a chromogenic substrate, 150 ~1 per well of 1 mg ml-’ mtrophenyl phosphate (Merck) m 0 1 M hydrogencarbonate buffer-0 001 M MgCl, (pH 9 8) were added and mcubated for 1 h at 37°C The absorbance was measured at 405 run (SLT 400 AT ELISA reader)

Four thorough washmgs of the rmcrotltre plate were performed between each step of the proce- dure usmg 0 5 M NaCl and 0 05 mM Tween 20 m Qstllled water

The tltres of the antisera were determmed usmg a direct ELISA with tiydrostreptomycm as coatmg and the anti-rabbit-antlbody enzyme coqugate for antibody detection as mdlcated above Speclficlty was tested wth the same direct ELISA usmg different mhibltors as coatmg sub- stance All serum fractions durmg the rmmumza- tlon schedule were tested m the same way

Evaluatwn To compensate for fluctuations in dtierent

tests, the relative absorbance (% absorbance) was calculated (absorbance of zero sample = 100%) Posltlve and negative samples were dlscnmmated by usmg three tnnes the standard deviation (3s) of analflical results referrmg to zero samples

RESULTS

Ant&&es Antibodies used m the test reached tltres up to

1 64000 after seven booster mjectlons The specticlty was enhanced by boostmg Inhlbltors tested for cross-reactlvlty were as follows (cross- reactlvlty m %) dlhydrostreptomycm 1000, streptomycin 90 0, tobramycm 17, paromamycm 18, oxytetracyclme 17, polymyxm B 19, genta- mlcin 18, neomycm 17, kanamycm 18, pen&in G 17, amplcti 18, tetracyclme 19, ery- thromycm 18, chloramphemcol 2 0 and sul-

P Hammer et al /And Chm Acta 275 (1993) 313-316 315

TABLE 1 TABLE 2

Repeatabdlty determmed usmg 20 mdependentiy prepared nulk samples contammg dtfferent concentrations of dlhydro- streptomycm (pg kg-‘) (all results % absorbance)

Parameter ’ Concentration (pg kg-l)

0 16 31 63 125 25 50 100

PA 989 506 295 205 155 117 86 62 SA 57 31 22 16 15 11 10 08 cv (%I 58 61 75 78 97 94 116 129

a ZA = anthmetlc mean, sA = standard dewatlon, C V = coefficient of vanation

Dwnmmatlon ranges for posItwe and negatwe results appiy- mg three tunes the standard devlatlon (all results 56 ab- sOrbaI&

Parameter * Dlhydrostreptomycm Streptomycm (n-20) (?I = 12)

0 16 0 16 tigk8-1 FBW’ pgkB-l c1gkB-’

2, 989 506 1000 663 I,-3s 818 413 829 582 x,+3s 116 0 599 117 1 744

B fA = anthmefic mean, s = standard devlatlon

phaduIlldme 18 The cross-reactrvrty of drhydro- streptomycm and streptomycm was 90% The other substances did not react at a stgmficant level

ELBA The repeatabmty of the ELBA was tested

with rdenttcaily prepared and repeatedly exam- med raw rmlk samples contammg different con- centrations of drhydrostreptomycm (n = 20 of each) (Table 1)

The dlscrlmmatlon range for posmve and neg- ative results was defined usmg samples contam-

mg 16 pg kg-’ of dmydrostreptomycm (n = 20) or streptomycm (n = 121, compared wrth corre- spondmg numbers of zero samples (Table 2) by applying the 3s crttenon. It was found that there 1s a range of relatrve absorbance values between posrtrve and negative results of 818-59 9% ab- sorbance for dthydrostreptomycm and 82 9- 74 4% absorbance for streptomycm Accordmg to the low concentratrons detectable and the ex- pected MRL of 100 pg kg-‘, rt was decided to

TABLE 3

Quantitative evaluation of 120 blmd samples spiked anth different concentrations of dlhydrostreptomycm

Spdced concentration (pg kg- ‘1

n Classdication ranges (pg kg-‘)

0 16 31 63 125 25 50 100

0 30 30 - 16 21 - 21 - 31 15 14 1 - 63 12 - - 2 9 1

125 12 - - - 9 3 250 9 6 3 - 500 12 - - - 11 1

1000 9 - - - 9

TABLE 4

Evaluation of field samples, performed either for streptomycm or for dlhydrostreptomycm as analyte

Proposed Negattves analyte

Positwes (rg kg-‘)

<16 -16 -31 -63 -125 -25 _ 50

Streptomycm 135 14 18 12 2 7 8 1 Dlhydrostreptomycm 135 31 13 3 11 4 -

316 P Hammer et aL /Ad Chm Acta 275 (1993) 313-316

assign samples fittmg these ranges as probably being negatwe

To confirm the accuracy of the test, spiked samples were used because of the lack of a physlco-chemical method of the required sensltlv- sty One milk sample was dlwded mto 120 ahquots and spiked with Merent concentrations of dlhy- drostreptomycm (30 negatnres and 90 positIves) All ahquots were prepared as bhnd samples Quahtatrve evaluation showed no false-posltlve or false-negative results Quantltatlve evaluation of the samples 1s given m Table 3

Fwt2.i samples In October 1991, 197 bulk rmlk samples were

taken and exammed by the described ELISA Evaluation was performed either for strepto- mycm or for dlhydrostreptomycm as analyte (Ta- ble 4) Because of the cross-reactlvlty of these two substances, the content of one or the other can only be proposed by comparison with a homolo- gous standard dllutron

DISCUSSION

The method described allows a clear posltlve- negative classlficatlon of raw nulk samples con- taming streptomycms at the level of 16 pg kg-l

Quantlficatlon IS possible up to 100 pg kg-’ without sample dllutlon

Because of the cross-reactlvrty of streptomycm and dlhydrostreptomycm it 1s nnposslble to de- cide whether the component m a sample 1s one or the other The positwe-negatrve classtication 1s not mfluenced, however, but for quantification the result for a sample has to be compared with a homologous standard dllutlon Hence the quan- tlflcatlon results wfi &ffer d either streptomycm or dlhydrostreptomycm 1s the proposed analyte Concerning the field samples, the results for dl- hydrostreptomycm are more probable consldermg the application of the two antiblotlcs m vetermary medlcme m Germany

This work was supported by an EEC research grant (Contract No 1001/90-111)

REFERENCES

Int Dauy Fed Bull, No 258/91, 1991 38th Report of the Jomt FAO/WHO Expert Comnuttee on Food Addltnres, World Health Orgamzatlon, Geneva, 1991 I R Bishop, S E Duncan, GM Jones and W D WhIttier, Vlrgmla Polytechnic Institute and State Umverstty, Blacks- burg, VA, unpubhshed work, 1991 G A Hebert, P L Pelham and B Plttman, Appl Mlcroblol , 25 (1973) 26