Determination of clenbuterol in bovine liver by enzyme immunoassay

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  • Analy8ca Chumca Acta, 275 (1593) 227-230 Elsewer Science Pubhshers B V , Amsterdam

    227

    Determination of clenbuterol in bovine liver by enzyme immunoassay

    S D Bucknall, AL MacKenzie, M J Sauer, D J Everest, R Newman and R Jackman

    Bwchemutry Dzscq~he, Central Vetennary Laboratory, New Haw, Weybrtdge, Surrey KT15 3NB (UK)

    (Received 20th May 1992)

    Abstract

    Procedures are described for optmusmg and vabdatmg an ELISA method for measurmg clenbuterol residues m bovme bver wlthout pnor sample ennchment Optunal assay conddlons are obtamed by premcubatmg bver supematant wllth an unmoblhsed antibody rmed to clenbuterol-ovalbumen After further mcubatlon wrth a salbutamol-peroxldase coqlugate, &our IS developed usmg a tetramethylbenzldene substrate The assay wll pemut measurement of clenbuterol residues m hver at the maxmmm residue level of 05 ng g- Hnth a confidence of > 99%

    I(eywords Enzymatx methods, Immunoassay, &Agomsts, Bovme bver, Clenbuterol, ELISA method

    Clenbuterol 1s a @agonist which 1s licensed only for the treatment of respiratory disorders m cattle and horses and as a tocolytlc At high dose It can be used as a repartltlonmg agent promotmg the conversion of fat to muscle and therefore mcreasmg carcase value and m this respect its use has been banned As clenbuterol is toxic it is necessary to monitor levels of the residue m ammal tissue destmed for human consumption to ensure the maxnnum residue lumt (MRL) for this compound 1s not exceeded To support this labo- ratorys vetermaly drug residue momtormg pro- gramme an ELISA method was developed for clenbuterol m urme [l] It was necessary to optl- muse and validate this assay for rapidly and reh- ably measuring residues of clenbuterol m hver

    Correspondence to SD Buck&l, Bmchermstry Dwlplme, Central Vetermary Laboratory, New Haw, Weybndge, Surrey KT15 3NB (UK)

    EXPERIMENTAL

    An ELISA method for clenbuterol m urme usmg an alkalme phosphatase enzyme system was performed m the presence of either urme or 10% bovme liver homogenate to assess the matrvr ef- fect of the hver (Fig 1) In an attempt to de- crease assay tune a horseradish peroxldase (HRP) enzyme label was substituted for the alkalme phosphatase label (Fig 2) The antisera obtamed from an ammal before (248/5) and after (248/6) rechallengmg mth clenbuterol-ovalbumen un- munogen were compared (Fig 3) Utihsmg the 4% cross-reactlvrty of the clenbuterol antibody wth salbutamol, salbutamol-HRP was prepared (by the conjugation of salbutamol henuscuccmate 121 to ammated HRF usmg the rmxed anhydnde method based on [3]) and substituted for the clenbuterol-HRP conjugate (Fig 4) Sample vol- ume was then mcreased from 10 to 200 ~1 and sample concentration doubled from 10 to 20%

    Elsevler Science Publishers B V

  • 228 SD Bucknall et aL /Anal Chun Acta 275 (1993) 227-230

    7

    Fig 1 Alkaline phosphatase clenbuterol ELBA m urme ( -_) vs 10% hver (- - - - - -1 homogenate

    (w/v) (Fig 5) Figure 6 shows the effect of prem- cubatlon of the test samples and standards m the wells at approxunately lo-mm mtervals up to 1 h, pnor to addition of salbutamol-HRP

    loo,

    90

    60 F t

    t 20

    10 1 Fig 2 Clenbuterol peroxldase ( -_)/alkahne phosphatase (- - - - - -1 conlugates Assay tmes are 0 5 and 4 5 h, respec- tlve1y

    100

    60

    Be0 aR

    40

    20

    0

    Fig 3 Anttsera bleed 24S/5 (- - - - - -1 vs 248/6 (-_)

    The followmg ELISA procedure evolved from these mvestlgatlons Mlcrotltre plates were coated vvlth clenbuterol-spectilc antisera usmg partial denaturatlon of the antibody with glycme to m-

    loo,

    Ftg 4 Salbutamol peroxtdase (- - - - - -1 vs clenbuterol peroxl- dase ( -_) coqugate

  • SD B&t&l et aL /AnaL Ch Acta 275 (1993) 227-230 229

    (5%), bovme serum albumm (BSA, l%), and dned pnor to storage at 4C Bovme hver test samples and negative control hver were prepared by ho- mogenlzatlon 111 PBS (005 M, pH 7 0) to @ve 20% (w/v> suspensions which were centrfiged (2500 g, 4C, 15 mm) The supematant was re- tamed for assay Standards were prepared as above usmg control negative bovme liver fortlfied with clenbuterol HCl to provide a range between 0 25-8 ng g-l

    c

    m-

    !:: 40-

    30-

    20-

    Ftg 5 Clenbuterol standard curves III 10% (- - - - - -1 and 20% ( -_) her homogenate Sample volume, 200 pl

    crease bmdmg [41 The plates were then washed, 6 x with phosphate buffered salme (PBS, 0 1 M, pH 7 0) contammg Tween 20 (0 05%), sucrose

    loo,

    90

    80 I

    so

    20

    10

    0 05 1 ~'twa 8 16

    Fig 6 SIX clenbuterol standard curves w~tb premcubatlon tlmcs -, 60, ------, 45, , 30; ---1 20, - -,lO, - - ,Omm

    TABLE 1

    Assays of control her forttied wth clenbuterol HCI

    Standard concentration (ng g-1

    0 05 10 20

    Inter Mean (%I?/&) 96 65 47 34 SD 125 18 19 16 cv 13 21 42 46 n 87 32 32 32

    Intra SD 37 21 24 15 CV 38 33 51 44 n 8 8 8 8

    120

    100

    40

    OLD I I I I 001 01 10 100 500 900 995 999

    PROBABImn

    F@ 7 Clenbuterol probabhty curves 0, Negative samples, q ,05 ngg- standard, A, 10 ng g- standard

  • 230

    100

    90

    30

    70

    60

    30

    2oc

    10 -

    0 I I I I I 0.3 05 1 2 3 5 10

    Fug 8 Composite clenbuterol standard curve f 2s D -, Intra-, - - - - - -, Inter-assay rz = 0 98

    200 ~1 ahquots of samples, controls and stan- dards were added to each well and the plate premcubated (40 mm, room temperature), 50 ~1 of salbutamol-HRP was added and the plate was shaken for 15 mm

    After mcubatlon the plate was washed with Tween 20 (0 05%) and 200 ~1 of TMB-per- oxldase substrate added to each well After shak- mg the plate for 15 mm, colour development was stopped by the addition of 50 ~1 of 10% H,SO, Absorbance was read at 450 nm on a Dynatech MR700 ELISA plate reader

    Usmg this procedure a number of assays were performed using negative control hver fortified with clenbuterol HCl at 0,O 5, 10 and 2 0 ng g-l, to assess both inter- and mtra-assay variation (Table 1) A probability curve was constructed by plottmg percentage cumulative frequency of B/B, (probablhty) agamst B/B, for 176 negative tissue samples and homogenates sp&ed at 0 5 and 10 ng g- (Fig 7)

    S D 3ucknall et aL /Am! Chm Acta 275 (1993) 227-230

    RESULTS AND DISCUSSION

    The followmg conclusions were drawn from the optumsatlon procedures described Although liver matnx had a negligible effect on linear re- sponse the assay was not sensitive enough to detect the MRL of 0 5 ng g- liver The mtroduc- tlon of clenbuterol-HRP conjugate gave a marginal increase m sensltlvlty and reduced assay time from 4 5 to 0 5 h Rechallengmg the nnmu- msed sheep with clenbuterol-ovalbumen resulted m a doubled sensltlvlty Assay sensltnrlty was fur- ther improved by the substltutlon of a salbuta- mol-HRP conjugate owing to the antibodies lower affinity for salbutamol than for the clen- buterol m the test sample By mcreasmg sample volume and sample concentration, sensltlvlty was doubled without srgnlficantly increasing back- ground colour After premcubatlon of the sample with unmobllrsed antibody the B/B, at 0 5 ng g -I was lowered from 62 to 44% At the MRL of 0 5 ng g- the mter-assay coefficient of variation (CV > was 2 7% when the number of samples tested was 32 and mtra-assay C V was 3 3% when the number of batches tested was 8 At a B/B, value of approxunately 65% there 1s a 1% probablhty of producmg a false positive result and less than 0 5% probablhty of producing of false negative result at the 0 5 ng g- detection level Cross-reactlvlty with other @agonists was found to be salbutamol 4%, terbutahne 2%, lso- proterenol, ractopamme, DL-4-hydroxy-3-methoxy mandehc acid, dlhydroxymadalelc acid and nor- adrenaline less than 1%

    The optmused assay provides a robust, sensl- tlve and specific analytical procedure wrth a lm- ear range between 0 25 and 8 ng g- (Fig 8) suitable for the screenmg of liver samples

    REFERENCES

    1 R J H Plckett and M J Sauer, Anal Glum Acta, 275 (1993) 269

    2 N Beauheu, C Charette, J C K Loo, N Jordan and I J McGdveray, J Pharm Blamed Anal, 3 (1985) 575

    3 B F Erlanger, Methods Enzymol , 20 (1980) 85 4 J F Conradle, M Govender and L VLsser J Immunol

    Methods, 59 (1983) 285

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