Enzyme immunoassay for the determination of hexestrol in meat

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  • ISSN 00036838, Applied Biochemistry and Microbiology, 2011, Vol. 47, No. 1, pp. 7781. Pleiades Publishing, Inc., 2011.Original Russian Text M.M. Vdovenko, C.F. Peng, C.L. Xu, E.S. Vylegzhanina, A.A. Komarov, I.Yu. Sakharov, 2011, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2011,Vol. 47, No. 1, pp. 8489.

    77

    Illegal use of hormonal growth stimulators sometimes occurs in highly intensive animal breeding,because this technology violation allows for a significant increase of production at minimum cost. Products thus obtained are extremely dangerous for thehealth of humans, since the hormonal growth stimulators are carcinogenic and can disrupt sexual maturation and reproduction in humans [14]. Nowadaysthe use of hormonal growth stimulators in animalbreeding is prohibited in most countries. According tothe EC directive 96/23 meat and animal feed in thecountries of the European Union are monitored fortraces of prohibited anabolic growth stimulators, suchas stilbenes, steroids, lactones of resorcinic acid, andbetaadrenomimetics. These substances belong togroup A of the most dangerous medicines. The mainobjective of the control of the preparations of thisgroup is the detection of any concentrations of the illegally used preparations.

    The presence of hormonal growth stimulators infoods is most often detected by chromatographicmethods [57]. Notwithstanding the high sensitivity,reproducibility, and reliability of these methods, theyhave significant drawbacks, such as the necessity to useexpensive equipment operated by highly qualified specialists; serial chromatographic analysis is laborintensive and take a lot of time. Immunochemicalmethods are devoid of these drawbacks; therefore,much effort is put into the development of immunochemical assays for the detection of hormonalgrowth stimulators [8].

    The aim of the present study was to develop anindirect competitive enzymelinked immunosorbentassay for the analysis of hexestrol (HES), one of theanabolic preparations of the stilbene group, and toapply this method for the determination of hexestrol inthe meat of animals.

    METHODS

    Materials. A horseradish peroxidase preparation(RZ 3.0) from Sigma (United States) was used in thepresent work without further purification.

    Hexestrol, 3,3',5,5'tetramethylbenzidine (TMB)and caseine sodium salt were from Sigma (UnitedStates), 30% 22 was from ChemMed (Russia), andTween 20 was from Ferak (Germany). All the salts usedwere of analytical or chemically pure grade. Theconcentration of hydrogen peroxide was assessed spectrophotometrically by measuring the optical density ofthe solutions at 240 nm (240 = 43.6

    1 cm1) [9].Immunoreagents. A conjugate of hexestrol and

    ovalbumin (HESOVA) was obtained as described previously [10]. Briefly, 5 ml trimethylamine and 20 mlisobutylchloroformiate were added to 10 mg of 3carboxypropylhexestryl ester dissolved in 0.4 ml of 1 : 1(vol : vol) mixture of dioxane and dimethylsulfoxideand incubated for 1 h at 48 (solution A). Simultaneously, 20 mg of ovalbumin were dissolved in 0.8 mldistilled water and the solution was mixed with 0.8 mldimethylsulfoxide. This solution (solution B) was alsoincubated for 1 h at 48. The solution A was slowlydropwise added to the solution B and the mixture was

    Enzyme Immunoassay for the Determination of Hexestrol in MeatM. M. Vdovenkoa, C.F. Pengb, C.L. Xub, E. S. Vylegzhaninac,

    A. A. Komarovc, and I. Yu. Sakharovaa Faculty of Chemistry, Moscow State University, Moscow, 119991 Russia

    b School of Food Chemistry and Technology, South Yangtze University, 214122 Chinac AllRussia State Center for Quality Control and Standardization of Veterinary Therapeutics and Animal Feed,

    Moscow, 123022 Russia email: sakharovivan@gmail.com

    Received December 4, 2009

    AbstractAn indirect competitive enzymelinked immunosorbent assay (ELISA) of hexestrol (HES), ananabolic hormone forbidden for use in livestock farming, has been developed. Conditions of ELISA havebeen optimized by varying the concentrations of the coating conjugate (HESovalbumin), antiHES antiserum, casein, and Tween 20. In the absence of Tween 20 in the reaction mixture, the detection limit (IC10)equaled 0.01 ng/ml, IC50 equaled 0.17 ng/ml, and the working range (IC20IC80) equaled 0.030.86 ng/ml,while, in the presence of 0.05% Tween 20, these values equaled 0.05, 2.9, and 0.2632.0 ng/ml, respectively.Standard deviation of the analysis results did not exceed 5.4%. If ELISA was performed in the absence ofdetergents, the recovery value upon HES determination in spiked beef samples ranged from 74 to 147%.

    DOI: 10.1134/S0003683811010182

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    APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 47 No. 1 2011

    VDOVENKO et al.

    incubated for 4 h at 48. The HESOVA conjugateobtained was dialyzed against 10 mM Kphosphatebuffer, pH 7.4, containing 0.15 M NaCl (PBS) andstored at 20. The conjugate of HES and bovineserum albumin (BSA) was synthesized according to asimilar procedure.

    Polyclonal antiserum to HES was obtained by subcutaneous immunization of rabbits (body mass 1.52.5 kg) with the HESBSA conjugate during 6 months,as described previously [11]. The antibody titers weredetermined by ELISA. The specific antisera werestored in 50% (vol : vol) glycerol solutions at 20.

    The conjugate of polyclonal sheep antirabbit IgGantibodies and horseradish peroxidase was synthesizedas described [12]: briefly, 5 mg of horseradish peroxidase (HRP) were dissolved in 0.5 ml distilled water andsodium periodate was added to the solution to the finalconcentration of 17 mM. HRP oxidation was performed for 20 min at room temperature in the dark,and after this, the reaction mixture was dialyzedagainst 1 mM sodium acetate buffer, pH 4.9, overnightat 4 for the elimination of excess oxidizer and theproducts of its decomposition. The peroxidase solution obtained was mixed with a solution of 0.5 mg ofpurified polyclonal sheepantirabbit antibodies in75 l of 50 mM sodium carbonate buffer, pH 9.6, andincubated at room temperature for 2 h with constantstirring. The Schiff bases formed were reduced by adding 10 l of a freshly prepared 10% solution of NaBH4.The reaction was performed for 30 min at room temperature. The conjugate was dialyzed against PBS andstored in 50% (vol : vol) glycerol solution at 20.

    Immunoenzymatic determination of hexestrol. TheHESOVA conjugate was preadsorbed in the wells of96well microplates with a high absorbing capacity(Costar, United States) by incubating 100 l of theconjugate solution (0.252.00 mg/ml) in 50 mMbicarbonate buffer, pH 9.6, in the wells at 4 overnight. After the removal of the unadsorbed conjugate100 l of 0.1% casein solution in 50 mM phosphatebuffer, pH 7.4, containing 0.15 M NaCl (PBS) wereadded to each well and incubated for 1 h at 37.Excess casein was removed by four washes with PBS

    containing 0.05% Tween 20 (PBST). After this, 50 lof HES solution (0.003600 ng/ml) and antiHESserum were added to the wells. The antiserum wasdiluted with PBS or PBST, and HES solutions werediluted with a 0.2% casein solution in 20% methanolin PBS or PBST. The competitive immunologicalreaction was performed for 1 h at 37. The unboundantibodies were washed away and 100 l of the solutionof the HRPsecondary antibody conjugate (diluted 1 : 6000)in PBST were added to the wells and incubated for 1 hat 37. The working dilution of the conjugate usedwas determined in a separate experiment. After washingthe microplate, 100 l of a freshly prepared substratesolution containing 0.42 mM TMB and 1.2 mM H2O2 in100 mM acetate buffer, pH 4.3, were added to eachwell. The enzymatic reaction was stopped with 50 l of2 M H2SO4, and the optical density of the solutions inthe wells was measured at 450 nm.

    Beef sample preparation. Five grams of beef notcontaining fat or connective tissue were ground, mixedwith 5 ml of 0.067 M phosphate buffer, pH 7.2, andhomogenized for 2 min in a knife homogenizer. Onegram of the homogenate obtained was mixed with 3 mlof methanol and sonicated (Elmasonic S, Germany)at the power of 80 W for 20 min, and then centrifugedfor 10 min at 4000 g. The pellet was extracted withmethanol again. The pellet obtained after the secondextraction was mixed with 3 ml of 1 : 3 watermethanol mixture and sonicated for 20 min. The homogenate was centrifuged for 10 min at 4000 g. The extraction with the watermethanol mixture was performedtwice. After four rounds of extraction, the supernatants from one meat sample were combined, mixedwith 2 ml hexane, and shaken intensively. The hexanelayer was removed and the hexane extraction wasrepeated. The methanol from the bottom (watermethanol) layer was removed by evaporation at 50C.The aqueous solution was mixed with 5 ml of tertbutyl methyl ether and the extraction was performedfor 20 min with manual shaking once in 5 min. Thebottom layer was extracted with tertbutyl methylether again. The organic fractions were pooled andevaporated in a rotary evaporator (45C). Before theanalysis, the dried sample was dissolved in 0.5 ml ofthe working buffer (PBS or PBST) and centrifuged for10 min at 7000 g. At the last stage of sample preparation, the supernatant was diluted tenfold with theworking buffer. HES solutions with the concentrationscorresponding to IC25, IC50, and IC75 in the meatextract sample were prepared by mixing an ethanolsolution of HES (1 mg/ml) and beef extract.

    HO OH

    H5C2 H

    H C2H5

    +

    ovalbumin

    antigen

    antibody

    secondaryantibody

    peroxidase

    +

    Fig. 1. The scheme of the indirect competitive enzymelinked immunosorbent assay for hexestrol.

    Fig. 2. The chemical structure of hexestrol.

  • APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 47 No. 1 2011

    ENZYME IMMUNOASSAY FOR THE DETERMINATION OF HEXESTROL IN MEAT 79

    RESULTS AND DISCUSSION

    Although the major factors determining the qualityof immunoenzymatic assay are the characteristics ofthe immunoreagents (antibodies, coating conjugates,and enzyme conjugates) used, the conditions of theanalysis also have a significant influence on theparameters of this analytical method. Taking this intoaccount, we optimized the concentration of antiHESantibodies in the reaction medium on the first stage ofthe development of the indirect competitive ELISA(Fig. 1) for the detection of HES (the structural formula is given in Fig. 2) in the meat of animals.

    As shown in Fig. 3, the use of a low dilution of thespecific antiserum (dilution 1 : 800) resulted in a calibration curve with a high background value of theoptic density (Fig. 3, curve 1). On the other hand, thevalue of the optic density did not depend on the concentration of HES added to the reaction mediumwhen a highly diluted antiserum (dilution 1 : 6400) wasused, and, therefore, the concentration of HES couldnot be measured (Fig. 3, curve 4). Calibration curvesof high quality were obtained when the HES antiserumwas diluted 1 : 1600 or 1 : 3200 (Fig. 3, curves 2 and 3).

    Detergents are used in conventional ELISA to prevent unspecific sorption on polystyrene plates. Inaddition, the sorption of a blocking protein is often

    A450

    3.0

    2.5

    2.0

    1.5

    1.0

    0.5

    0.01 0.1 1 10 100

    1

    2

    3

    [HES], ng/ml

    A450

    1.2

    0.9

    00.01 0.1 1 10 100

    1

    2

    [HES], ng/ml

    0.6

    0.3

    A450

    2.5

    2.0

    1.5

    1.0

    0.5

    0.01 0.1 1 10 100

    1

    2

    3

    [HES], ng/ml

    4

    A450

    1.0

    0.8

    0.6

    0.4

    0.20.01 0.1 1 10 100

    1

    2

    [HES], ng/ml

    4

    Fig. 3. Selection of the optimal dilution of the antiHESantiserum in the ELISA of hexestrol. The following dilutions were tested for the optimization: 11 : 800, 21 :1600, 31 : 3200, and 41 : 6400.

    Fig. 4. Calibration curves for the immunoenzymatic assayfor hexestrol with 10% methanol in PBST (2) with or (1)without 0.1% casein as the working buffer.

    Fig. 5. Selection of the optimal concentration of HESOVA used as the coating conjugate in the ELISA of hexestrol. The following concentrations (g/ml) were used forthe optimization: 10.25, 20.5, 31.0, and 42.0.

    Fig. 6. Calibration curves for the immunoenzymatic assayfor hexestrol with 10% methanol and 0.1% casein in PBST(2) with or (1) without 0.05% Tween 20 as the workingbuffer.

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    VDOVENKO et al.

    performed at a certain stage of the assay. On the firststage of our investigation, we used both of these procedures in the detection of HES, with Tween 20 as thedetergent and casein as the blocking protein. However,the curve 1 in Fig. 4 shows that the optical density corresponding to the nonspecific reaction was relativelyhigh in this version of the analysis. Therefore, caseinwas used not only as blocking protein but also as one ofthe components of the reaction mixture on the stage ofcompetition. The addition of 0.1% casein into the

    reaction mixture allowed for the successful suppression of the nonspecific adsorption of the antibodies inthe microplate wells (Fig. 4, curve 2). It is also necessary to note that the introduction of casein into thereaction mixture caused a decrease of the efficiency ofthe specific immunological reaction.

    In further experiments, the influence of the concentration of the HESOVA conjugate used as thecoating antigen on the analytical characteristics of theELISA was studied. As shown in Fig. 5, curve 1, thecalibration curve can not be constructed if the concentration of the coating antigen is low. Increasing theHESOVA concentration allowed for the constructionof highquality calibration curves for the determination of HES (Fig. 5, curves 2 and 3). However, a further increase of HESOVA concentration (to 2 g/ml)had negative consequences, namely, a dramatic deterioration of the analytical characteristics of ELISA,such as an increase of the detection limit value and adramatic narrowing of the working range of HES concentrations (Fig. 5, curve 4). The effect detected isobviously due to the formation of inactive HESOVAaggregates due to the hydrophobic interaction of HESfragments of different molecules of the coating conjugate. Comparison of such analytical characteristics asIC10, IC50 and the working range (IC20IC80) for fourcalibration curves constructed using different HESOVA concentrations showed that the best results wereobtained at a HESOVA concentration of 0.5 g/ml(Table 1).

    It was found during the development of ELISA forthe detection of azinphosphomethyl in water samples[13] that the omission of Tween 20 from the reactionmixture at the competition stage caused an increase ofthe assay sensitivity. We have tried an analogousapproach in HES determination. As shown in Fig. 6,the omission of the detergent from the reaction mixture caused a shift of the calibration curve to the lower

    Table 1. The influence of the coating conjugate (HESOVA)concentration on the analytic parameters of the ELISA forhexestrol

    HESOVA, g/ml IC10, ng/ml IC50, ng/ml

    Working range, ng/ml

    0.25 0.4 3.6 0.716

    0.5 0.1 3.4 0.430

    1.0 0.7 11.5 1.598

    2.0 8.0 34.6 1692

    Table 2. The effect of Tween 20 in the reaction medium at thecompetition stage on the analytic parameters of the ELISA forhexestrol

    Reaction medium composition

    IC10, ng/ml

    IC50, ng/ml

    Working range, ng/ml

    PBS with 0.1% casein, 10% methanol and 0.05...

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