Chemiluminescence enzyme immunoassay for the determination of sulfamethoxydiazine

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Spectrochimica Acta Part A 81 (2011) 544 547Contents lists available at ScienceDirectSpectrochimica Acta Part A: Molecular andBiomolecular Spectroscopyjou rn al hom epa ge: www.elsev ier .coChemil r thsulfamYongjun onCollege of Publa r t i c lArticle history:Received 2 MaAccepted 18 JuKeywords:SulfamethoxyChemiluminesQuantitations oftectedhputis pa. For e witas froresult1. IntroductionSulfamethoxydiazine (SMD) has effectiveness on urinary andrespiratory tract infections and is often used for animal diseasetreatment and prevention [1]. It takes a long time to metabolizeSMD completely. Therefore, there is SMD residue in meat, dairy andeggs if the ahealth. Codmum residu[2]. In USA exit in foodSMD residuIt has bfor detectiomatography[4], gas chspectrometmass spectetc. These consuming,of samples.tages of hipollution tofore, it is sig CorresponE-mail addenvironment-protection and accurate method for the determina-tion of the content of SMD in food.The goal of this paper is to establish a method, chemilumines-cence enzyme immunoassay, which is suitable for high-throughputdetermination of the content of SMD in food.1386-1425/$ doi:10.1016/j.nimal has taken in SMD. Such food is harmful to humanex Alimentarius Commission provides that the maxi-e limit for sulfonamides in animal tissues is 100 g/kgand Japan, there is policy that no SMD residue should. Thus, it is signicant to carry out the determination ofal in the meat, dairy, eggs and other kinds of food.een reported that various methods had been appliedn of sulfonamides chemical, including thin layer chro- [3], high performance liquid chromatography (HPLC)romatography (GC) [5], gas chromatography masser (GC/MS), high performance liquid chromatographyrometer (HPLC/MS) [6], capillary electrophoresis [7],methods had disadvantages, e.g. high-cost and time- and were not suitable for fast screening large number Another method, radioimmunoassay [8], had advan-gh sensitivity, specicity and good accuracy, but its the environment hindered its wide application. There-nicant to develop a high-throughput, high-sensitivity,ding author.ress: (Y. Wu).2. Experimental2.1. ChemicalsAll the reagents were of analytical grade. SMD monoclonalantibody was purchased from Guangzhou MeiJin BiotechnologyCo., Ltd. SMD was purchased from SIGMA with optical purities of99.5%. Horseradish peroxidase (HRP) was purchased from ROCHEInc. (Specic Activity > 250 u/mg). Deionized water was puried byMilli Q system (Waters, Milford, MA). In this method, the follow-ing buffers were used. (A) Coating buffer, 0.05 mol/L carbonate:bicarbonate buffer solution, pH 9.6, (B)incubation buffer, 0.01 mol/Lsodium phosphate buffered saline (PBS), pH 7.27.4, (C) washingbuffer, buffer B with 0.05% Tween 20, (D) dilution buffer, buffer Bwith 1% BSA.2.2. InstrumentsMP280-based chemiluminescence immunoassay analyzer wasfrom Beijing Tai Geke letter Biological Technology Co., Ltd.Microplate Reader (Tecan Sunrise). HPLC was purchased from the see front matter 2011 Elsevier B.V. All rights reserved.saa.2011.06.047uminescence enzyme immunoassay foethoxydiazineWu , Songcheng Yu, Fei Yu, Nali Yan, Lingbo Qu, Hic Health, Zhengzhou University, No. 100 of Science Avenue, Zhengzhou 450001, China e i n f oy 2011ne 2011diazinecence enzyme immunoassaya b s t r a c tSulfamethoxydiazine (SMD), which ihealth. No SMD residue should be deteis signicant to develop a high-througof the content of SMD in food. In thdeveloped for quantication of SMDrange was from 10 to 2000 pg/ml, th18%, respectively, and the recovery wexamined with this method, and the and quality control of food.m/locate /saae determination ofgquan Zhangn used for animal disease treatment, is harmful to human in food in some countries, such as USA and Japan. Therefore, it, high-sensitivity and accurate method for the determinationper, chemiluminescence enzyme immunoassay (CLEIA) wasthis method, the limit of detection was 3.2 pg/ml, the linearhin-day and inter-day precision were below 13% and belowm 85% to 105%. Milk and egg were selected as samples to be indicated that this CLEIA method was suitable for screening 2011 Elsevier B.V. All rights reserved.Y. Wu et al. / Spectrochimica Acta Part A 81 (2011) 544 547 545United States Diane (P680 HPLC Pump, ASI-100 Automated SampleInjector, Thermostatted Column Compartment Tcc-100, UVD170).2.3. Procedures2.3.1. SynthTwo meliminary exestablishedlet spectrosof them waMethod 2.5% glutarfree from ligincubation with incubaMethod solved withconcentratiincubated fSMD solutiowith 1 mol/ble at 9.5. Abuffer was Calculataccording tBindingratioAccording twould mee2.3.2. ProceIn this Cmolecules ibody coatedunits (RLU)tive correlaestablisheda serial of SAfter op100 l SMDincubated itemperatursolution antively, and and the subthat, chemicence immwas issued a2.3.3. MethThe perfof detectionAccording tThen the avwere calculLOD = X 2day variatiotests in a sthree tests expressed bexperimentrate amounvalue), andFig. Thea:ry (%thodwithnalyults enti bind 1 aate s resolet and. Those for SMD were 241 nm and 256 nm. And peak at presented HRPSMD conjugate. Thus, from Fig. 1, it wased that HRPSMD conjugate was prepared successfully. AndFig. 2. The infrared spectra of HRP, SMD and conjugate HRPSMD.esis of HRPSMD conjugatesthods were used to label SMD with HRP. After pre-periments, the optimal condition parameters were. The HRPSMD conjugate was identied by ultravio-copy and infrared spectroscopy. Then the binding ratios calculated according to the literature.1 [9]: SMD was mixed with HRP by a mass ratio of 1:1.aldehyde was added in the mixture and stirred for 4 hht. Then 100 l ethanolamine of 1 mol/L was added, andat room temperature for 2 h. After that it was dialysedtion buffer for 5 days.2 [10]: 1.0 mg HRP was weighed accurately and dis- 250 l incubation buffer. NaNO2 was added to aon of 1 mol/L. Then 300 l 1 mol/L HCl was added andor 20 min at 4 C. This mixed solution was then added ton (7.0 mg SMD was weighed accurately and dissolvedL of NaOH solution) slowly until the pH value was sta-fter incubated at 4 C overnight, dialysis with incubationkept on for 5 days to obtain HRPSMD conjugates.ion of binding ratio: the binding ratio was calculatedo the following formula: = conjugat HRPSMD= Aconjugate/Cconjugate AHRP/CHRPASMD/CSMDo literature [11], the binding ratio between 5 and 25t the requirement and the conjugate could be applied.dure for measurement of CLEIALEIA, the HRPSMD conjugate, competed by the SMDn the sample to be analyzed, combined to the SMD anti- on the microplate. In this case, the relative luminescent and the concentration of SMD in sample were nega-ted. Therefore, a quantitative mathematical model was by the relationship between RLU and concentrations ofMD standard solutions.timization, the optimal condition was established. monoclonal antibody in dilution buffer (2.5 g/ml) wasn a polypropylene microplate with 96 wells at roome. After washed with washing buffer, 100 l sampled HRPSMD conjugate solution were added, respec-incubated for 1 h. Then washed with washing buffer,strates of HRP (luminol and H2O2) were added. Afterluminescent signal was measured by chemilumines-unoassay analyzer. The light between 425 and 430 nmnd the relative luminescent unit (RLU) was determined.od evaluationormance of this CLEIA was evaluated, including limit (LOD), precision, recovery and method comparison.o literature [11], ten blank solutions were analyzed.erage value (X) and relative standard deviation (RSD)ated. LOD could be calculated by the formula below:RSD. The precision was determined by intra- and inter-ns. The intra-day precision was determined by threeingle day. The inter-day precision was determined bycarried out in three different days. The variations werey the relative standard deviations (RSD). The recovery was used to evaluate the accuracy of this method. Accu-t of SMD (spiked value) was added to sample (original then the content of SMD (observed value) was deter-minedformulrecoveThe meCLEIA were a3. Res3.1. IdThemethoconjugTheultraviFigs. 1406 nm233 nmindicat. 1. The UV spectra of HRP, SMD and conjugate HRPSMD. average recovery was determined by the following) = observed value original valuespiked value 100 comparison experiment was carried out by comparing ELISA and HLPC. Two kinds of samples, milk and egg,zed.and discussioncation of HRPSMD conjugatesding ratio of HRPSMD conjugates synthesized withnd 2 were four and six, respectively. It was shown thatynthesized with method 2 could be used in CLEIA.ults of the identication of HRPSMD conjugate byspectroscopy and infrared spectroscopy are shown in 2. The absorption peaks for HRP were 277 nm and546 Y. Wu et al. / Spectrochimica Acta Part A 81 (2011) 544 547Fig. 3. Kinetics curves of chemiluminescence reaction.this was in aabsorptionsand 12501the absorptcomparisonHRP, as wetained HRP 3.2. KineticAccordinluminescenFrom the rewas fast andreaction tim3.3. CalibraA series of SMD werwere prepawas drawnrithm of theregression ey = 0.006The lineacoefcient wTable 1The precision of CLEIA at different concentrations of SMD.SMD (pg/ml) Within-run precision (n = 10) Between-run precision (n = 10)X SD RSD% X SD RSD%10 11.8 1.5 12.8 11.3 1.9 17.2100 98.0 11.1 11.3 107.4 14.7 13.71000 924.2 115.0 12.4 1041.8 168.2 16.2Table 2Linear range, coefcient and limit of detection of CLEIA, ELISA and HPLC (n = 5).Method Linear range Coefcient Limit of detectionCLEIA 102000 pg/ml 0.9952 3.2 pg/mlELISA 10300 ng/ml 0.9970 4.5 ng/mlHPLC 0.051.25 g/ml 0.9996 0.01 g/mlTable 3The precision d ies of e Tc(250 42.4 84.7200 175.8 87.92000 1824 91.2mit of detection, precision and recovery analytical limit of detection was 3.2 pg/ml. The within-d between-run precisions were below 13% and below 18%,tively (Table 1). The recoveries were between 91.0% and in milk and between 84.0% and 92.0% in egg. The perfor- of this method showed that it was suitable for carrying outtative determination of SMD in food.ethod comparisonhis part, CLEIA was compared with ELISA and HPLC by deter-on of SMD in milk and egg. The sample preparation for ELISAccord with the result of infrared spectrum in Fig. 2. The in 35004000 cm1 region, 22502500 cm1 region750 cm1 region were for the amino acids in HRP andion in 8501200 cm1 region was for SMD. Through between the infrared spectra between conjugate andll as SMD, the result indicated that the conjugate con-and SMD.s of chemiluminescence reactiong to literature [12], an experiment of kinetics of chemi-ce reaction was carried out. The result is shown in Fig. 3.sult it could be seen that chemiluminescence reaction longtime-last. And 1.5 min was selected as the optimale in CLEIA.tion curveof standard SMD solution, in which the concentrationse 10, 50, 100, 200, 500, 1000, 2000 pg/ml, respectively,red and determined by CLEIA. Calibration curve (Fig. 4) by the logarithm of light intensity (y) versus the loga- concentration of standard SMD solution (x). Standardquation was:4x + 6.6846r range was from 10 to 2000 pg/ml and the correlationas 0.9952.MethoCLEIA ELISAHPLC Table 4RecoverSamplMilk Egg3.4. LiTherun anrespec104.0%mancequanti3.5. MIn tminatiFig. 4. Calibration curve of CLEIA.and CLEIA wwas in lineTables 26.Table 5Recoveries of Sample Milk Eggsof CLEIA, ELISA and HPLC (n = 10).Within-run precision (%) Between-run precision (%)11.312.8 13.717.212.014.5 15.617.62.44.2 4.95.9SMD by CLEIA in milk and egg (n = 10).heoreticaloncentrationpg/ml)Detectableconcentration(pg/ml)Average recovery(%)50 45.9 91.8200 188 94.0000 2078 103.9as according to references [1317]. And that for HPLC to the literatures [1821]. The results are shown inSMD by ELISA in milk and egg (n = 10).Theoreticalconcentration(ng/ml)Detectableconcentration(ng/ml)Average recovery(%)10 9.02 90.2100 91.7 91.7300 321.9 107.310 8.34 83.4100 88.9 88.9300 240.6 80.2Y. Wu et al. / Spectrochimica Acta Part A 81 (2011) 544 547 547Table 6Recoveries of SMD by HPLC in milk and egg (n = 10).Sample Theoreticalconcentration(ng/ml)Detectableconcentration(ng/ml)Average recovery(%)Milk 0.10 0.0891 89.11.25 0.9684 77.5Eggs 0.25 0.2246 89.91.25 1.0127 88.2The results showed that the detection limit of CLEIA was low-est, that its recovery was better than those of HLPC and ELISA, andthat the precision was suitable for quantication determination.Although the precision of CLEIA was not as well as HPLC, it wassimple-sample-preparing, high throughput, low-cost. Therefore, itis a promising method.3.6. DiscussionAs a kind of widely used drug in curing urinary and respira-tory tract infection, sulfamethoxydiazine was widespread in foodrelated to animal. Due to the low concentration of sulfamethoxy-diazine in food, a high sensitivity of determination method wasrequired for quality control. In order to develop a highly sensitive,well reproducible, and high throughput assay for the determinationof sulfamethoxydiazine, CLEIA, horseradish peroxidase, luminoland hydrogen peroxide as chemiluminescence reaction system,was established in this paper. This assay decreased the limit ofdetection by thousands of times, compared with the reportedmethods for analyzing sulfamethoxydiazine. This increased thereliability of quality control of food.4. ConclusA CLEIA establishedsimple samthe potentiAcknowledgmentThis work is supported by Henan Province Major Public ResearchProject.References[1] R. Gao, Y.-Z. Zhao, J.-Z. Zhao, Chinese Journal of Biologicals 21 (2008) 338342.[2] Y.-N. Wu, B. Shao, J.-Z. Shen, Veterinary Drug Residue Detection and MonitoringTechnology, Chemical Industry Press, Beijing, 2007.[3] WTO, 21 Century Food Safety, Foreign Medical Health Volumes, 2002,pp. 68.[4] S.C. Susan, C.H. Nancy, Journal of the Association of Ofcial Analytical Chemists73 (1990) 368373.[5] L. Lyse, C. Germain, S. Stephen, Journal of the Association of Ofcial AnalyticalChemists 13 (1990) 365367.[6] L.V. Bui, Journal of AOAC International 76 (1993) 966976.[7] S. Porter, Analyst, 1994, Cambridge, UK.[8] M. Fu, S.Y. Chu, Analytica Chimica Acta 499 (2003) 215221.[9] K. Jiao, S. Zhang, Application of Enzyme-Linked Immunoassay, Chemical Indus-try Press, Beijing, 2004.[10] J.-M. Lin, L.-X. Zhao, Y. Wang, Chemiluminescence Immunoassay, ChemicalIndustry Press, Beijing, 2008.[11] G.-X. Shen, R.-L. Zhou, Experimental Techniques of Modern Immunology, HuBeiScience and Technology Press, Wuhan, 1998.[12] A. Paul Schaap, H. Akhavan-Tafti, R. de Silva, S. Siripurapu, R.S. Handley, C.R.Endreszl, B.A. Schoenfelner, Eleventh International Symposium on 2000 Biolu-minescence and Chemiluminescence, 2000, p. 221.[13] General Administration of Quality Supervision, Inspection and Quarantine ofthe Peoples Republic of China, GB/T 213302007 Determination of Strep-tomycin Residues of Animal Origin-Enzyme-Linked Immunosorbent Assay,Standards Press of China, Beijing, 2007.[14] B.-H. Liu, N. Xiong, X.-L. Wang, D.-S. Shi, X.-Y. Li, D.-P. Peng, D.-R. Bi, ChineseJournal of Veterinary Drug 42 (2008) 1619.[15] L.-m. Wu, J.-z. Zhang, G.-F. Li, J.-H. Zhou, X.-F. Xue, J. Zhao, Food Science 29(2008) 535537.[16] D.-G. Zhou, H. Fang, W.-X. Zuo, Z.-H. Guo, X.-F. Yan, Chinese Journal of Veteri-nary Drug 37 (2003) 2831.[17] G.-A. Chang, L. Li, P. Chen, R.-P. Xu, Y.-M. Liu, F. Li, Chinese Journal of Healthoratory Technology 13 (2003) 422423.. Wu, Chuan ides in. TsaomatSavic,35.ionmethod to determine sulfamethoxydiazine in food was in this paper. Due to its advantages of good precision,ple preparation, high-throughput and low cost, it hadal of wide application for quality control of food.Lab[18] Y.L[19] Sicam[20] W.HChr[21] G. 29. Li, Y.J. Liu, J.Z. Shen, Chromatographia 66 (2007) 191195.Animal Husbandry and Food Bureau, Determination of Five Sulfon- Eggs by HPLC, 2009.i, H.Y. Chuang, H.H. Chen, Y.W. Wu, S.H. Cheng, T.C. Huang, Journal ofography 1217 (2010) 78127815. M. Zecevic, B. Jocic, L. Zivanovic, Chromatographia 66 (2007)Chemiluminescence enzyme immunoassay for the determination of sulfamethoxydiazine1 Introduction2 Experimental2.1 Chemicals2.2 Instruments2.3 Procedures2.3.1 Synthesis of HRPSMD conjugates2.3.2 Procedure for measurement of CLEIA2.3.3 Method evaluation3 Results and discussion3.1 Identification of HRPSMD conjugates3.2 Kinetics of chemiluminescence reaction3.3 Calibration curve3.4 Limit of detection, precision and recovery3.5 Method comparison3.6 Discussion4 ConclusionAcknowledgmentReferences


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