JZUS - Journal of Zhejiang University SCIENCE

Journal of Zhejiang University SCIENCE B 2014 Vol.15 No.2 P.165-172

Production of a monoclonal antibody against oxytetracycline and its application for oxytetracycline residue detection in shrimp*

Author(s): Tossapon Wongtangprasert¹, Wirongrong Natakuathung¹, Umaporn Pimpitak², Anumart Buakeaw², Tanapat Palaga³, Kittinan Komolpis², Nanthika Khongchareonporn²
Affiliation(s): 1. Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand; more
Corresponding email(s): nanthika.k@chula.ac.th
Key Words: Oxytetracycline, Monoclonal antibody, Enzyme-linked immunosorbant assay (ELISA), Shrimp

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Tossapon Wongtangprasert, Wirongrong Natakuathung, Umaporn Pimpitak, Anumart Buakeaw, Tanapat Palaga, Kittinan Komolpis, Nanthika Khongchareonporn. Production of a monoclonal antibody against oxytetracycline and its application for oxytetracycline residue detection in shrimp[J]. Journal of Zhejiang University Science B, 2014, 15(2): 165-172.

@article{title="Production of a monoclonal antibody against oxytetracycline and its application for oxytetracycline residue detection in shrimp",
author="Tossapon Wongtangprasert, Wirongrong Natakuathung, Umaporn Pimpitak, Anumart Buakeaw, Tanapat Palaga, Kittinan Komolpis, Nanthika Khongchareonporn",
journal="Journal of Zhejiang University Science B",
volume="15",
number="2",
pages="165-172",
year="2014",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1300181"
}

%0 Journal Article
%T Production of a monoclonal antibody against oxytetracycline and its application for oxytetracycline residue detection in shrimp
%A Tossapon Wongtangprasert
%A Wirongrong Natakuathung
%A Umaporn Pimpitak
%A Anumart Buakeaw
%A Tanapat Palaga
%A Kittinan Komolpis
%A Nanthika Khongchareonporn
%J Journal of Zhejiang University SCIENCE B
%V 15
%N 2
%P 165-172
%@ 1673-1581
%D 2014
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1300181

TY - JOUR
T1 - Production of a monoclonal antibody against oxytetracycline and its application for oxytetracycline residue detection in shrimp
A1 - Tossapon Wongtangprasert
A1 - Wirongrong Natakuathung
A1 - Umaporn Pimpitak
A1 - Anumart Buakeaw
A1 - Tanapat Palaga
A1 - Kittinan Komolpis
A1 - Nanthika Khongchareonporn
J0 - Journal of Zhejiang University Science B
VL - 15
IS - 2
SP - 165
EP - 172
%@ 1673-1581
Y1 - 2014
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1300181

Abstract
Chinese Summary
Academic Network
Reviewer Comment

Abstract: A novel monoclonal antibody (MAb) against oxytetracycline (OTC) was generated and characterized. The MAb was used in the development of an enzyme-linked immunosorbant assay (ELISA)-based detection system. An OTC-bovine serum albumin (BSA) conjugate was prepared and used in the immunization of mice. A conventional somatic cell fusion technique was used to generate MAb-secreting hybridomas denoted 2-4F, 7-3G, and 11-11A. An indirect competitive ELISA (icELISA) was applied to measure the sensitivity and specificity of each MAb in terms of its 50% inhibitory concentration (IC₅₀) and percentage of cross-reactivity, respectively. MAb 2-4F exhibited the highest sensitivity, with an IC₅₀ of 7.01 ng/ml. This MAb showed strong cross-reactivity to rolitetracycline, but no cross-reactivity to other unrelated antibiotics. When MAb 2-4F was used to detect OTC from shrimp samples, the recoveries were in the range of 82%–118% for an intra-assay and 96%–113% for an inter-assay. The coefficients of variation of the assays were 3.9%–13.9% and 5.5%–14.9%, respectively.

抗土霉素单克隆抗体的制备及其在虾中土霉素残留的检测应用

研究目的：抗土霉素单克隆抗体的制备和特性分析，并用于间接竞争酶联免疫吸附测定法（icELISA）分析虾样品中土霉素的残留。
创新要点：本研究建立分泌抗土霉素单克隆抗体的杂交瘤细胞株，筛选出对虾中土霉素残留检测具有较高灵敏度的单克隆抗体2-4F。
研究方法：用细胞杂交技术使土霉素-牛血清白蛋白偶联物免疫的BALC/c雌性小鼠的脾细胞与骨髓瘤细胞融合，建立三株分泌抗土霉素单克隆抗体的杂交瘤细胞株（2-4F、7-3G和11-11A），并制备它们的单克隆抗体。通过icELISA法分析单克隆抗体对土霉素的半抑制质量浓度（IC₅₀）和交叉反应率，明确其灵敏度和特异性。
重要结论：实验结果发现单克隆抗体2-4F具有最高的灵敏度，对土霉素的IC₅₀为7.01 ng/ml，且该单抗特异性强，仅与罗利环素之间有强烈的交叉反应，而与非相关抗生素不发生交叉反应。当利用单克隆抗体2-4F检测虾样品中的土霉素含量时，其批内和批间回收率分别为82%~118%和96%~113%，变异系数分别为3.9%~13.9%和5.5%~14.9%。因此，单克隆抗体2-4F可用于虾产品中土霉素残留分析试剂盒的开发。

关键词：土霉素；单克隆抗体；酶联免疫吸附测定法（ELISA）；虾

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References

[1] Abbott, M., Hayward, S., Ross, W., 2010. Validation procedures for quantitative food allergen ELISA methods: community guidance and best practices. J AOAC Int, 93(2):442-450.

[2] Carson, M.C., Ngoh, M.A., Hadley, S.W., 1998. Confirmation of multiple tetracycline residues in milk and oxytetracycline in shrimp by liquid chromatography-particle beam mass spectrometry. J Chromatogr B Biomed Sci Appl, 712(1-2):113-128.

[3] Chfer-Pericsa, C., Maquieiraa, ., Puchadesa, R., 2010. Immunochemical determination of oxytetracycline in fish: comparison between enzymatic and time-resolved fluorometric assays. Anal Chim Acta, 662(2):177-185.

[4] Cherlet, M., de Baere, S., de Backer, P., 2003. Quantitative analysis of oxytetracycline and its 4-epimer in calf tissues by high-performance liquid chromatography combined with positive electrospray ionization mass spectrometry. Analyst, 128(7):871-878.

[5] Cinquina, A.L., Longo, F., Anastasi, G., 2003. Validation of a high-performance liquid chromatography method for the determination of oxytetracycline, tetracycline, chlortetracycline and doxycycline in bovine milk and muscle. J Chromatogr A, 987(1-2):227-233.

[6] Cliquet, P., Cox, E., Haasnoot, W., 2003. Extraction procedure for sulfachloropyridazine in porcine tissues and detection in a sulfonamide-specific enzyme-linked immunosorbent assay (ELISA). Anal Chim Acta, 494(1-2):21-28.

[7] The Council of the European Communities, 1990. Council Regulation (EEC) No. 2377/90 laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Off J Eur Commun, L224:22-23.

[8] Croubels, S.M., Vanoosthuyze, K.E.I., van Peteghem, C.H., 1997. Use of metal chelate affinity chromatography and membrane-based ion-exchange as clean-up procedure for trace residue analysis of tetracyclines in animal tissues and egg. J Chromatogr B Biomed Sci Appl, 690(1-2):173-179.

[9] Faraj, B.A., Ali, F.M., 1981. Development and application of a radioimmunoassay for tetracycline. J Pharmacol Exp Ther, 217(1):10-14.

[10] Frey, A., Meckelein, B., Externest, D., 2000. A stable and highly sensitive 3,3',5,5'-tetramethylbenzidine-based substrate reagent for enzyme-linked immunosorbent assays. J Immunol Methods, 233(1-2):47-56.

[11] Furusawa, N., 1999. High-performance liquid chromatographic determination/identification of oxytetracycline and sulphadimidine in meat and eggs. Chromatographia, 49(7-8):369-373.

[12] Gao, F., Zhao, G.X., Zhang, H.C., 2013. Production of monoclonal antibody against doxycycline for immunoassay of seven tetracyclines in bovine muscle and milk. J Environ Sci Health B, 48(2):92-100.

[13] Gmez-Jimenez, S., Espinosa-Plascencia, A., Valenzuela-Villa, F., 2008. Oxytetracycline (OTC) accumulation and elimination in hemolymph, muscle and hepatopancreas of white shrimp Litopenaeus vannamei following an OTC-feed therapeutic treatment. Aquaculture, 274(1):24-29.

[14] Harlow, E., Lane, D., 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory,New York :

[15] Hock, B., Dankwardt, A., Kramer, K., 1995. Immunochemical techniques: antibody production for pesticide analysis. A review. Anal Chim Acta, 311(3):393-405.

[16] Howard, G.C., Kaser, M.R., 2007. Making and Using Antibodies: A Practical Handbook. CRC Press Taylor & Francis Group,Florida :80-92.

[17] Huang, T.S., Du, W.X., Marshall, M.R., 1997. Determination of oxytetracycline in raw and cooked channel catfish by capillary electrophoresis. J Agric Food Chem, 45(7):2602-2605.

[18] Le, H., Szurdoki, F., Szekacs, A., 2003. Evaluation of an enzyme immunoassay for the detection of the insect growth regulator fenoxycarb in environmental and biological samples. Pest Manag Sci, 59(4):410-416.

[19] Le, T., Yu, H., Guo, Y., 2009. Development of an indirect competitive ELISA for the detection of doxycycline residue in animal edible tissues. Food Agric Immunol, 20(2):111-124.

[20] Le, T., Yu, H., Wang, X., 2011. Development and validation of an immunochromatographic test strip for rapid detection of doxycycline residues in swine muscle and liver. Food Agric Immunol, 22(3):235-246.

[21] Le, T., Yi, S.H., Zhao, Z.W., 2011. Rapid and sensitive enzyme-linked immunosorbent assay and immunochromatographic assay for the detection of chlortetracycline residues in edible animal tissues. Food Addit Contam Part A, 28(11):1516-1523.

[22] Nogueira-Lima, A.C., Gesteira, T.C.V., Mafezoli, J., 2006. Oxytetracycline residues in cultivated marine shrimp (Litopenaeus vannamei Boone, 1931) (Crustacea, Decapoda) submitted to antibiotic treatment. Aquaculture, 254(1-4):748-757.

[23] Serrano, P.H., 2005. Responsible Use of Antibiotics in Aquaculture. Food and Agriculture Organization of the United Nations,Rome :

[24] Shah, V.P., Midha, K.K., Dighe, S., 1992. Analytical methods validation: bioavailability, bioequivalence, and pharmacokinetic studies. J Pharm Sci, 81(3):309-312.

[25] Srisomboon, P., Poomchatra, A., 1995. Oxytetracycline residues in farmed shrimp: the re-emerging threatening issue to human health and economics. J Health Sci (Thailand), 4(2):163-170.

[26] Wang, W., Lin, H., Xue, C., 2004. Elimination of chloramphenicol, sulphamethoxazole and oxytetracycline in shrimp, Penaeus chinensis following medicated-feed treatment. Environ Int, 30(3):367-373.

[27] Yuan, S., Wang, Q., Yates, S.R., 2010. Development of an efficient extraction method for oxytetracycline in animal manure for high performance liquid chromatography analysis. J Environ Sci Health B, 45(7):612-620.

[28] Yuwono, M., Indrayanto, G., 2005. Oxytetracycline: analytical profile. Profiles Drug Subst Exc Relat Methodol, 32:97-117.

[29] Zhao, C.B., Peng, D.P., Wang, Y.L., 2008. Preparation and validation of the polyclonal antibodies for detection of chlortetracycline residues. Food Agric Immunol, 19(2):163-174.

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抗土霉素单克隆抗体的制备及其在虾中土霉素残留的检测应用

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