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1 7 2 Vol. 7 No Journal of Food Safety and Quality Feb., 2016 苏明明 1, 王翛 2, 王晓薇 2, 王琦 2, 曹际娟 (1., ; 2., ) 1* 摘 要 : 目的 Charm II 方法 6,,,, 结果, 20 μg/kg, 结论,,,, 关键词 : ; ; Detection of tetracycline in bovine serum with radioimmunoassay method SU Ming-Ming 1, WANG Xiao 2, WANG Xiao-Wei 2, WANG Qi 2, CAO Ji-Juan 1* (1. Liaoning Entry-Exit Inspection and Quarantine Bureau, Dalian , China; 2. Dalian Entry-Exit Inspection and Quarantine Bureau, Dalian , China) ABSTRACT: Objective To detect the sulfonamide residues in bovine serum by Charm II radioimmunoassay method. Methods Six serum samples were collected for the addition test. The control point was set. The results from the sample test were compared with the control point. The sensitivity and specificity of the immune reaction system was evaluated. The measured value was compared with the control point. The sample was determined to be negative if the measured value was greater than the control point, whereas, the sample was positive if the measured value was less than the control point. Results Test results can be obtained in short time and the limit of tetracycline detection was 20 μg/kg. The method was highly specific, easy and convenient to operate. Conclusion Other test methods are still needed to further confirm the positive results as the method proposed in this paper is only a preliminary method. This method can keep livestock alive and reduce losses of merchants. It can quickly give out test results and meet detection requirements of tetracycline residues in bovine serum. KEY WORDS: radioimmunoassay method; tetracycline; bovine serum 1 引言 WTO,,,,, * 通讯作者 :,, caojijuanlnciq@163.com *Corresponding author: CAO Ji-Juan, Professor, Technical Center of Liaoning Entry-Exit Inspection & Quarantine Bureau, No.60, Changjiang East Road, Zhongshan District, Dalian , China. caojijuanlnciq@163.com

2 630 7 (tetracyclines), (chlotetracycline) (oxytetracycline) (tetracycline),,,, [1,2],,, ( ),,,,,,,,,, (maximum residue limit, MRL): 100 μg/l [3,4],,,,, (ELISA) [5-10] (high performance liquid chromatography-tandem mass spectrometry, HPLC-MS/MS) [11-15], HPLC,, ; ELISA, Charm Ⅱ,,,, FDA [16] Charm Ⅱ [17,18] 2 材料与方法 2.1 仪器与设备 Charm II6600/7600 ( Charm ); ( Sigma ); ( IKA ); (65±1) ; ; 50 ml ; ; ( Charm ); ( Charm ) 2.2 样品与试剂 检测基质 :,, 18~25, 8~12 h 检测试剂 Charm Ⅱ (Optifluor) ( Charm ) G ( Sigma ) 2.3 实验方法 测试原理,, [3H], [3H] [3H] cpm (count per minute, ), cpm 样品制备 1 g( 0.1 g) 50 ml, 30 ml MSU, 5 min (80±2) 45 min, 10 min, 1750 g 10 min,, ph ph 7.5, M2 1 mol/l ph7.5, 测定步骤,, 300 μl,

3 2, : s, 4 ml,, 15 s, 15, (65±1), 3 min, 1750 g 5 min,,,,, 300 μl,, 3 ml, Charm Ⅱ6600/7600, [3H] cpm 阳性结果的确定 cpm,, : (1) ±20%(, 3 cpm ) (2) cpm, (1) (2), 控制点的建立 6 ( ) ( ) 6 cpm, 20% 1, cpm, 20 μg/kg, cpm cpm, 20 μg/kg μg/l, μg/kg, cpm,, cpm ( cpm cpm, B/B 0 ), Y= X+ 0.92, ~50 μg/kg, ,, cpm 0.6, 20 μg/kg 3.2 检测限的验证 μg/kg,, μg/kg cpm 1919, 6 6, 20 μg/kg,, 20 μg/kg 2 3 结果与分析 3.1 抗原抗体竞争性反应标准曲线 MSU, MSU μg/kg, cpm, 1 Table 1 表 1 不同药物浓度四环素测定结果 The results of different concentrations tetracycline 1 RRA Fig. 1 The competitive RRA semilogarithmic curve of chlortetracycline (µg /kg) cpm 表 2 牛血清中四环素类抗生素残留量测定 ( 控制点 : 1919) Table 2 The determination of chlortetracycline antibiotic residues in bovine serum(control point: 1919) 6 cpm ( ) ( 20 μg/kg)

4 特异性验证实验, 4,, 20 μg/kg, cpm 1919, 4 100% G, 100 μg/kg, cpm, cpm 1919, Charm II 4 结论 Charm II, 20 μg/kg,, ;, 0%,,,,,,,,,, 参考文献 [1] Jacobsen AM, Halling-Sørensen B. Multi-component analysis of tetracyclines, sulfonamides and tylosin in swine manure by liquid chromatography tandem mass spectrometry [J]. Anal Bioanal Chem, 2006, 66(8): [2] Himmelsbach M, Buchberger W. Residue analysis of oxytetracycline in water and sediment samples by high-performance liquid chromatography and immunochemical techniques [J]. Microchim Acta, (7): 1 2. [3] Aga DS, O Connor S, Ensley S, et al. Determination of the persistence of tetracycline antibiotics and their degradates in manure-amended soil using enzyme-linked immunosorbent assay and liquid chromatography-mass spectrometry [J]. J Agric Food Chem, 2005, 25(9): [4] Arikan OA. Degradation and metabolization of chlortetracycline during the anaerobic digestion of manure from medicated calves [J]. J Hazard Mater, 2008, 16(3): [5] Carlson JC, Mabury SA. Dissipation kinetics and mobility of chlortetracycline,tylosin,and monensin in an agricultural soil in Northumberland County, Ontario, Canada [J]. Environ Toxicol, 2006, 20(5): [6] Yan DD, He LM, Zhang GJ, et al. Simultaneous determination of cyadox and its metabolites in chicken tissues by LC-MS/MS [J]. Food Anal Methods, 2012, 60(2): 6 9. [7] Liubavina IA, Valiakina TI, Grishin EV. Monoclonal antibodies labeled with colloidal gold for immunochromatographic express analysis of diphtheria toxin [J]. Bioorganicheskaia Khimiia, 2011, 42(3): [8] Salter, Robert, Douglas, et al. Intel-laboratory study of the charm ROSA safe level aflatoxin M 1 quantitative lateral flow test for raw bovine milk [J]. J AOAC Int, 2006, 17(10): [9] Li H, Sun ZY, Zhong WY, et al. Ultrasensitive electrochemical detection for dna arrays based on silver nanoparticle aggregates [J]. Anal Biochem, 2010, 25(3): [10] Sarmah AK, Meyer MT, Boxall ABA. A global perspective on the use, sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment [J]. Chemosphere, 2006, 39(4): [11] Cliquet P, Cox E, Haasnoot W, et al. Generation of group-specific antibodies against sulfonamides [J]. J Agric Food Chem, 2003,14(9): [12] Pastor-navarro N, Garcia-rover C, Maquieira A, et al. Specific polyclonal-based immunoassays for sulphathiazole [J]. Anal Bioanal Chem, 2004, 15(3): [13] Jeon M, Kim J, Paeng KJ, et al. Biotin-avidin mediated competitive enzyme-linked immunosorbent assay to detect residues of tetracyclines in milk [J]. Microchem J, 2008, 57(5): [14] Lamar J, Petz M. Development of a receptor-based microplate assay for the detection of beta-lactam antibiotics in different food matrices [J]. Anal Chim Acta, 2007, 43(12): [15] Korpimaki T, Hagren V, Brockmann EC, et al. Generic lanthanide fluoroimmunoassay for the simultaneous screening of 18 sulfonamides using an engineered antibody [J]. Anal Biochem, 2004, 36(7): [16] Franek M, Diblikova I, Cernoch I, et al. Broad-specificity immunoaseay for sulfonamide detection: immunochemical strategy for generic antibodies and competitors [J]. Anal

5 2, : 633 Biochem, 2006, 30(5): [17] Spinks CA, Wyatt GM, Everest S, et al. Atypi-cal antibody specificity: advancing the development of a generic assay for sulfonamides using heterolo- gous ELISA [J]. J Sci Food Agric, 2002, 15(10): [18] Goldman ER, Clapp AR, Anderson GP, et al. Multiplexed toxin analysis using four colors of quantum dot fluororeagents [J]. Anal Biochem, 2004, 30(3): ( 责任编辑 : 杨翠娜 ) 作者简介 苏明明, 工程师, 硕士, 主要研究方向为农残兽残分析 meng8135@163.com 曹际娟, 研究员, 硕士, 主要研究方向为农残兽残分析 caojijuanlnciq@163.com