牛尿中玉米赤霉醇残留酶联免疫检测方法的研究
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摘要
本研究采用混合酸酐法制备了玉米赤霉醇(Zeranol,ZER)免疫原，免疫BALB／c小鼠，用间接酶联免疫吸附法(ELISA)对抗体效价进行了检测，并建立了ELISA检测方法。ZER免疫原(ZER-7-半琥珀酸酯-BSA，以下简称Z7BSA)中半抗原与载体蛋白的偶联比为10∶1，包被原(ZER-7-半琥珀酸酯-OVA，以下简称Z70VA)中半抗原与载体蛋白的偶联比为8∶1。抗血清效价为1∶8000。利用杂交瘤技术获得了两株稳定分泌ZER单克隆抗体(以下简称单抗)的杂交瘤细胞株IC12和9B4。经鉴定两株单抗亚型均为IgG_1，杂交瘤细胞染色体数目为95～102条，间接ELISA测定腹水效价为1∶3.2×10~5，单抗分子量为151.8KD。该单抗与ZER同系物β-玉米赤霉醇、α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉酮、玉米赤霉烯酮的交叉反应率分别为35.5％，47.4％，7.1％，73.0％，56.3％；与己烯雌酚、雌二醇、睾酮、克伦特罗的交叉反应率均小于0.1％。该细胞株体外传代和冻存复苏后抗体分泌稳定。
用方阵滴定法确定包被原最适工作浓度为0.5μg／ml，单抗最适稀释倍数为1∶5000，辣根过氧化物酶标记羊抗鼠IgG(以下简称酶标二抗)最适稀释倍数为1∶5000。采用间接竞争ELISA方法建立检测ZER的标准曲线，在0.5ng／ml～100ng／ml范围内呈线性相关，回归方程为y=-66.844x+424.15，相关系数R~2=0.996，50％抑制浓度(IC_(50))为3.0ng／ml，以10％抑制率对应的浓度计算，则该方法对ZER的检测限为0.6ng／ml。
以该单抗为基础，进行牛尿中玉米赤霉醇酶联免疫检测试剂盒的研制。确定了试剂盒中各种溶液的配方。试剂盒最低检测限为0.60ng／ml，试剂盒对空白牛尿的最低检测限为7.74ng／ml。以10ng／ml，21ng／ml，35ng／ml 3个浓度添加空白牛尿，回收率在70.0％～116.0％之间，板内变异系数在6.0％～15.9％之间，批内变异系数为15.3％，批间变异系数为14.3％，均低于20％。试剂盒于2～8℃下可保存6个月。
In this study, Zeranol(ZER) was coupled to carrier protein bovine serumalbumin(BSA) and ovalbumin(OVA) using the mixed anhydride method. The ZER-BSA was used as immunogen and the ZER-OVA used as coating antigen. The coupling ratio of ZER to BSA was 10:1 and to OVA was 8:1. BALB/c mice were immuned by the immunogen and the titer of anti-serum was 1:8000 by two-dimensional titration method.
Two hybridomas producing anti-ZER monoclonal antibodies were obtained by limited dilution, named 1C 12 and 9B4. The characteristics of monoclonal antibody was performed by EL1SA test, chromosomal counting and SDS-PAGE electrophoresis. The results indicated that the subclass of the McAb was IgGl, the titer of ascitic fluid was 1: 3.2×105, the molecular weight was 151.8KD .the chromosomal numbers were 95-102. The cross-reactivities of McAb to zeranol, β-zearalanol,α-zearalenol, β-zearalenol, zearalanone, zearalenone were 100%, 35.5%, 47.4%, 7.1%, 73.0%, 56.3% respectively, and to diethylstilbestrol, 17 β-estradiol, testosterone, clenbuterol were all below 0.1% . The indirect competitive Enzyme Linked Immunosorbent Assay(ELISA) for ZER was developed. The best concentration of coating antigen was 0.5ug/ml, the optional working dilution of the McAb and enzyme -conjugate were all 1:5000 , The linear detecting range of calibration curves to detect ZER was 0.5ng/ml~100ng/ml , The formular was y = -66.844x+424.15, R2 = 0.996, the 50% inhibition concentration(IC50) was 3.0ng/ml. The limit of detection was 0.6ng/ml.
The enzyme immunoassay kit for the quantitative analysis of zeranol was developed with McAb against ZER. The lower detection limit of ZER test was 0.60ng/ml. According to the sample preparation protocol, the detection limit was 7.74ng/ml in bovine urine. When the spiked concentration of ZER were 10ng/ml, 21ng/ml and 35ng/ml, the recovery range was all from 70.0% to 116.0%, the coefficient range of variation was from 6.0% to 15.9%. The ELISA kit could be reserved for 6 months at 2~8℃.
用方阵滴定法确定包被原最适工作浓度为0.5μg／ml，单抗最适稀释倍数为1∶5000，辣根过氧化物酶标记羊抗鼠IgG(以下简称酶标二抗)最适稀释倍数为1∶5000。采用间接竞争ELISA方法建立检测ZER的标准曲线，在0.5ng／ml～100ng／ml范围内呈线性相关，回归方程为y=-66.844x+424.15，相关系数R~2=0.996，50％抑制浓度(IC_(50))为3.0ng／ml，以10％抑制率对应的浓度计算，则该方法对ZER的检测限为0.6ng／ml。
以该单抗为基础，进行牛尿中玉米赤霉醇酶联免疫检测试剂盒的研制。确定了试剂盒中各种溶液的配方。试剂盒最低检测限为0.60ng／ml，试剂盒对空白牛尿的最低检测限为7.74ng／ml。以10ng／ml，21ng／ml，35ng／ml 3个浓度添加空白牛尿，回收率在70.0％～116.0％之间，板内变异系数在6.0％～15.9％之间，批内变异系数为15.3％，批间变异系数为14.3％，均低于20％。试剂盒于2～8℃下可保存6个月。
In this study, Zeranol(ZER) was coupled to carrier protein bovine serumalbumin(BSA) and ovalbumin(OVA) using the mixed anhydride method. The ZER-BSA was used as immunogen and the ZER-OVA used as coating antigen. The coupling ratio of ZER to BSA was 10:1 and to OVA was 8:1. BALB/c mice were immuned by the immunogen and the titer of anti-serum was 1:8000 by two-dimensional titration method.
Two hybridomas producing anti-ZER monoclonal antibodies were obtained by limited dilution, named 1C 12 and 9B4. The characteristics of monoclonal antibody was performed by EL1SA test, chromosomal counting and SDS-PAGE electrophoresis. The results indicated that the subclass of the McAb was IgGl, the titer of ascitic fluid was 1: 3.2×105, the molecular weight was 151.8KD .the chromosomal numbers were 95-102. The cross-reactivities of McAb to zeranol, β-zearalanol,α-zearalenol, β-zearalenol, zearalanone, zearalenone were 100%, 35.5%, 47.4%, 7.1%, 73.0%, 56.3% respectively, and to diethylstilbestrol, 17 β-estradiol, testosterone, clenbuterol were all below 0.1% . The indirect competitive Enzyme Linked Immunosorbent Assay(ELISA) for ZER was developed. The best concentration of coating antigen was 0.5ug/ml, the optional working dilution of the McAb and enzyme -conjugate were all 1:5000 , The linear detecting range of calibration curves to detect ZER was 0.5ng/ml~100ng/ml , The formular was y = -66.844x+424.15, R2 = 0.996, the 50% inhibition concentration(IC50) was 3.0ng/ml. The limit of detection was 0.6ng/ml.
The enzyme immunoassay kit for the quantitative analysis of zeranol was developed with McAb against ZER. The lower detection limit of ZER test was 0.60ng/ml. According to the sample preparation protocol, the detection limit was 7.74ng/ml in bovine urine. When the spiked concentration of ZER were 10ng/ml, 21ng/ml and 35ng/ml, the recovery range was all from 70.0% to 116.0%, the coefficient range of variation was from 6.0% to 15.9%. The ELISA kit could be reserved for 6 months at 2~8℃.
引文
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[2] 林书康，游存华．牛羊增重剂——玉米赤霉醇．黄牛杂志，1992，4：62-66
[3] Baldwin, R. S., Williams, R. D., Terry. M. K. Zeranol: A review of the metabolism, toxicology,and analytical methods for detection of tissue residues, Regul. Toxicol. Pharmacol., 1983, 3:9-25
[4] 李宗燕．肉牛育肥九十天．黄牛杂志，1999，01
[5] Migdalof, B. H., Dugger, H. A., Heider, J. G.,Coombs, R. A., and Terry, M. Biotransformation of zeranol: Disposition and metabolism in the female rat, rabbit, dog, monkey, and human. Xenobiotica, in press, 1983
[6] Ingerowski, G. H., and Stan, H.-J. In vitro metabolism of the anabolic drug zeranol. J. Environ.Patrol. Toxicol. 1977, 2:1173-1182
[7] Powell-Jones, W., Raeford, S., and Lucier, G. W. Binding properties of zearalenone mycotoxins to hepatic estrogen receptors. Mol. Pharmacol. 1981, 20:35-42
[8] Brown, R. G. Toxicology and tissue residues of zeranol. In An Foras Taluntais, Proceedings,Conferenec on the Use, Residues and Toixicology of Growth Promoters, Dubin, April 9, 1980:31-37
[9] Sharp, C. D., and Dyer, I. A. Zearalanol metabolism in steers. J. Anita. Sci. 1972, 34:176-179
[10] Hidy, E H., Baldwin, R.S., Greashara, R. L., Keith, C. L. and McMullen, J. R. Zearalenone and some derivatives: Production and biological activities. Advan. Appl. Microbiol. 1977, 22:59-82
[11] Food and Drug Administration. Part 135b-New Animal Drugs for Implantation or Injection. Part 135g-Tolerances for Residues of New Animal Drugs in Food. Federal Register. 1970, 35, (168):13727-13728
[12] 蒋学之，环境雌激素对人类健康的潜在影响，中国公共卫生，1997，13(4)：251-252
[13] 农业部第235号公告《动物性食品中兽药最高残留限量》
[14] 李俊锁主编．同化激素类药物残留分析．上海科学技术出版社，2002，578
[15] 朱蓓蕾主编．动物性食品药物残留．上海科学技术出版社，1994，72
[16] Medina, M. B. and Schwartz, D. P., Thin-layer chromatographic detection of zeranol and estradiol in fortified plasma and tissue extracts with Fast Corinth V. Journal of Chromatography,1992, 581:119-128
[17] Medina,M. B. and Nagdy, N., Improved thin-layer chromatographic detection of diethylstilbestrol and zeranol in plasma and tissues isolated with alumina and ion-exchange membrane columns in tandem. Journal of Chromatography, 1993, 614:315-323
[18] Lynda J. James, Larry G. Mcgirr, and Trevor K Smith, High pressure liquid chromatography of zearalenone and zearalenols in rat urine and liver. J. Assoc. Off. Anal. Chem. 1982, V(65):9-13
[19] Trenholm, H. L., Warner, R. M., Farnworth, E. R. J.Assoc. Off. Anal Chem. 1981, 64:302
[20] Renzo Bagnati, Manrizio Paleologo Oriundi, Vincenzo Russo and Marisa Danese, et al,Determination of zeranol and β-zearananol in calf urine by immunoaffinity extraction and gas chromatography-mass spectrometry after repeated administration of zeranol, Journal of Chromatography, 1991, 564:493-502
[21] Hyeong L. Kim, Allen C. Ray and Robert D. Stipanovic, Rapid separation and identification of urinary metabolites of zeranol by HPLC-UV spectrophotometry. J.Agric. Food. Chem. 1986, 34:312-315
[22] Dixon, S.N., Radioimmunoassay of the anabolic agent zeranol. Journal of Veterinary Pharmacology and Therapeutics, 1980, 3:177-181
[23] Carter, A. P., Dixon S. N. and Bew, M. H., Preparation and properties of monoclonal antibodies to the anabolic agent zeranol. Journal of Veterinary Pharmacology and Therapeutics, 1984, 7:17-21
[24] Patel P.J. and Dixon S.N., An Enzyme immunoassay for the anabolic agent zeranol. Food Addit,Contain, Anal Surveillance. Eval Control 1986 V(6):91-102
[25] Jansen J. M., A chemiluminescent immunoassay for zeranol and its metabolites, Journal of Veterinary Pharmacology and Therapeutics, 1986, 9:101-108
[26] 彭涛．玉米赤霉醇分子印迹聚合物的制备：[硕士学位论文]．北京：中国农业大学，2002，摘要
[27] Start, H. J., and Abraham, B. Determination of residues of anabolic drugs in meat by gas chromatography-mass spectroscopy. J. Chromatogr. 1980, 195:231-241
[28] Erlanger, B.E, Brek, F., Beiser, S.M. and Lieberman, S. Steroid-Protein Conjugates. Journal of Biological chemistry. 1959, 234:1090-1094
[29] 黄宪，陈振初等．有机合成化学．化学工业出版社，1983，312-491
[30] (美)帕克．生物活性化合物的放射免疫测定法．科学出版社，1981，4-29
[31] 张立明．现代生物化学分析原理．中国科技大学出版社，1991，21-34
[32] 杨利国．酶免疫测定技术．南京：南京大学出版社，1998，242．286
[33] 何继红，沈建忠．磺胺二甲嘧啶单克隆抗体的研制．中国兽医杂志， 2003，1：8-10
[34] 王建平．β_2-兴奋剂酶联免疫吸附检测法的研究：[硕士学位论文[．北京：中国农业大学，2003，13．15
[35] 徐志凯．实用单克隆抗体技术．陕西科学技术出版社，1992，27-125
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