克伦特罗、青霉素和庆大霉素抗体的制备与间接竞争ELISA检测克伦特罗残留的初步研究
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摘要
克伦特罗具有典型的β—肾上腺素活性和对β_2受体的选择性亲和力，它在兽医临床上被用作支气管扩张剂用于治疗气管炎和哮喘。但若在养殖业非法用作营养再分配剂以提高瘦肉率，可造成其在动物性产品中的残留而危害消费者健康。庆大霉素作用于多种革兰氏阳性和阴性菌，在兽医临床上，庆大霉素主要通过注射途径给药，用于呼吸道感染以及乳腺感染等。但该药的排泄较慢，如果使用不当，容易造成在动物性产品中的残留蓄积。青霉素G既能被青霉素酶灭活，也能被胃肠道的酸性环境降解，兽医临床上多以注射方法用于革兰氏阳性敏感菌引起的感染性疾病(如奶牛乳腺炎等)的治疗，青霉素G是牛奶中比较常见的残留药物之一，可以引起过敏反应，抑制酸奶加工过程中乳酸菌的生长。因此，建立针对这些药物残留的快速检测方法对控制动物性食品的卫生质量、保障消费者健康具有比较重要的社会经济意义。
上述三种药物为小分子半抗原，缺乏免疫原性。将半抗原与合适的载体蛋白偶联后，半抗原可以成为全抗原并具有较好的免疫原性。碳二亚胺(EDC)是一种同型双功能交联剂，可以通过一步反应形成稳定的半抗原—载体复合物。免疫原EDC偶联试剂盒可用于克伦特罗、青霉素和庆大霉素的偶联。按照半抗原—载体EDC标准偶联程序，我们偶联了克伦特罗—BSA、克伦特罗—OVA、青霉素—BSA、青霉素—OVA、庆大霉素—BSA、庆大霉素—KLH。偶联物4℃透析3天后，用BSA偶联物加佐剂免疫新西兰白兔。从第三次免疫后7天心脏采血。
放血分离血清后，以间接ELISA测抗体效价，用OVA和KLH偶联物包被酶标板，1％酪蛋白封闭1小时，一抗(倍比稀释的相应血清)和酶标二抗(500×稀释的羊抗兔IgG/HRP)分别在37℃反应2小时。PBST洗3次后用OPD(邻苯二胺)显色，最后用2M硫酸终止反应，酶标仪测490nm OD值。根据OD值(Y-轴)和稀释度(X—轴)的曲线，找出免疫血清曲线中的拐点及其对应X-轴的数值确定抗体效价。方案一中青霉素抗体效价为1600×，方案二中克伦特罗抗体效价为400×。在方案三中，克伦特罗、青霉素和庆大霉素抗体效价分别为400×，400×和200×。在间接竞争ELISA和硝酸纤维素膜试验中，我们同时比较了1％OVA、1-3％明胶、
l％BSA的封闭效果，结果表明 1％酪蛋白是最优的封闭液。
我们用间接竟争ELISA对尿液中克伦特罗残留进行了模拟检测。方法是：用克
伦特罗－OVA 200 X稀释包被酶标板，l％酪蛋白封闭，然后用 10 X稀释的亲和层析
纯化兴奋剂抗体IgG和含有兴奋剂的样品同时竞争，最后加酶标二抗，OPD底物显
色。同时，用不同浓度的克伦特罗标准品同时进行检测，并制作参比曲线进行回归
分析。初步结果表明：间接竟争ELISA的检测下限可达到10ng／ffil。为进一步优化
并建立克伦特罗的ELISA检测方法奠定了基础。
Clenbuterol is a sympathomimetic agent with principally p-adrenergic activity and a selective action on p-receptors. It is used in veterinary medicine as bronchodilatator and tocolytic. It has also been illegally appled in animal feed additives as a repartitioning agent which would lead to resiudes in the animal products with the potential of food-borne poisoning. Gentamycin is active against many gram-positive and gram-negative bacteria. It is administered by intramuscularly or intravenously for general infections or orally for gastro-intestinal infection. It is also used intramammarily for bovine mastitis and topically in ocular and auricular infections. Improper use of the drug may result in residual problems because of its slow elimination. Penicillin G is inactivated by penicillinases and is unstable in gastric acid. It is often used for the treatment of infections by susceptible bacteria through parenteral application or intramammary infusion for bovine mastitis. Its reside in milk is of great concern
to the consumers for its potential of allergic reactions as well as to the dairy industry for its inhibitory effect on the starter cultures. Therefore,development of fast methods for detection of residues of these drugs in the foods of animal origins is of great public health significance and socio-economic importance.
The above three drugs are haptens. Their immunogenicity can be imporved by coupling them to suitable carrier protein molecules. N-ethyl-N'-[3-(dimethylamino)propyl] carbodiimide hydrochloride (EDC) is a bifunctional coupling reagent. It is efficient for one-step coupling to form a wide variety of hapten-carrier protein immunogens. Conjugation may occur at either the C- or N-terminal of the hapten or at any carboxyl- or amine-containing side chains. Imject Immunogen EDC Conjugation Kits from Pierce are designed to simplify the production of clenbuterol-BSA,clenbuterol-OVA,penicillm-BSA,penicillin-OVA and gentamycin-BSA,gentamycin-KLH. After Dialysis for 3 days at 4"C for desalting,the BSA conjugates were immunized to rabbits after mixing with the aluminum adjuvant. Blood samples were taken 7 days after the third vaccination. Serum samples were prepared and stored at -20 C.
Antibody titers were measured by indirect Enzyme-Linked Immunosorbant Assay (ELISA). The antigen (hapten-OVA or -KLH conjugates) was immobilized overnight at 4 C to the polystyrene microtiter plate. Unbound sites were blocked by 1% casein for 1 hr at 37C. Serial dilutions of anti-serum samples were added to the wells and allowed to bind for 2 hrs at 37C,followed by three times of washing. Goat anti-rabbit HRP-IgG conjugate (1:500) was added and incubated for 2 hrs at 37 C. After another cycles of washing,100 uL of the substrate o-phenylenediamine solution was added for color development at 37 C for 20 minutes. The reaction was stoped by addition of 50 uL of 2 M H2SO4. Absorbance was measured at 490 nm on a microplate-based ELISA reader. Antibody titers were approximated by plotting dilutions of the anti-sera against OD490nm.
The antibody titers of penicillin (Scheme 1) and clenbuterol (Scheme 2) are 1600 X and 400 X respectively while the titers of penicillin,clenbuterol and gentamycin in Scheme 3 are 400 X,400 X and 200 X respectively. We also compared the blocking effects of 1% ovoalbumin,1-3% gelatin,1% bovine serum albumin and 1% casein. Casein at 1% was
found the most satisfactory blocking solution.
Indirect competitive ELISA was examined for detection of clenbuterol resides in rabbit urine. Clebuterol-OVA conjugate was immobilized onto the microplate wells and unbound sites blocked by 1% casein. Affinity-purified antibody (10 x dilution) was added together with different concentrations of clenbuterol. The remaining procedures were same as above. Good linear relationship was found between the concentration of added clenbuterol and OD490nm values. This approach could detect as low as 10 ng/ml of clenbuterol residue in rabbit urine. Further efforts are needed to optimize the detecting system.
上述三种药物为小分子半抗原，缺乏免疫原性。将半抗原与合适的载体蛋白偶联后，半抗原可以成为全抗原并具有较好的免疫原性。碳二亚胺(EDC)是一种同型双功能交联剂，可以通过一步反应形成稳定的半抗原—载体复合物。免疫原EDC偶联试剂盒可用于克伦特罗、青霉素和庆大霉素的偶联。按照半抗原—载体EDC标准偶联程序，我们偶联了克伦特罗—BSA、克伦特罗—OVA、青霉素—BSA、青霉素—OVA、庆大霉素—BSA、庆大霉素—KLH。偶联物4℃透析3天后，用BSA偶联物加佐剂免疫新西兰白兔。从第三次免疫后7天心脏采血。
放血分离血清后，以间接ELISA测抗体效价，用OVA和KLH偶联物包被酶标板，1％酪蛋白封闭1小时，一抗(倍比稀释的相应血清)和酶标二抗(500×稀释的羊抗兔IgG/HRP)分别在37℃反应2小时。PBST洗3次后用OPD(邻苯二胺)显色，最后用2M硫酸终止反应，酶标仪测490nm OD值。根据OD值(Y-轴)和稀释度(X—轴)的曲线，找出免疫血清曲线中的拐点及其对应X-轴的数值确定抗体效价。方案一中青霉素抗体效价为1600×，方案二中克伦特罗抗体效价为400×。在方案三中，克伦特罗、青霉素和庆大霉素抗体效价分别为400×，400×和200×。在间接竞争ELISA和硝酸纤维素膜试验中，我们同时比较了1％OVA、1-3％明胶、
l％BSA的封闭效果，结果表明 1％酪蛋白是最优的封闭液。
我们用间接竟争ELISA对尿液中克伦特罗残留进行了模拟检测。方法是：用克
伦特罗－OVA 200 X稀释包被酶标板，l％酪蛋白封闭，然后用 10 X稀释的亲和层析
纯化兴奋剂抗体IgG和含有兴奋剂的样品同时竞争，最后加酶标二抗，OPD底物显
色。同时，用不同浓度的克伦特罗标准品同时进行检测，并制作参比曲线进行回归
分析。初步结果表明：间接竟争ELISA的检测下限可达到10ng／ffil。为进一步优化
并建立克伦特罗的ELISA检测方法奠定了基础。
Clenbuterol is a sympathomimetic agent with principally p-adrenergic activity and a selective action on p-receptors. It is used in veterinary medicine as bronchodilatator and tocolytic. It has also been illegally appled in animal feed additives as a repartitioning agent which would lead to resiudes in the animal products with the potential of food-borne poisoning. Gentamycin is active against many gram-positive and gram-negative bacteria. It is administered by intramuscularly or intravenously for general infections or orally for gastro-intestinal infection. It is also used intramammarily for bovine mastitis and topically in ocular and auricular infections. Improper use of the drug may result in residual problems because of its slow elimination. Penicillin G is inactivated by penicillinases and is unstable in gastric acid. It is often used for the treatment of infections by susceptible bacteria through parenteral application or intramammary infusion for bovine mastitis. Its reside in milk is of great concern
to the consumers for its potential of allergic reactions as well as to the dairy industry for its inhibitory effect on the starter cultures. Therefore,development of fast methods for detection of residues of these drugs in the foods of animal origins is of great public health significance and socio-economic importance.
The above three drugs are haptens. Their immunogenicity can be imporved by coupling them to suitable carrier protein molecules. N-ethyl-N'-[3-(dimethylamino)propyl] carbodiimide hydrochloride (EDC) is a bifunctional coupling reagent. It is efficient for one-step coupling to form a wide variety of hapten-carrier protein immunogens. Conjugation may occur at either the C- or N-terminal of the hapten or at any carboxyl- or amine-containing side chains. Imject Immunogen EDC Conjugation Kits from Pierce are designed to simplify the production of clenbuterol-BSA,clenbuterol-OVA,penicillm-BSA,penicillin-OVA and gentamycin-BSA,gentamycin-KLH. After Dialysis for 3 days at 4"C for desalting,the BSA conjugates were immunized to rabbits after mixing with the aluminum adjuvant. Blood samples were taken 7 days after the third vaccination. Serum samples were prepared and stored at -20 C.
Antibody titers were measured by indirect Enzyme-Linked Immunosorbant Assay (ELISA). The antigen (hapten-OVA or -KLH conjugates) was immobilized overnight at 4 C to the polystyrene microtiter plate. Unbound sites were blocked by 1% casein for 1 hr at 37C. Serial dilutions of anti-serum samples were added to the wells and allowed to bind for 2 hrs at 37C,followed by three times of washing. Goat anti-rabbit HRP-IgG conjugate (1:500) was added and incubated for 2 hrs at 37 C. After another cycles of washing,100 uL of the substrate o-phenylenediamine solution was added for color development at 37 C for 20 minutes. The reaction was stoped by addition of 50 uL of 2 M H2SO4. Absorbance was measured at 490 nm on a microplate-based ELISA reader. Antibody titers were approximated by plotting dilutions of the anti-sera against OD490nm.
The antibody titers of penicillin (Scheme 1) and clenbuterol (Scheme 2) are 1600 X and 400 X respectively while the titers of penicillin,clenbuterol and gentamycin in Scheme 3 are 400 X,400 X and 200 X respectively. We also compared the blocking effects of 1% ovoalbumin,1-3% gelatin,1% bovine serum albumin and 1% casein. Casein at 1% was
found the most satisfactory blocking solution.
Indirect competitive ELISA was examined for detection of clenbuterol resides in rabbit urine. Clebuterol-OVA conjugate was immobilized onto the microplate wells and unbound sites blocked by 1% casein. Affinity-purified antibody (10 x dilution) was added together with different concentrations of clenbuterol. The remaining procedures were same as above. Good linear relationship was found between the concentration of added clenbuterol and OD490nm values. This approach could detect as low as 10 ng/ml of clenbuterol residue in rabbit urine. Further efforts are needed to optimize the detecting system.
引文
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4 Yan Y T, McEllgoti M L. Multiple action of beta-2 agonists skeletal and adipose tissue. Journal of Biochemistry. 1989, 261: 1-10
5 L. O. Fiems, B. G. Cottyn, C. V. Boucque, in: L. O. Fiems, B. G. W. Cottyn, D. l. Demeyer (Eds.), Animal Biotechnology and the Quality of Meat Production, Elsevier, Oxford, 1991, 31-48.
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7 C. Ayotte, M. Donike, H. Geyer, A. Gotzmann, U. Mareck-Engelke, S. Rauth (Eds.), Proceedings of the 10th Cologne Workshop on Dope Analysis, Verlag Sport und Buch Strauss, Edition Sport, Cologne, 1993, 185-196.
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11 周光宏 韩正康。日粮中添加克伦特罗对肉鸭胴体组成的影响。中国畜牧杂志。1993，29(1)，14-16
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2 G. Engelhardt, Arzneiforschung (Drug Res.) 1976, 26, 1404.
3 K. Meinen, M. Rahn, M. Hermer, K. L. Rominger, T. Kanitz, Z. Geburtshilfe Perinatol. 1988, 192, 163-168.
4 Yan Y T, McEllgoti M L. Multiple action of beta-2 agonists skeletal and adipose tissue. Journal of Biochemistry. 1989, 261: 1-10
5 L. O. Fiems, B. G. Cottyn, C. V. Boucque, in: L. O. Fiems, B. G. W. Cottyn, D. l. Demeyer (Eds.), Animal Biotechnology and the Quality of Meat Production, Elsevier, Oxford, 1991, 31-48.
6 Anonymous, Musceling in on clenbuterol (Editorial), Lancet, 1992(340), 403.
7 C. Ayotte, M. Donike, H. Geyer, A. Gotzmann, U. Mareck-Engelke, S. Rauth (Eds.), Proceedings of the 10th Cologne Workshop on Dope Analysis, Verlag Sport und Buch Strauss, Edition Sport, Cologne, 1993, 185-196.
8 周光宏 孙晨华。克伦特罗对鸭的产肉性能和有关代谢指标的影响。南京农业大学学报。1991，14(4)，81-86
9 许志锁 田家良。克伦特罗对肉仔鸡生产性能及胴体品质的影响。吉林畜牧兽医。1991，13(5)，26-29
10 葛盛芳 陆天水。克伦特罗对红布罗肉鸡生长发育的影响。中国家禽。1992，(6)，25-27
11 周光宏 韩正康。日粮中添加克伦特罗对肉鸭胴体组成的影响。中国畜牧杂志。1993，29(1)，14-16
12 加大工作力度确保饲料安全——齐景发副部长在《饲料安全质量情况新闻发布会》上的讲话。饲料世界。2001，(4)，35-36
13 晓同。饲料安全 事关重大——农业部在京举行饲料安全质量情况新闻发布会。中国饲料。2001，(6)，2-4
14 陈茹 吴时清。克伦特罗及其检测技术。动植物检疫。1999，(2)，29-32
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