【作者】 吴宁鹏；

【导师】 沈建忠；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2004， 硕士

【摘要】 常山酮（Halofuginone,Hal）是一种应用广泛的抗球虫药，以3mg／kg的浓度添加到饲料中用于预防和治疗家禽的球虫病。本研究合成了常山酮免疫原，制备出针对常山酮的特异性多克隆抗体，建立了鸡组织中常山酮残留检测的ELISA方法和HPLC方法，并研究了鸡组织中常山酮的残留消除。 本研究建立了鸡肝脏和肌肉组织中常山酮残留检测的ELISA方法。用三甲基硅烷咪唑（TMSI）将常山酮结构中的羟基选择性保护，在温和的条件下使常山酮结构中的氨基琥珀酰化。最后在酸性条件下水解保护基团，得到半抗原常山酮琥珀酸衍生物。以NHS活性酯法制备了常山酮免疫原和包被原，偶联比率分别为9∶1和5∶1。ELISA方法的线性检测范围为0.1ng／ml—100ng／ml，以50ng／g、100ng／g和500ng／g的浓度水平添加时，鸡肝脏组织的平均添加回收率范围为74.2％—96.8％，日内变异系数范围为3.8％—12.8％，日间变异系数范围为8.8％—15.9％：鸡肌肉组织的平均添加回收率范围为74.3％-90.0％，日内变异系数为2.6％-16.6％，日间变异系数在5.4％—6.8％。本方法在鸡肝组织和肌肉组织中的检测限分别为23ng／g和15ng／g。 本研究建立了鸡肝脏和肌肉组织中常山酮残留检测的HPLC方法。用胰蛋白酶水解组织样品，乙酸乙酯提取，0.125mol／L乙酸铵缓冲液反提，Oasis^? HLB柱净化，HPLC测定。以50ng／g、100 ng／g和500 ng／g浓度水平添加时，鸡肝脏组织的平均添加回收率范围在81.3％-93.3％之间，日内变异系数范围在1.5％-10.9％之间，日间变异系数范围在2.7％-6.1％之间：鸡肌肉组织的平均添加回收率范围在88.4％-103.4％之间，日内变异系数范围在1.4％-11.6％之间，日问变异系数范围在4.6％-12.6％之间。本方法在鸡肝脏和肌肉组织中的检测限分别为19ng／g和23ng／g。 本研究对ELISA方法和HPLC方法进行了比较。ELISA方法和HPLC方法测定鸡肝脏和肌肉组织中常山酮残留有良好的相关性。在添加回收试验中，两种方法在鸡肝脏和肌肉组织中测得常山酮的实测值的相关系数分别为0.9760和0.9344。以3mg／kg剂量的氢溴酸常山酮混饲给予AA肉鸡10天，停药第0天时。ELISA方法测得鸡肝脏组织中常山酮的残留量为105.2ng／g，HPLC方法测得鸡肝脏组织中常山酮的残留量为84.6ng／g；停药第3天后，常山酮的残留量均低于两种方法的检测限。在停药第0天时，鸡肌肉组织中常山酮的残留量就低于检测限。用ELISA方法和HPLC方法检测鸡肝脏和肌肉组织中常山酮残留的结果基本相符。更多还原

【Abstract】 Halofuginone (Hal) is a widely used anti-coccidiosis agent, which is usually used as a feed additive at the medicated concentration of 3mg/kg for the prevention and treatment of the coccidiosis in poultry. In this study, halofuginone was modified to be linked with carrier protein to obtain a multiclonal antibodies. An ELISA method and a HPLC method for the determination of halofuginone in chicken tissues were developed, and the depletion of halofuginone residue from chicken tissue was investigated.An ELISA method for determination of halofuginone residue in chicken liver and muscle tissues was developed. The hydroxyl group on halofuginone was protected by N-trimethysilylimidazole, and the amino moiety readily reacted with succinic anhydride to produce the monosubstituted acid succinamide. Finally the trimethyisis moiety was removed by hydrolysis in acid condition to give a succinic acid derivative of halofuginone. This compound was coupled to BSA and OVA by NHS ester procedure,and the coupling ratio were 9.5:1 and 5:1, respectively.The linear detection of the ELISA method ranged from 0.1 ng/ml to 100 ng/ml. In the chicken liver tissue fortified at 50ng/g, 100ng/g, 500ng/g , the average recoveries ranged from 74.2% to 96.8%, with the coefficients of variation within-day were in the range of 3.8%-12.8%, and the coefficients of variation between-days were in the range of 8.8%-15.9%. In the chicken muscle tissue fortified at 50ng/g,100ng/g, 500ng/g, the average recoveries ranged from 74.3% to 90.0%, with the coefficients of variation within-day were in the range of 2.6%-16.6%, and the coefficients of variation between-days were in the range of 5.4%-6.8%.The detection limits in chicken liver tissue and in chicken muscle tissue were 23ng/g and I5ng/g, respectively.A HPLC method for determination of halofuginone residue in chicken liver and muscle tissue was developed. The tissue samples were hydrolyzed with trypsin, extracted with ethyl acetate, and re-extracted with 0.125mol/Lacetamide buffer. The extracts were then subjected to Oasis HLB cartridge for purification, and the elutes were determined by HPLC. In the chicken liver tissue fortified at 50ng/g,100ng/g, 500ng/g, the average recoveries ranged from 81.3% to 93.3%, with the coefficients of variation within-day were in the range of 1.5%-10.9%, and the coefficients of variation in between-days were in the range of 2.7%-6.9%. In the chicken muscle tissue fortified at 50ng/g,100ng/g, 500ng/g, the average recoveries ranged from 88.4% to 103.4%, with the coefficients of variation within-day were in the range of 1.4%-11.6%, and the coefficients of variation between-days were in the range of 9.0%-12.6%. The method detection limits in chicken liver tissue and in chicken muscle tissue were 19ng/g and 23ng/g, respectively.In this study, the ELISA method vs HPLC method for the determination of the halofuginone in chicken liver and muscle were compared. The coefficients of correlation for the detection of halofuginone in chicken liver and muscle by two methods were 0.9760 and 0.9344, respectively. Broiler chickens (AA) were fed at a medicated concentration of 3mg/kg of halofuginone-HBr for 10 days. Atwithdrawal time of 0 day, the residue levels of halofuginone in broiler chicken liver tissues detected by ELISA method and HPLC method were 105.2ng/g and 84.6ng/g, respectively. At and after 3 day, the residues were not detected by ELISA method or HPLC method. In the broiler chicken muscles, the residues were not detected by two methods at withdrawal time of 0 day. The ELISA method and the HPLC method for the determination of the halofuginone i n chicken liver and in muscle correlated well.更多还原