【作者】 郝凯；

【导师】 过世东； 胥传来；

【作者基本信息】 江南大学 ， 水产品加工及储藏工程， 2006， 博士

【摘要】 甲砜霉素（Thiamphenicol,TAP）是一种广谱抗菌素,其结构类似于氯霉素,能抑制革兰氏阳性细菌、阴性细菌和一些厌氧微生物,广泛应用于水产养殖当中并且会造成残留。因此,研究和建立检测方法成了当务之急。本研究以多克隆抗体和免疫分析技术为基础,根据酶联免疫原理,建立了快速简便的水产品中的酶联免疫检测方法。另外,还建立了相应的仪器检测方法。首先通过混合酸酐法合成甲砜霉素与牛血清蛋白偶联物,构建甲砜霉素完全抗原。通过紫外扫描、SDS-聚丙烯凝胶电泳和免疫动物实验,证明了产物合成成功。通过免疫新西兰白兔,得到抗TAP的抗血清,抗血清效价高达1×106。此抗血清分别经过饱和硫酸铵沉淀法、离子交换法和反向免疫亲和层析法纯化后,得到单一特异性的抗体,平均亲和常数为7.8×10-9 mol/L。以此抗体为基础,设计了间接竞争两步酶联免疫检测方法,并优化了缓冲液浓度、pH、吐温20浓度等参数。此酶联免疫检测试剂盒检测限为0.06 ng/mL,血液和组织回收率为80～110%。通过重复性、检测灵敏度、准确性等指标来评估此方法,并与气相色谱法进行了比较,结果表明,此间接竞争酶联免疫检测方法各项参数均能满足实际检测的需要。建立和优化了甲砜霉素残留的高效液相色谱法（HPLC）、高效液相色谱-质谱法（HPLC/MS）和气相色谱-质谱法（GC/MS）的检测方法。方法的回收率分别为:HPLC为78.6～95.4%;HPLC/MS为72.6～99.8%;GC/MS为85.6～101.5%,相对偏差均小于10% ,检出限分别为:HPLC为8.3μg/kg;HPLC/MS为0.1μg/kg;GC/MS为0.1μg/kg。结果表明,这些方法都能满足实际检测的需要。以高效液相色谱法（HPLC）为检测方法,研究了甲砜霉素在鲈鱼血液和可食性组织里的消除规律。鲈鱼单次口服5 mg/kg（鱼体）和30 mg/kg（鱼体）的甲砜霉素药物后,药物在血液中的达峰时间分别为6小时和8小时,药物平均滞留时间的分别为11.1小时和11.9小时;甲砜霉素在鲈鱼血液中的经时过程符合一级吸收二项指数方程:C=10.812(e^-0.087t– e_-0.274t),药物在鲈鱼血液中的吸收半衰期为1.997 h,消除半衰期为5.589 h;连续5d口服15和30 mg/kg（鱼体）的甲砜霉素,药物的吸收速率是一个常数,它在血液中的消除速率也是一个常数,这和单次口服结果一致,其消除速率方程分别为C_血液=0.849e^-0.494t ;C肌肉=1.461e^-0.682t,药物在鲈鱼血液和组织中的的消除半衰期分别为1.016d和1.403d;通过EMEA/CVMP/036/95所规定的统计方法计算休药期,根据95%的忍受限和最大残留限量,确定了5d和6d分别作为服用低剂量和高剂量药物的休药期。更多还原

【Abstract】 Thiamphenicol （TAP） is a broad-spectrum antimicrobial agent, structurally similar to chloramphenicol, used widely in aquiculture. It is bacteriostatic for both Gram-positive and Gram-negative aerobes and for some anaerobes. Thus it is important to develop detection method for TAP analysis in fishery now. We have developed a rapid ELISA detection method according to ELISA theory; in addition, apparatus detection methods have been developed.The conjugation of TAP and bovine serum albumin （BSA） （TAP-BSA, the immunoantigen of TAP） was produced according to the method of isobutyl chloroformate. The conjugation was identified by ultraviolet scan, SDS-PAGE and animal immunization test. The results demonstrated that the conjugation was synthesized successfully.According to immune protocol, three New Zealand white rabbits were immuned by TAP-BSA. The serum antiserum titre is as highly as 1×106. After purified by saturated ammonium sulphate, ion exchange and immunoaffinity chromatography in turn, monospecific antibody was obtained and its average affinity constant is about 7.8×10-9 mol/L. Based on this purified antibody, an indirect competitive ELISA was developed. After optimized some parameters such as the pH and ionic strength of buffers, concentration of Tween-20 and so on, the minimum detection limit of this ELISA arrived 0.06 ng/mL, and the recovery body of fluid （urine and serum） and tissue is about 80 to 110 percent. Besides, the ELISA kit was evaluated through following items: reproducibility, sensitivity, precision, and accuracy. Moreover, it was compared with the gas chromatography. All the results convinced us that this ELISA KIT can be applied to actual detection of thiamphenicol.The methods for the analysis of TAP were developed and optimized by HPLC、HPLC/MS and GC/MS. The recovery of HPLC、HPLC/MS and GC/MS were78.6～95.4%, 72.6～99.8% and 85.6～101.5%respectively;and the limits of quantitation were 8.3μg/kg, 0.1μg/kg and 0.1μg/kg respectively .The RSD of the methods were less than 10%. The results demonstrated that these methods were satisfied the requirement of practice detection.We have studied the depletion rule of TAP in Lateolabras janopicus’s serum and edible tissues by HPLC method. After both 5 mg/kg and 30 mg/kg oral single-dose administration , the peak serum were obtained 6h and 8h,mean residence time were 11.1 h and 11.9 h;the plasma concentration-time course of thiamphenicol can be describe by a one-compartment open model : C=10.812(e^-0.087t– e^-0.274t); the elimination half-time and absorption half-time were 5.589 h and 1.997 h. After both 5 mg/kg and 30 mg/kg for consecutive 5 days oral administration , the absorbed fraction and elimination rate of TAP remain constant ,this result更多还原