【作者】 彭池方；

【导师】 金征宇；

【作者基本信息】 江南大学 ， 食品科学， 2008， 博士

【摘要】 乙酰孕激素是一类人工合成的甾体药物,通过调节细胞的生长和分化功能,对人体及动物的生理功能具有十分重要的作用。在动物养殖中,它们广泛应用于动物的发情控制和调节、促生长和兽医临床。然而乙酰孕激素残留通过动物食品进入人体,累积作用可能会产生致畸性、致癌性等危害,因此,许多国家和地区都对严格控制乙酰孕激素用于动物生产中的促生长应用。目前对动物源食品中AGs残留进行监测是确保食品安全和人类健康的最后一道防线。本研究制备了AGs多克隆抗体,建立了可检测AGs多残留的酶联免疫分析法（ELISA）、液相色谱串联质谱法（LC-MS/MS）以及醋酸甲羟孕酮的毛细光电泳免疫分析方法（CEIA）,为监测动物源食品中AGs药物的残留提供了技术保障。制备并鉴定了半抗原3-羧甲基肟基-6α-甲基-17α-羟基孕甾-6-烯-20-酮醋酸酯（3-CMO-MPA）以及其与牛血清白蛋白的偶联物（3-CMO-MPA-BSA）,以此人工抗原免疫白兔,得到一种多克隆抗体的亲和常数为8.1×10⁸ L/mol。基于此抗体建立的ELISA方法,对MPA检测的IC₅₀值为5.8μg/L。交叉反应分析表明,此抗体能特异识别四种乙酰孕激素,醋酸甲羟孕酮（MPA）、醋酸氯地孕酮（CMA）、醋酸甲地孕酮（MEGA）及17α-羟基孕酮醋酸酯（HPA）,可应用于乙酰孕激素多残留分析的ELISA试剂盒开发。通过考察反应条件对ELISA方法灵敏度的影响,确定了ELISA方法的反应条件为:0.01 mol/L、pH7.5吐温-20含量为0.02%的磷酸盐缓冲液,37℃下竞争反应60min,酶标记物的反应30 min,显色30 min。合成了三个异源包被抗原,发现在采用3-羧甲基肟基-17α-羟基孕酮醋酸酯与卵清白蛋白偶联物（3-CMO-HPA-OVA）作为包被抗原时,比原同源方法更灵敏更高、群选择性更好。此异源免疫分析方法对醋酸甲羟孕酮（MPA）、醋酸氯地孕酮（CMA）、醋酸甲地孕酮（MEGA）和17α-羟基孕酮醋酸酯（HPA）的IC₅₀值分别为1.8、4.5、2.9、2.5μg/L;对MPA的灵敏度为0.05μg/L,线性范围为0.1-15μg/L,对CMA、MEGA和HPA的灵敏度为0.1μg/L,线性范围为0.2-30μg/L;对MPA、CMA、MEGA和HPA其交叉反应率分别为100%、40%、62%和72%。脂肪样品经过液液萃取、冷冻去脂、固相萃取净化,ELISA检测MPA、CMA、MEGA及HPA的检测限分别为0.6、1.2、1.2和1.0μg/kg。对空白猪脂肪中添加1-10μg/kg乙酰孕激素时,回收率在62.2-83.3%范围。肌肉、肝脏、肾脏等样品经过液液萃取、固相萃取净化,ELISA检测MPA、CMA、MEGA和的HPA的检测限在0.3～1.0μg/kg范围;在空白肌肉、肝脏和肾脏中添加1-10μg/kg乙酰孕激素时,回收率范围在62.2-91.5%范围内。饲料样品经过液液萃取、冷冻去脂,ELISA检测MPA的CMA、MEGA及HPA的检测限分别为2.0、3.0、3.0和2.5μg/kg。。在空白饲料中添加1-10μg/kg乙酰孕激素时,回收率在72.1-95.4%范围内。以0.8 mg/kg体重剂量连续喂服白兔5天,停药7天后,白兔的腿肌肉和肝脏中MPA平均残留量分别为7.2和5.6μg/kg。ELISA方法与气相色谱-质谱联用方法（GC-MS）确证方法的检测结果相关性良好。说明该试剂盒能满足乙酰孕激素残留筛选检测的要求。试剂盒保存实验证明,该试剂盒可在4℃下保存六个月后,或37℃下保存一周后,仍满足使用要求。采用溶剂提取、固相萃取净化等样品前处理方法,建立了脂肪中五种乙酰孕激,包括MPA、CMA、MEGA、HPA及甲烯雌醇醋酸酯（MLA）的液相色谱-串联质谱（LC-MS/MS）确证分析方法,该方法对五种乙酰孕激素的检出限为0.2～0.3μg/kg,定量限为0.5μg/kg。在空白猪脂肪样品中以0.5、1.0和5.0μg/kg三个水平添加时,五种乙酰孕激素的回收率范围在60.5～84.1%之间,RSD范围为8.9～16.8%之间。该方法能满足目前五种乙酰孕激素残留确证分析检测的要求。合成和鉴定了MPA荧光素标记物,通过毛细管电泳免疫分析（CEIA）激光诱导荧光（LIF）检测证明其具有免疫反应活性。在熔融毛细管中,建立了基于CE-LIF的竞争免疫分析方法。以0.2 mol/L Tis/0.1 mol/L硼酸缓冲（pH 9.0）和20 mM十二烷基磺酸钠（SDS）为运行缓冲液,检测脂肪中MPA的检测限为2.5μg/kg,对10μg/kg添加水平的样品,回收率为88%,变异系数为5.8%。此CEIA方法与ELISA方法相比,具有分析时间短、溶剂消耗小、样品前处理简单等优点。更多还原

【Abstract】 Acetylgestagens are a class of synthetic steroid drugs and very important for the physiological function of human and animal by regulate the growth and differentiation of cells.They are highly useful in oestrus regulation,growth-promoting and veterinary practice.However,the residues of AGs in animal derived food will represent potential hazard to human health,such as teratogenic and carcinogenic effect,by entering human body and accumulation.Thus,the use as growth-promoter has been prohibited in livestock by many countries.In this work,anti-acetylgestagens polyclonal antibody was produced and an ELISA method was developed for screening multi-residues of acetylgestagens.A LC-MS/MS method was also established for confirming them in animal fat tissues.Meanwhile,a CEIA with laser-induced fluorescence detection method was discussed.A hapten（3-CMO-MPA） and a conjugate of the hapten and bovine serum albumin were synthesized and identification.An antibody was produced by immunizing New Zealand rabbits with the conjugate,whose affinity constant was 8.1×10⁸ L/mol.The ELISA method against MPA was developed and the IC₅₀ was 5.8μg/L.The antibody was found specific for four acetylgestagens by analyzing cross-activity.The reaction conditions were selected by measuring the effect of them on the ELISA sensitivity.The buffer was 0.01 mol/L PBS（pH 7.5） with 0.02%Tween-20, the incubation time was 60 min at 37℃,the reaction time of enzyme labels was 30 min and the color developing time was 30 min.Three heterologous coating antigens were investigated after they were prepared.The conjugate of 3-CMO-HPA and ovalbumin（3-CMO-HPA-OVA） was found better than the homologous antigen （3-CMO-MPA-OVA） as coating antigen.The new heterologous ELISA method was more sensitive and class-selective than the homologous.The IC₅₀ for MPA,CMA, MEGA and HPA were 1.8,4.5,2.9 and 2.5μg/L,respectively.The sensitivity for MPA was 0.05μg/L and for CMA,MEGA and HPA 0.1μg/L.The linear range was 0.1-15μg/L for MPA and 0.2-30μg/L for CMA,MEGA and HPA,respectively.The cross-activities for MPA,CMA,MEGA and HPA were 100%,40%,62%and 72%, respectively.Pork fat samples were extracted with solvent,de-fatted through freezing and cleaned up with SPE column.The limits of detection（LODs） of the above ELISA method were 0.6,1.2,1.2 and 1.0μg/kg for MPA,CMA,MEGA and HPA, respectively.Mean recoveries were in the range 62.2-83.3%when blank pork fat samples were fortified range from 1 to 10μg/kg.Muscle,liver and kidney samples were extracted with solvent and cleaned up with SPE column.The LODs of the ELISA method were in the range 0.3-1.0μg/kg for the AGs.Mean recoveries were in the range 62.2-91.5%when blank samples were fortified range from 1 to 10μg/kg. After extracted with solvent and de-fatted through freezing,the LODs for in feed samples were 2.0,3.0,3.0 and 2.5μg/kg MPA,CMA,MEGA and HPA,respectively. Fortification at the range 1-10μg/kg,mean recoveries were 72.1-95.4%. Rabbits were administrated with MPA at 0.8 mg/kg.bw successively for 5 days and then withdrawl for 7 days.Then they were slaughtered and the incurred samples, muscle,liver and kidney,were obtained.The mean MPA residue in muscle and liver samples were 7.2 and 5.6μg/kg,respectively.The results by ELISA method had good linear correlation with GC-MS.These showed that the method could satisfy the requirements of AGs screening.The stability of the kits was investigated,and the results showed that they still kept good performance for application after they were stored for 6 months at 4℃or one week at 37℃.After solvent extraction and SPE clean-up,fat samples were detected by liquid chromatography tandem mass spectrometry.A confirming method for five AGs was established,the LODs for five AGs were verified from 0.2 to 0.3μg/kg and the limits of quantification（LOQ） were 0.5μg/kg.When blank fat samples were fortified AGs at 0.5,1.0 and 5.0μg/kg levels,the mean recoveries were in the range 60.5-84.1%and the relative standard deviation（RSD） were in the range 8.9-16.8%.A fluorescein label of MPA was synthesized and the immune reaction activity of the label was identified by capillary electrophoresis immunoassay（CEIA） with laser-introduced fluorescence（LIF） detector.The separation buffer was 0.2 mol/L Tis/0.1 mol/L Boric acid buffer（pH 9.0） containing 20 mM sodium dodecylbenzene sulfonate（SDS）.The LOD for MPA in fat was 2.5μg/kg,and mean recovery was 88%with RSD 5.8%at 10μg/kg fortification level.Compared with the above ELISA method,the CEIA could be applied with less solvent consumption after easier sample pretreatment.更多还原