【作者】 陈号；

【导师】 祁克宗；

【作者基本信息】 安徽农业大学 ， 基础兽医学， 2008， 硕士

【摘要】 青霉素类药物是一类高效广谱抗生素,在兽医临床上广泛应用于治疗细菌感染引起的畜禽肠道、呼吸道等疾病。由于能引起人的过敏反应和容易诱导细菌产生耐药性等原因,致使许多国家对动物用青霉素类抗生素和在食品中的残留进行了严格监控。又因青霉素类抗生素使用范围广、使用量大,其在动物性食品中的残留令人关注。因此,建立高效、灵敏的青霉素类药物残留检测方法在动物源性食品安全方面具有非常重要的意义。本研究以青霉素类药物母核6-氨基青霉烷酸（6-APA）偶联牛血清蛋白（BSA）和卵清白蛋白（OVA）分别合成两种免疫抗原和包被抗原;制备的高效价的抗6-氨基青霉烷酸（6-APA）的抗血清经活性检测后被纯化;纯化的IgG偶联CNBr活化的Sepharose 4B制备免疫亲和层析柱（IAC）,初步测定了免疫亲和层析柱对青霉素G的柱容量及添加回收率。结果如下:1.青霉素类药物族特异性人工抗原的合成及鉴定采用戊二醛法,将6-氨基青霉烷酸（6-APA）分别与牛血清蛋白（BSA）和卵清白蛋白（OVA）偶联,分别制备免疫抗原6-APA-BSA和包被抗原6-APA-OVA。合成的人工抗原经紫外扫描和红外扫描鉴定表明6-APA与蛋白载体偶联成功,且经紫外光谱扫描法计算出6-APA-BSA、6-APA-OVA的半抗原结合比分别为13.53、8.25。2.青霉素类药物族特异性抗体的制备用6-APA-BSA常规免疫实验兔制备抗血清。建立检测青霉素药物族特异性抗血清效价的间接ELISA法,其中包被抗原6-APA-OVA工作浓度为5μg/ml,阴性血清的工作浓度为4000倍稀释,酶标二抗的最佳稀释倍数为10000倍稀释。利用建立的间接ELISA法检测抗体效价,制备的三个抗血清的效价分别为5.12×10⁵、6.4×10⁴、3.2×10⁴。采用间接竞争ELISA法检测抗体的特异性。青霉素G、氨苄西林、邻氯西林、苯唑青霉素、阿莫西林的交叉反应率在13.5～101%之间,磺胺嘧啶钠、恩诺沙星、氯霉素、土霉素、林可霉素等非青霉素类抗菌药物的交叉反应率皆小于0.01%。说明抗血清族特异性良好;批内变异系数都在5.0%以内,批间变异系数都在10%以内。3.青霉素类药物族特异性IAC柱的制备及初步评价将制备的青霉素类药物族特异性抗血清通过硫酸铵粗提和DEAE-Sephadex A 50凝胶层析柱进一步纯化IgG,经10%PAGE-SDS电泳鉴定为目标蛋白。将所纯化蛋白与CNBr活化的Sepharose 4B琼脂糖偶联,偶联率为90.58%,装柱制备青霉素类药物族特异性IAC柱。采用HPCE检测方法对IAC柱柱效进行初步评价。建立青霉素G的HPCE检测标准曲线,青霉素G标准品浓度峰面积在1～100μg/mL范围内线性良好,标准曲线回归方程为y=468.66x -8.4299,R²=0.9971。峰面积日内、日间平均变异系数均低于4%;回收率在99.3～101.5%之间。IAC柱对青霉素G的动态柱容量为1433.8 ng ml^-1·gel,绝对柱容量为24.42ng/mg IgG。在一星期内重复使用5次,青霉素G的动态柱容量仍能达到600 ng ml^-1·gel,并且不影响使用。青霉素G样品添加回收率为94.67%～76.38%,变异系数5.67%～15.91%。淋洗液选用甲醇-乙腈-醋酸/醋酸钠缓冲液（60:25:15）为淋洗液。方法在准确度和精密度上均达到了残留分析的要求。更多还原

【Abstract】 Penicillins,the high efficient antibiotics,are widely used in veterinary medicine for animal bacterial infections which caused by intestinal and respiratory diseases,for producting resistant strains and allergic reactions,many countries monitor penicillins strictly which used in animals and residued in food.As a class of antibacterial drugs widely and the biggest used in veterinary medicine,the residue of penicillins was a very outstanding problems to the food safty. It is very necessary for the developments of adequate analytical procedures to determine the residues in products of animal Origin to penicillin drugs.Group-specificity synthetic antigens against penicillins were prepared by choosing the 6-Aminopenicillanic Acid,are immunoantigens and coating antigens.and polyclonal antiserums were prepared by immunization of rabbits.Then,Biological activities of the antiserums were determined and IgG was isolated and purified from the antiserum.Then,which was coupled to Sepharose 4B activated by CNBr to develope immonoaffinity chromatography column,and the preliminary study on its Evaluation has been carried out.The results are as follows.1.The Preparetion and identifition of artificial Group-specificity antigenics against penicillins6-Aminopenicillanic Acid was coupled to BSA and OVA by a glutaraldehyde method, 6-APA-BSA was immunoantigen and 6-APA-OVA was artificial coating antigen.The synthetic antigens were scanned and identied by UV spectrum and Fourier transform infrared spectrometry（FTIR）.which showed that the couplings were successed and the rates of coupled antigens were 13.53,8.25,calculated by UV method.2.The Preparation of Group-specificity polyclonal antibody against penicillins and Detecting for immunological activitiesGroup-specificity polyclonal antibodies against penicillins were prepared by immunization of rabbits with artificial 6-APA-BSA.Indirect ELISA was established to screen antiserums,by optimization,optimal reactive condition of I-ELISA for detecting coated with artificial 6-APA-OVA was determined as follows,the quantity of antigen coated was 5μg/ml,the positive serum dilution and the negative control were both 1:4000,and the dilution of IgG—HRP was 1:10000.and the Titre of three antiserum were 5.12×10⁵、6.4×10⁴、3.2×10⁴ in the method of I-ELISA.In constrast,Cross-reactivity rates of Penicillin G,Ampicillin,Cloxacillin,Oxacillin, Amoxicillin were in the range from 13.5%to 101%.The rates of Other antibiotics （Enrofloxacin,Sulfadiazine,Chloramphenicol,Terramycin and Lincomycin） were all very low.The coefficients of variation for the Ci-ELISA were all low 5%in intra-assay and 10%in inter-assay.3.The preparation for Immunoaffmity Column against penicillins and The dynamic capacity of IAC was preliminarily evaluatedIgG was isolated and purified from the antiserum by a series procedure of precipitation with saturated ammonium sulfate and chromatography with DEAE-Sephadex A-50.After SDS-PAGE electrophoreisis,purified IgG was reach the electrophoretic purity.The purified IgG was coupled to sepharose 4B activated by CNBr to prepare multi- immuno -affinity chromatography columns.the rate of coupling was 90.58%.The regression equation of Penicillin G in HPCE was y = 468.66x-8.4299,The liner range was 1～100μg·ml^-1,and the coefficient was 0.9971.The recovery is within 99.3%to 101.5%. The relative standard deviation（RSD） of within day and between day were within 4%,The dynamic capacity of the resulting immunosorbents was 1433.8 ng ml^-1 gel,and the absolute capacity of IAC was 24.42 ng/mg IgG,respectively.The recovery of penicil -lin G was 94.67%～76.38%,with CV of 5.67%～15.91%.and the eluent was Methanol- Acetonitrile-Acetate /acetate buffer（60:25.15）.The Accurate and Precision were all meet the requireements of the detection to the residue of penicillins.更多还原