同步检测头孢类抗生素与链霉素胶体金免疫层析法的建立

发布时间：2018-01-03 05:28

本文关键词：同步检测头孢类抗生素与链霉素胶体金免疫层析法的建立　出处：《安徽农业大学》2012年硕士论文　论文类型：学位论文

【摘要】：头孢类抗生素和链霉素都具有抗菌谱广、杀菌能力强的特点，因而在兽医临床上被广泛应用于治疗禽畜细菌感染等疾病，这两类药物又经常联用以增强治疗效果，这样增加禽畜机体内药物的残留，通过食物链蓄积在人体内，给人类的健康造成严重的危害。因此，为减少这两类药物残留对人健康的危害，必须要加强对动物源性食品中这两类药物残留的检测，建立一种灵敏度高，操作简便的方法具有十分重要的意义。 1.人工抗原的合成及多克隆抗体的制备选取7-氨基头孢烷酸（7-ACA）、头孢拉定(CED)、头孢氨苄(CEX)采用戊二醛法分别与牛血清蛋白（BSA）相结合制备免疫抗原、与卵清白蛋白（OVA）偶联制备包被抗原。采用化学交联法将链霉素（SM）分别与BSA和OVA偶联制备免疫抗原SM-BSA与包被抗原SM-OVA。人工抗原经紫外扫描和SDS-PAGE蛋白电泳鉴定阳性，紫外光谱扫描法计算7-ACA-BSA、CED-BSA、CEX-BSA以及SM-BSA结合比分别为14.34、15.62、12.29、15.21。用此四种抗原免疫新西兰白兔得到抗血清，经过ELISA测定抗血清效价，，抗7-ACA-BSA IgG、抗CED-BSA IgG、抗CEX-BSAIgG以及抗SM-BSAIgG的效价分别为：12800、64000、64000、64000。结果表明：偶合成功，结合比恰当。所得抗血清效价符合实验要求。 2.抗体交叉反应性的测定经过优化建立了间接ELISA的检测方法，通过交叉反应性的测定筛选出抗CEX-BSAIgG对头孢类药物有较好的交叉反应性，交叉反应率达到53.4%~101%，而抗SM IgG对硫酸链霉素及双氢链霉素有较好的交叉反应性交叉反应率达到85%~101%。链霉素与头孢类抗生素之间的交叉反应率却小于0.1%。因此选择抗CEX-BSA IgG为研制检测头孢类抗生素试纸条的抗体。 3.金标抗体的制备及单一检测试纸条的制备将纯化后的抗体蛋白与制备的粒径为18nm的胶体金相结合制备成金标蛋白，并且对试纸条的组成材料和各条件进行优化后，将试纸条各组分按顺序组装好，并对试纸条性能进行测试，头孢氨苄检测试纸条在PBS和牛奶基质中对头孢氨苄的最低检测限分别为10ng/mL和80ng/mL，在牛奶基质中对其他头孢类抗生素的最低检测限都在500ng/mL之内；链霉素检测试纸条在PBS和牛奶基质中对链霉素的最低检测限分别为150ng/mL和400ng/mL，牛奶基质中对硫酸链霉素和双氢链霉素的最低检测限在1000ng/mL以内。表明研制的试纸条的特异性和灵敏度等性能基本符合检测要求。 4.双元检测试纸条的组装及性能测定将头孢类抗生素及链霉素双元检测免疫层析试纸条组装好，其在PBS及牛奶基质中对头孢氨苄的最低检测限分别为20ng/mL和100ng/mL，对链霉素的最低检测限分别为200ng/mL和500ng/mL。在牛奶中对其他β-内酰胺类抗生素的最低检测限都在1000ng/mL之内；对双氢链霉素和硫酸链霉素的检测最低检测线都在1000ng/mL之内。表明研制的双元检测试纸条基本符合生产中用于检测这两类药物残留的要求。
[Abstract]:Both cephalosporins and streptomycin have wide antibacterial spectrum and strong bactericidal ability, so they are widely used in veterinary clinic to treat livestock bacterial infection and other diseases. These two kinds of drugs are often combined to enhance the therapeutic effect, so as to increase the residue of drugs in the livestock body, through the food chain accumulation in the human body, causing serious harm to human health. In order to reduce the harm to human health caused by these two kinds of drug residues, it is necessary to strengthen the detection of these two kinds of drug residues in animal-derived food. It is of great significance to establish a method with high sensitivity and simple operation. 1. Synthesis of artificial antigen and preparation of polyclonal antibody. The immune antigens were prepared by glutaraldehyde (Glutaraldehyde) method and bovine serum protein (BSA), respectively, in which 7-ACAA, CEDX, CEDX and CEDX were combined with bovine serum protein (BSA) by glutaraldehyde (Glutaraldehyde) method. The coated antigen was prepared by coupling with ovalbumin ovalbumin (OVA). Streptomycin (SMN) was prepared by chemical crosslinking method. Immune antigen SM-BSA and coated antigen SM-OVA were prepared by coupling with BSA and OVA respectively. The artificial antigen was identified by UV scanning and SDS-PAGE protein electrophoresis. The binding ratios of 7-ACA-BSAD-BSAD-BSAX-BSA and SM-BSA were 14.34 ~ 15.62 ~ 12.29, respectively. 15.21.The antiserum was obtained by immunizing New Zealand white rabbits with these four antigens. The titer of antiserum was determined by ELISA, and the antibody against 7-ACA-BSA IgG and anti-#en1# IgG was also determined. The titers of anti CEX-BSAIgG and anti SM-BSAIgG were: 1: 12800, 644000, 644000, respectively. The results showed that the coupling was successful. The titer of the antiserum obtained met the requirements of the experiment. 2. Determination of cross-reactivity of antibodies The indirect ELISA detection method was established by optimization, and the cross reactivity of anti CEX-BSAIgG to cephalosporins was screened out by cross reactivity determination. The cross reaction rate was 53.4% and 101%. And anti-SM. The cross reaction rate of IgG to streptomycin sulfate and dihydrostreptomycin reached 8510 1. The cross reaction rate between streptomycin and cephalosporins was less than 0.1. Anti CEX-BSA. IgG was used to develop an antibody for the detection of cefosporins. 3. Preparation of gold labeled antibody and preparation of single test strip The purified antibody protein was combined with the colloidal metal with the diameter of 18 nm to prepare the gold standard protein, and the composition and conditions of the test strip were optimized. The test strips were assembled in sequence, and the performance of the test strips was tested. The minimum detection limits of cefalexin in PBS and milk substrates were 10 ng / mL and 80 ng / mL, respectively. The minimum detection limit for other cephalosporins in milk substrates was within 500 ng / mL; The minimum detection limits for streptomycin in PBS and milk substrates were 150 ng / mL and 400 ng / mL, respectively. The minimum detection limit for streptomycin sulfate and dihydrostreptomycin in milk matrix is less than 1000 ng / mL, which indicates that the specificity and sensitivity of the test strip are basically in line with the detection requirements. 4. The assembly and performance measurement of duplex test strip The cefalexin and streptomycin double component immunochromatographic test strips were assembled. The minimum detection limits of cefalexin in PBS and milk matrix were 20 ng / mL and 100 ng / mL, respectively. The minimum detection limits for streptomycin were 200 ng / mL and 500 ng / mL, respectively. The minimum detection limits for other 尾 -lactam antibiotics in milk were within 1000 ng / mL. The minimum detection lines for both dihydrostreptomycin and streptomycin sulfate are within 1000 ng / mL. It is shown that the developed double-component test strip basically meets the requirements for the detection of the residue of these two kinds of drugs in production.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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