本文選題：鏈霉素 + 殘留�。� 參考：《天津科技大學(xué)》2013年碩士論文

【摘要】：本文針對動物源性食品和飼料中鏈霉素殘留潛在的安全問題,研究建立了間接競爭酶聯(lián)免疫檢測方法(icELISA)。本研究采用環(huán)氧樹脂法將鏈霉素(STR)與牛血清蛋白(BSA)偶聯(lián)制得免疫原,免疫新西蘭白兔,得到鏈霉素多克隆抗體。通過鏈霉素(STR)與O-羧甲基羥胺(CMO)連接,引入羧基活性基團(tuán)成功得到半抗原STR-CMO,并利用碳二亞胺法將半抗原與卵清蛋白(OVA)偶聯(lián)制得包被抗原。通過優(yōu)化包被原包被量,抗體稀釋倍數(shù),離子強(qiáng)度,pH值等參數(shù),建立了間接競爭酶聯(lián)免疫檢測方法。最終選擇抗體稀釋倍數(shù)為1：1000；包被抗原的包被量為0.5 μg/well;封閉液為0.5%脫脂乳粉/PBS；樣品稀釋液為0.01 mol/L PBS,pH 7.4;抗體稀釋液為0.1%BSA/PBS。所建立的icELISA方法靈敏度(IC50)為2.00±0.24 ng/mL,檢測限(IC15)為0.24±0.08 ng/mL。所制備的鏈霉素抗體對雙氫鏈霉素的交叉反應(yīng)為95.50%,對其他六種結(jié)構(gòu)類似的氨基糖苷類抗生素沒有交叉反應(yīng),證明此抗體具有很高的特異性。選取了雞肉、雞肝、豬肉、豬肝、豬腎、魚肉、蝦肉、牛奶、雞飼料和豬飼料作為樣品進(jìn)行添加回收實(shí)驗(yàn)。樣品在15ng/g、30ng/g、120ng/g、300ng/g、600ng/g五個添加水平上的回收率在71.32-106.94%之間,板內(nèi)及板間變異系數(shù)均小于18%�；厥章实姆讲罘治霰砻�,除肝臟樣品外,對于不同的鏈霉素殘留量以及不同類型的檢測樣品,方法的回收率之間沒有顯著性差異。通過與商品化試劑盒進(jìn)行比較,二者的檢測結(jié)果具有較好的相關(guān)性,線性相關(guān)系數(shù)R2為0.9854,且本實(shí)驗(yàn)所建立的方法針對動物組織樣品的檢出限優(yōu)于商品化試劑盒。說明本方法具有很好的準(zhǔn)確度和廣泛的應(yīng)用性。
[Abstract]:An indirect competitive enzyme-linked immunosorbent assay (Elisa) for the detection of streptomycin residues in animal derived food and feed was developed in this paper. In this study, the immunogen was prepared by coupling streptomycin (STR) with bovine serum protein (BSA) by epoxy resin method. New Zealand white rabbits were immunized with streptomycin polyclonal antibody. The hapten STR-CMOs were successfully obtained by linking streptomycin (STR) with O-carboxymethyl hydroxylamine (CMO), and the coated antigen was prepared by coupling hapten with ovalbumin (OVA) by carbodiimide method. An indirect competitive enzyme-linked immunosorbent assay (Elisa) was established by optimizing the parameters such as the original coating volume, the dilution multiple of antibody and the pH value of ionic strength. Finally, the dilution ratio of antibody was 1: 1000, the coating amount of antigen was 0.5 渭 g / wellwell, the blocking solution was 0.5% defatted milk powder / PBS, the sample diluent was 0.01 mol/L PBSs pH 7.4, and the antibody dilution solution was 0.1 BSA-PBSs. The sensitivity of the icELISA method is 2.00 鹵0.24 ng / mL, and the detection limit is 0.24 鹵0.08 ng / mL. The cross reaction of streptomycin antibody against dihydrostreptomycin was 95.50, and there was no cross reaction with other six similar aminoglycoside antibiotics, which proved that the antibody had a high specificity. Chicken, chicken liver, pork, pig liver, pig kidney, fish meat, shrimp meat, milk, chicken feed and pig feed were selected as samples. The recoveries of the samples were 71.32-106.94% at the five additive levels of 15ng / g ~ 30ng / g ~ 120ng / g ~ 300ng / g / g ~ 600ng / g, and the coefficients of variation were less than 18% both intra- and inter-plate. The variance analysis of recovery showed that there was no significant difference between the recovery rates for different streptomycin residues and different types of samples except liver samples. The linear correlation coefficient R2 was 0.9854, and the detection limit for animal tissue samples was better than that of commercial kit. It shows that this method has good accuracy and wide application.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R155.5

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相關(guān)期刊論文 前2條

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本文編號：1870027