本文選題：鏈霉素 + 快速檢測(cè)�。� 參考：《中國(guó)計(jì)量學(xué)院》2012年碩士論文

【摘要】：鏈霉素（Streptomycin，SM）具有性質(zhì)穩(wěn)定、抗菌譜廣、生產(chǎn)工藝簡(jiǎn)單、療效好等特點(diǎn)，尤其對(duì)結(jié)核分枝桿菌有強(qiáng)大抗菌作用，是一種廣泛應(yīng)用于動(dòng)物疾病治療、預(yù)防的氨基糖苷類抗生素。但鏈霉素具有嚴(yán)重的耳毒性和腎毒性，它在動(dòng)物源性食品中的殘留已引起國(guó)內(nèi)外的普遍關(guān)注。開發(fā)快速、準(zhǔn)確、經(jīng)濟(jì)的檢測(cè)鏈霉素類抗生素殘留的方法在生產(chǎn)和生活中具有重要意義。 本文采取當(dāng)前普遍應(yīng)用的免疫檢測(cè)法——酶聯(lián)免疫檢測(cè)法（ELISA）和一種新型的檢測(cè)方法——基于核酸適配子快速檢測(cè)法，對(duì)食品中鏈霉素殘留檢測(cè)技術(shù)進(jìn)行研究，本研究獲得以下結(jié)果： 1. SM人工抗原的制備與鑒定：采用碳二亞胺法（EDC）和戊二醛法（GA）將分別與牛血清白蛋白（BSA）、卵清白蛋白（OVA）偶聯(lián)，制得免疫抗原（SM-cBSA）和包被抗原（SM-OVA）。經(jīng)紫外掃描和SDS-PAGE電泳鑒定，SM成功偶聯(lián)到蛋白質(zhì)載體上，偶聯(lián)比分別為7.6:1和17.7:1。 2. SM多克隆抗體和單克隆抗體的制備與鑒定：將SM-cBSA分別免疫新西蘭大白兔6只和Balb/C小鼠3只，用間接ELISA和間接競(jìng)爭(zhēng)ELISA測(cè)定抗血清效價(jià)和半數(shù)抑制濃度，并在小鼠多抗的基礎(chǔ)上篩選最佳融合小鼠，制備單克隆抗體。 經(jīng)8次免疫新西蘭大白兔，，獲得了兔多抗（rSM pAb），經(jīng)鑒定，其效價(jià)可達(dá)為1:8000，阻斷ELISA測(cè)定半數(shù)抑制濃度（IC50）為3.32ng/mL，與鏈霉素和雙氫鏈霉素的交叉反應(yīng)率分別為100%與120.78%，與其它化合物的交叉反應(yīng)率（CR%）＜0.1%。經(jīng)5次免疫Balb/C小鼠，獲得效價(jià)可達(dá)1:80000，IC50為3.44ng/mL的鼠多抗（mSM pAb）；根據(jù)間接ELISA結(jié)果，選擇2號(hào)小鼠細(xì)胞做細(xì)胞融合，篩得一株敏感特異的雜交瘤細(xì)胞1G1-E3，用體內(nèi)誘生腹水法制備鼠單抗（mSM mAb）；經(jīng)間接ELISA鑒定，細(xì)胞培養(yǎng)上清和腹水的效價(jià)分別為1:1280和1:1×106，阻斷ELISA測(cè)定其IC50為2.91ng/mL，與其他化合物的CR%均＜0.01%。本實(shí)驗(yàn)制備多抗和單抗均可用于SM殘留檢測(cè)的免疫學(xué)試驗(yàn)，但mSM mAb的性能更好。 3.應(yīng)用mSM mAb研制SM殘留快速檢測(cè)阻斷ELISA試劑盒（SM-Kit）。SM-Kit的標(biāo)準(zhǔn)曲線呈S型，線性檢測(cè)范圍為0.5~40.5ng/mL，IC50為3.60ng/mL；牛奶、蜂蜜樣品的平均添加回收率均在80%~120%之間；批內(nèi)和批間變異系數(shù)均小于15%；除與雙氫鏈霉素有交叉反應(yīng)，與其他化合物的交叉反應(yīng)率均小于0.01%，不同生物基質(zhì)對(duì)SM-Kit的檢測(cè)結(jié)果影響小；穩(wěn)定性試驗(yàn)表明SM-Kit在4℃可保存6個(gè)月以上。 4.建立了基于兩種基于適配子的SM殘留快速檢測(cè)方案，兩種方案的檢測(cè)IC50分別為160.83ng/mL和168.16ng/mL，初步證明方案的可行性，并為進(jìn)一步研究奠定良好基礎(chǔ)。
[Abstract]:Streptomycinia Streptomycinae (SMSM) is a kind of aminoglycoside antibiotic which is widely used in animal disease treatment and prevention because of its stable properties, wide antibacterial spectrum, simple production process and good curative effect, especially to Mycobacterium tuberculosis. However, streptomycin has serious ototoxicity and nephrotoxicity, and its residues in animal-derived foods have attracted widespread attention at home and abroad. It is of great significance to develop a rapid, accurate and economical method for detecting streptomycin residues in production and life. In this paper, the method of detection of streptomycin residues in food was studied by using Elisa (enzyme linked immunosorbent assay) and a new method of rapid detection based on aptamer of nucleic acid. The results of this study are as follows: 1. Preparation and identification of SM artificial antigen: the immune antigen (SM-cBSAA) and coated antigen (SM-OVA) were prepared by coupling bovine serum albumin (BSA) and ovalbumin (OVA) with carbodiimide (EDCA) and glutaraldehyde (GA) respectively. The results of UV scanning and SDS-PAGE electrophoresis showed that SM was successfully coupled to the protein vector, and the coupling ratios were 7.6: 1 and 17.7: 1, respectively. 2. Preparation and identification of SM polyclonal antibody and monoclonal antibody: 6 New Zealand white rabbits and 3 Balb/C mice were immunized with SM-cBSA. The antiserum titers and half inhibitory concentrations were determined by indirect ELISA and indirect competitive ELISA. The best fusion mouse was selected on the basis of mouse polyclonal antibody to prepare monoclonal antibody. New Zealand white rabbits were immunized for 8 times. Its titer was 1: 8 000, IC50 was 3.32 ng / mL, the cross reaction rates with streptomycin and dihydrostreptomycin were 100% and 120.78, respectively, and the cross reaction rate with other compounds was less than 0.1%. After 5 times Balb/C mice were immunized, the titer of mouse polyclonal antibody mSM pAbhe was 1: 80000 and IC50 was 3.44ng/mL. According to the results of indirect ELISA, mouse cell line 2 was selected for cell fusion. A sensitive and specific hybridoma cell line 1G1-E3 was screened, and mouse mSM mAb1 was prepared by in vivo induced ascites method, the titers of the supernatant and ascites were 1: 1280 and 1:1 脳 106, respectively, and the IC50 of blocking ELISA was 2.91 ng / mL, and the CR% of the other compounds was less than 0.01%. In this experiment, polyclonal antibodies and monoclonal antibodies can be used for the detection of SM residues in immunological tests, but the performance of mSM mAb is better. 3. The standard curve of SM-Kitt SM-Kit kit developed by mSM mAb was S-shaped, the linear detection range was 0.5 ~ 40.5ng / mL ~ (-1) IC50 was 3.60 ng / mL, the average recovery of milk and honey samples was between 80 ~ 120%. The coefficient of variation within and between batches was less than 15. The cross reaction rate with other compounds was less than 0.01 except for the cross reaction with dihydrostreptomycin. Different biological substrates had little effect on the detection results of SM-Kit. The stability test shows that SM-Kit can be stored for more than 6 months at 4 鈩

本文編號(hào)：1804874