發(fā)布時(shí)間：2018-05-29 18:28

  本文選題：三聚氰胺 + 單克隆抗體��； 參考：《重慶理工大學(xué)》2010年碩士論文

【摘要】： 三聚氰胺(Melamine,Mela)是一種化工原料,由于其含氮量高達(dá)66.6 %,常被非法添加到牛乳及飼料中,嚴(yán)重威脅人類和動(dòng)物的生命安全。目前的檢測方法大多需要專門的儀器設(shè)備并且操作繁瑣,已無法滿足現(xiàn)場快速檢測的要求。因此,研究和建立一種方便、安全和快速的檢測方法意義深遠(yuǎn)。本實(shí)驗(yàn)利用蛋白質(zhì)連接技術(shù)合成了人工抗原,通過雜交瘤技術(shù)制備了抗Mela單克隆抗體,利用金標(biāo)免疫層析技術(shù)成功研制了Mela金標(biāo)免疫層析快速檢測試劑盒,該試劑盒的靈敏度、特異性、穩(wěn)定性等均符合檢測要求,其規(guī)�；Y選特點(diǎn)更適用于常規(guī)檢測需要,具有重要的現(xiàn)實(shí)意義和極高的商業(yè)價(jià)值。 首先以多元酸酐與混合酸酐聯(lián)用法將半抗原Mela分別與載體牛血清白蛋白(BSA)和卵清白蛋白(OVA)偶聯(lián)制成人工抗原,以Mela-BSA免疫BALB/c小鼠,免疫小鼠脾細(xì)胞與SP2/0細(xì)胞進(jìn)行融合,獲得雜交瘤細(xì)胞。以Mela-OVA作為包被抗原用ELISA方法對(duì)雜交瘤細(xì)胞進(jìn)行篩選,采用有限稀釋法,經(jīng)過對(duì)雜交瘤細(xì)胞的克隆和亞克隆,獲得1株能穩(wěn)定分泌抗Mela單克隆抗體的雜交瘤細(xì)胞株H2C6。以間接ELISA方法測定腹水單抗效價(jià)為1:204800,免疫層析實(shí)驗(yàn)(GICA)鑒定單抗與Mela特異性結(jié)合;H2C6單抗Mela的半數(shù)抑制濃度(IC50)為4.15μg/mL,與三聚氰酸、氯霉素、青霉素、鏈霉素、磺胺類藥物等沒有交叉反應(yīng);該細(xì)胞株體外多次傳代培養(yǎng)和反復(fù)凍存復(fù)蘇后抗體分泌穩(wěn)定。 采用檸檬酸三鈉還原法制備直徑20nm的膠體金并標(biāo)記抗Mela單克隆抗體作為檢測試劑,單抗加入量為20μg/mL,標(biāo)記pH控制在9.0左右,以1% PEG20000作穩(wěn)定劑。采用美國Millipore公司的FB-08玻璃纖維膜作為金標(biāo)結(jié)合墊,Sartorius公司的CN-140硝酸纖維素膜做為層析膜制成金標(biāo)免疫層析試劑盒,試劑盒的檢測靈敏度為1μg/mL,在室溫干燥條件下可保存18個(gè)月以上。 本實(shí)驗(yàn)研制的膠體金免疫層析試劑盒最大的優(yōu)點(diǎn)是無需借助檢測儀器,檢測快速、特異、靈敏、簡便,5min內(nèi)可出檢測結(jié)果,適用于對(duì)Mela殘留進(jìn)行現(xiàn)場檢測。本研究也為其他動(dòng)物藥物殘留檢測膠體金試紙的研制提供了借鑒。
[Abstract]:Melamine (melamine) is a kind of chemical raw material. Because of its nitrogen content as high as 66.6, it is often illegally added to milk and feed, which seriously threatens the safety of human and animal life. At present, most of the detection methods need special instruments and equipments, and the operation is tedious, which can not meet the requirements of field rapid detection. Therefore, it is of great significance to study and establish a convenient, safe and fast detection method. In this experiment, artificial antigens were synthesized by protein-binding technique, monoclonal antibodies against Mela were prepared by hybridoma technique, and the rapid detection kit for Mela gold-labeled immunochromatography was successfully developed by using gold-labeled immunochromatography, and the sensitivity of the kit was obtained. The specificity, stability and so on are in line with the requirements of detection, and the characteristics of large-scale screening are more suitable for routine detection, which has important practical significance and high commercial value. At first, the hapten Mela was conjugated with the carrier bovine serum albumin (BSA) and ovalbumin (ovalbumin) to form artificial antigens by using polyanhydride and mixed anhydride respectively. BALB/c mice were immunized with Mela-BSA, and spleen cells were fused with SP2/0 cells. Hybridoma cells were obtained. The hybridoma cell line H2C6 was obtained by using Mela-OVA as the coating antigen and ELISA method. The hybridoma cell line H2C6 was obtained by using the limited dilution method and the cloning and subcloning of the hybridoma cells. Indirect ELISA assay was used to determine the titer of ascites monoclonal antibody (1: 204800). The half inhibitory concentration (IC50) of McAb and Mela specific binding mAb Mela was 4.15 渭 g / mL with tripolycyanic acid, chloramphenicol, penicillin and streptomycin, and the specific inhibitory concentration of Mela was 4.15 渭 g 路mL ~ (-1) with tripolycyanic acid, chloramphenicol, penicillin and streptomycin. There was no cross reaction between sulfanilamides and other drugs, and the antibody secretion of the cell line was stable after repeated cryopreservation and resuscitation. Colloidal gold with diameter 20nm was prepared by trisodium citrate reduction method and labeled with monoclonal antibody against Mela as detection reagent. The amount of monoclonal antibody was 20 渭 g / mL, the labeling pH was about 9.0, and 1% PEG20000 was used as stabilizer. The gold standard immunochromatographic kit was made by using the FB-08 glass fiber membrane of Millipore Company as the gold standard binding pad and the CN-140 nitrocellulose membrane of Sartorius Company as the chromatography membrane. The sensitivity of the kit was 1 渭 g / mL, and the kit could be preserved for more than 18 months under the condition of room temperature drying. The greatest advantage of the colloidal gold immunochromatographic kit developed in this experiment is that it is rapid, specific, sensitive and simple to detect Mela residues in 5 minutes without the aid of detection instruments. It is suitable for the field detection of Mela residues. This study also provides a reference for the development of colloidal gold test paper for the detection of drug residues in other animals.
【學(xué)位授予單位】：重慶理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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本文編號(hào)：1951937