本文選題：稻米 + 鎘��； 參考：《長(zhǎng)沙理工大學(xué)》2014年碩士論文

【摘要】：65%的中國人以稻米為絕對(duì)主食,2002年,農(nóng)業(yè)部稻米及制品質(zhì)量監(jiān)督檢驗(yàn)測(cè)試中心曾對(duì)全國市場(chǎng)稻米進(jìn)行安全性抽檢,結(jié)果顯示,稻米中鎘超標(biāo)率為10.3%。未來中國農(nóng)產(chǎn)品安全問題中,鎘等重金屬污染將取代農(nóng)藥,成為事故多發(fā)地帶。傳統(tǒng)的檢測(cè)方法已不能滿足需求,急需找到快速、靈敏、高效的新型檢測(cè)方法,本論文以稻米中重金屬鎘為研究對(duì)象,以制備單克隆抗體為基礎(chǔ),進(jìn)一步組裝金標(biāo)試紙條,以期建立現(xiàn)場(chǎng)、快速鎘的免疫檢測(cè)方法。采用大分子雙功能螯合劑1-(4-異硫氰芐基)乙烯基二胺-N,N,N',N'-四乙酸(isothiocyano benzy-EDTA, iEDTA)螯合重金屬鎘離子形成六齒配合物,再分別偶聯(lián)載體OVA和BSA,合成人工免疫抗原Cd-iEDTA-OVA和檢測(cè)抗原Cd-iEDTA-BSA。石墨爐原子吸收分光光度計(jì)測(cè)得Cd-iEDTA-OVA和Cd-iEDTA-BSA中鎘含量分別為174.6230μg-L-1和48.1881μg·L-1；紫外分光光度掃描結(jié)果表明完全抗原同時(shí)具備載體蛋白與半抗原的吸收特性,并測(cè)得OVA和BSA含量分別為1.7892mg-mL-/1和1.8065mg·mL-1,結(jié)果說明鎘完全抗原合成成功,可用于后續(xù)試驗(yàn)研究。采用Cd-iEDTA-OVA多點(diǎn)少量注射BALB/c小鼠,獲得抗血清,通過細(xì)胞融合、雜交瘤及克隆技術(shù)獲得7F4和7E8兩株特異性較好的雜交瘤細(xì)胞；通過體內(nèi)誘生腹水法及辛酸-硫酸銨鹽析、差量離心法純化技術(shù),獲得7E8E5、7E8G9、7F488、7F4D6和7F4H85株單克隆抗體。酶聯(lián)免疫吸附法(ELISA)結(jié)果表明,5株單抗均屬IgGl型；親和力均高,數(shù)量級(jí)達(dá)108；僅與Hg2+有較強(qiáng)交叉,與其他離子幾乎無交叉；初步建立了Cd2+間接競(jìng)爭(zhēng)ELISA法,最低檢測(cè)限為260ng·mL-1,在260～5000ng·mL-1范圍內(nèi)趨近直線。SDS-PAGE法結(jié)果顯示有且只有兩條清晰的區(qū)帶(重鏈帶和輕鏈帶),說明單抗純度高。其中7E8E5和7E8G9效價(jià)相對(duì)高,分別達(dá)1：512000和256000,親和常數(shù)也最高,分別為4.55×1081·mmoL-1和2.20×1081·moL-1,因此可從該兩株單抗擇優(yōu)制備金標(biāo)試紙條。采用檸檬酸三鈉還原法制備13nm金顆粒,通過目測(cè)、可見-紫外分光光度法、透射電鏡法鑒定,結(jié)果表明金溶液穩(wěn)定于酒紅色,且呈球形、大小均一,可用于標(biāo)記7E8G9。通過條件優(yōu)化得到：最佳標(biāo)記pH為8.2,最小抗體用量為每1mL膠體金溶液中6.0μg單抗,T線包被濃度為1.0mg·mL-1,C線包被濃度為1.5mg·mL-1,且包被條件為37℃條件下干燥60min,最佳金標(biāo)抗體稀釋度為0.75倍,最佳封閉溫度及時(shí)間為25℃干燥60min,最佳離子濃度為O.Olmol·L-1。將完全抗原Cd-iEDTA-BSA作為T線,羊抗鼠二抗作為C線,包被于硝酸纖維素膜上,納米金顆粒標(biāo)記的7E8G9單克隆抗體包被于金標(biāo)墊上,組裝成定性檢測(cè)金標(biāo)試紙條。試紙條的靈敏度為0.2 mg·kg-1、重復(fù)性良好、與Hg2+有較強(qiáng)交叉,最高共存濃度不得高于1.0mg·kg-1；與Zn2+有一定交叉,最高共存濃度不得高于10 mg·kg-1;與Cu2+、Fe2+、Ca2+、Mg2+、Al3+、.Pb2+幾乎無交叉；貯存期約為1年。試紙條檢測(cè)稻米中鎘與GFAAS結(jié)果基本一致。
[Abstract]:65% of Chinese people take rice as the absolute staple food. In 2002, the quality Supervision and Test Center of Rice and its products of the Ministry of Agriculture carried out a sampling inspection on the safety of rice in the national market. The results showed that the cadmium excess rate in rice was 10.3%.In the future, cadmium and other heavy metal pollution will replace pesticides and become accident prone areas.The traditional detection method can not meet the demand, so it is urgent to find a new rapid, sensitive and efficient detection method. In this paper, the heavy metal cadmium in rice is taken as the research object, and the gold standard test strip is further assembled based on the preparation of monoclonal antibody.In order to establish a field, rapid immunoassay method for cadmium.Using macromolecular bifunctional chelating agent 1-chelating agent 1-chelate 4-isothiocyano benzy-EDTA (iEDTAA) to form hexadentate complexes, the artificial immune antigen (Cd-iEDTA-OVA) and the detection antigen (Cd-iEDTA-BSA) were synthesized by chelating the heavy metal cadmium ions. The artificial immune antigen (Cd-iEDTA-OVA) and the detection antigen (Cd-iEDTA-BSA) were synthesized by using the macromolecular bifunctional chelating agent 1-chelating agent 4-isothiocyano benzy-EDTA-EDTA-EDTA-EDTA-EDTA-EDTA.The content of cadmium in Cd-iEDTA-OVA and Cd-iEDTA-BSA was 174.6230 渭 g-L-1 and 48.1881 渭 g / L ~ (-1), respectively, by graphite furnace atomic absorption spectrophotometer.The contents of OVA and BSA were 1.7892mg-mL-/1 and 1.8065mg mL-1, respectively. The results showed that cadmium complete antigen was successfully synthesized and could be used for further study.The antiserum was obtained by injecting a small amount of Cd-iEDTA-OVA into BALB/c mice. Two hybridoma cell lines, 7F4 and 7E8, were obtained by cell fusion, hybridoma and cloning techniques, and ascites were induced in vivo and the octanoic acid-ammonium sulfate saltout was used.The monoclonal antibodies of 7E8E5O7E8G9, 7F488C7F4D6 and 7F4H85 strain were obtained by differential centrifugation.The results of Elisa showed that all of the 5 McAbs belonged to IgGl type, with high affinity and order of magnitude of 108. They only had strong cross with Hg2 and almost no cross with other ions. The indirect competitive ELISA method of Cd2 was established preliminarily.The lowest detection limit was 260ng mL-1. The results of the method showed that there were only two distinct bands (heavy chain band and light chain band) in the range of 260~5000ng mL-1. The results of SDS-PAGE showed that the purity of the McAb was high.The titer of 7E8E5 and 7E8G9 was relatively high, reaching 1: 512000 and 256000, respectively, and the affinity constant was the highest (4.55 脳 1081 mmoL-1 and 2.20 脳 1081 mol ~ (-1), respectively).13nm gold particles were prepared by tri-sodium citrate reduction method. The results showed that the gold solution was stable in wine red, spherical in size and uniform in size, and could be used to label 7E8G9 by visual measurement, visible ultraviolet spectrophotometry and transmission electron microscopy.The optimal labeling pH was 8.2, the minimum antibody dosage was 6.0 渭 g / 1mL colloidal gold solution and the concentration of 1.0mg mL-1C line coating was 1.5mg mL-1, and the best dilution of gold-labeled antibody was 0.75 times at 37 鈩，

本文編號(hào)：1759531