本文選題：甲基對硫磷 + 單克隆抗體��； 參考：《山東農(nóng)業(yè)大學》2017年碩士論文

【摘要】：甲基對硫磷(O,O-dimethyl-O-4-nitrophenyl phosphorothioate,PM)是一種高毒有機磷酸酯類農(nóng)藥,主要用于防治棉花、水稻、果樹害蟲。甲基對硫磷在食品中的殘留會通過食物鏈進入人體,對神經(jīng)系統(tǒng)、內(nèi)分泌系統(tǒng)、免疫系統(tǒng)及生殖系統(tǒng)等產(chǎn)生毒害作用。為保障食品安全,建立快速、簡單、準確的甲基對硫磷殘留檢測方法是非常必要的。相對于傳統(tǒng)的酶抑制法、儀器分析法等,酶聯(lián)免疫吸附測定(ELISA)具有操作簡單、成本低廉、特異性強、可批量檢測多個樣品等優(yōu)勢。本研究制備了甲基對硫磷特異性單克隆抗體和單鏈抗體,并使用制備的單克隆抗體和單鏈抗體分別建立了甲基對硫磷的免疫檢測方法。主要研究結(jié)果如下:(1)首先,使用甲基對硫磷特異性免疫原免疫BALB/C小鼠,通過細胞融合,雜交瘤細胞篩選及腹水的制備和純化獲得了甲基對硫磷特異性單克隆抗體(PM-4G6)。(2)利用改進的高碘酸鈉法將單克隆抗體(PM-4G6)與辣根過氧化物酶(HRP)偶聯(lián),獲得酶標抗體(PM-4G6-HRP)。分別利用PM-4G6和PM-4G6-HRP建立了甲基對硫磷的的間接競爭酶聯(lián)免疫吸附測定法(IC-ELISA)和直接競爭酶聯(lián)免疫吸附測定法(DC-ELISA)。在最佳分析條件下,IC-ELISA的檢測限(LOD,IC10)為0.6ng/m L,半抑制濃度(IC50)為6.6ng/m L,線性檢測范圍是1.4-28.0ng/mL;DC-ELISA的檢測限為0.6ng/m L,IC50為3.6ng/m L,線性檢測范圍是1.2-12.4ng/mL。交叉反應(yīng)測定結(jié)果表明所建立的ELISAs除了與殺螟硫磷、對硫磷、甲基對氧磷、對氧磷這4種農(nóng)藥有一定的交叉反應(yīng)外,與其余用于分析的農(nóng)藥無明顯交叉反應(yīng),對甲基對硫磷的特異性較好。(3)采用樣品添加回收實驗對建立的檢測方法進行評價。對面粉和大米樣品的甲基對硫磷添加回收實驗結(jié)果顯示,IC-ELISA的加標回收率在85.2%-128.9%之間,變異系數(shù)范圍為2.6%-7.1%;DC-ELISA的加標回收率在101.9%-116.3%之間,變異系數(shù)范圍為3.8%-10.6%。結(jié)果證明了這兩種方法在樣品中應(yīng)用具有較高的可靠性。(4)使用甲基對硫磷特異性免疫原免疫BALB/C小鼠,成功構(gòu)建了甲基對硫磷的噬菌體單鏈抗體庫。通過固相淘選和噬菌體單克隆篩選,獲得兩種不同的甲基對硫磷單鏈抗體PM-30和PM-25,其中PM-30的靈敏度較高。將PM-30的基因插入到pET-28a(+)載體,并轉(zhuǎn)化E.coli BL21(DE3),進行原核表達。最佳表達條件為25℃,0.1mmol/L IPTG條件下誘導表達8h。表達后利用生物素連接酶(BirA)對單鏈抗體進行生物素化,經(jīng)純化,生物素化單鏈抗體產(chǎn)量可達59.2±3.7mg/L。(5)使用獲得的生物素化PM-30單鏈抗體建立了甲基對硫磷的IC-ELISA檢測方法。經(jīng)測定,在最佳分析條件下,IC-ELISA的檢測限為0.9ng/m L,IC50為14.5ng/m L,線性檢測范圍是5.9-191.4ng/m L。交叉反應(yīng)測定結(jié)果顯示該方法除了與對硫磷、殺螟硫磷、甲基對氧磷3種農(nóng)藥有較弱的交叉反應(yīng)外,與其余用于分析的農(nóng)藥無明顯交叉反應(yīng),對甲基對硫磷的特異性較好。
[Abstract]:Methyl parathion (O, O-dimethyl-O-4-nitrophenyl phosphorothioate, PM) is a highly toxic organophosphate pesticide, which is mainly used to prevent cotton, rice and fruit tree pests. Methyl parathion residues in food will enter the human body through food chain, and have toxic effects on the nervous system, the internal secretion system, the immune system and the reproductive system. In order to ensure food safety, it is very necessary to establish a rapid, simple and accurate methyl parathion residue detection method. Relative to traditional enzyme inhibition method, instrument analysis method, enzyme linked immunosorbent assay (ELISA) has the advantages of simple operation, low cost, strong specificity, and batch detection of multiple samples. This study prepared methyl parathion Specific monoclonal antibodies and single chain antibodies were used to establish an immunoassay for methyl parathion using the monoclonal antibody and single chain antibody prepared. The main results are as follows: (1) first, using methyl parathion specific immunogenic immunization BALB/C mice, screening of hybridoma cells, preparation and purity of ascites through cell fusion, hybridoma cells screening. The methyl parathion specific monoclonal antibody (PM-4G6) was obtained. (2) using the improved sodium iodate method, the monoclonal antibody (PM-4G6) and horseradish peroxidase (HRP) were coupled to obtain the enzyme labeled antibody (PM-4G6-HRP). The indirect competitive enzyme linked immunosorbent assay (IC-ELISA) for methyl parathion (IC-ELISA) was established by PM-4G6 and PM-4G6-HRP respectively. Direct competitive enzyme linked immunosorbent assay (DC-ELISA). Under the optimal analysis conditions, the detection limit of IC-ELISA (LOD, IC10) is 0.6ng/m L, and the semi inhibitory concentration (IC50) is 6.6ng/m L, and the linear detection range is 1.4-28.0ng/mL. The ELISAs, which is established in Ming Dynasty, has a certain cross reaction to 4 kinds of pesticides, parathion, methyl parathion, methyl parathion, and oxygen and phosphorus. There is no obvious cross reaction with the other pesticides used for analysis, and the specificity of methyl parathion to methyl parathion is better. (3) using sample adding recovery experiment to evaluate the established detection methods. The results of the methyl parathion recovery experiment showed that the recovery rate of IC-ELISA was between 85.2%-128.9%, the range of variation coefficient was 2.6%-7.1%, the recovery rate of DC-ELISA was 101.9%-116.3%, and the range of variation coefficient was 3.8%-10.6%.. The results showed that the two methods were highly reliable in the application of the samples. (4) the use of armour was used. The phage single chain antibody library of methyl parathion was constructed successfully in BALB/C mice based on the specific immunogenic immunogenicity of parathion. Through solid-phase selection and phage screening, two different methyl parathion single chain antibodies PM-30 and PM-25 were obtained, in which the sensitivity of PM-30 was higher. PM-30 gene was inserted into pET-28a (+) vector and E. was transformed into E.. Coli BL21 (DE3) was used for prokaryotic expression. The optimum expression condition was 25 C, 0.1mmol/L IPTG induced expression of 8h. expression by biotin ligase (BirA) for biotinylation of single chain antibody. After purification, the production of biotinylated single chain antibody could reach 59.2 + 3.7mg/L. (5), and a methyl pair was established by using the obtained biotinylated PM-30 single chain antibody. IC-ELISA detection method of phosphorous phosphorus. Under the optimum analysis conditions, the detection limit of IC-ELISA is 0.9ng/m L, IC50 is 14.5ng/m L, and the linear detection range is 5.9-191.4ng/m L. cross reaction determination results show that the method has a weak cross reaction to 3 kinds of pesticides, such as parathion, Pho, methyl parathion, and the rest of the method. The pesticides had no obvious cross reaction, and the specificity for methyl parathion was good.

【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S481.8

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