重金屬汞酶聯(lián)免疫檢測方法的研究
發(fā)布時(shí)間：2019-03-03 16:08
【摘要】：本實(shí)驗(yàn)建立了重金屬汞離子的間接競爭酶聯(lián)免疫(IC-ELISA)分析方法。由于汞離子不具有免疫原性,因此以異硫氰酸芐基乙胺四乙酸(ITCBE)為雙功能螫合劑連接汞離子和鑰孔血藍(lán)蛋白(KLH)制備免疫原,在此基礎(chǔ)上免疫新西蘭大白兔獲得特異性抗體,抗體效價(jià)為1：70000。用獲得的抗體建立汞的間接競爭酶聯(lián)免疫分析方法。 本實(shí)驗(yàn)在優(yōu)化了包被抗原包被量、抗體稀釋倍數(shù)、稀釋液離子強(qiáng)度、稀釋液pH值和封閉液的基礎(chǔ)上,建立了重金屬汞間接競爭ELISA的抑制率標(biāo)準(zhǔn)曲線。建立的IC-ELISA檢測方法的檢測限為0.45μg/L,靈敏度為4.10μg/L。特異性實(shí)驗(yàn)表明,抗體對(duì)鎳和銅有一定的交叉反應(yīng),交叉反應(yīng)率分別為10.8%和13.5%,與其他金屬均無明顯交叉反應(yīng)。本實(shí)驗(yàn)選取自來水、海河水、白芷、胡蘿卜和蝦喤為檢測樣本,通過基質(zhì)影響消除實(shí)驗(yàn)確定樣品前處理方法,同時(shí)進(jìn)行添加回收實(shí)驗(yàn)。自來水和海河水過0.22μm水膜后用雙蒸水稀釋即可消除基質(zhì)影響,白芷、胡蘿卜和蝦蛄采用硝酸和高氯酸體積比為5:1的硝化體系進(jìn)行硝化處理,然后用雙蒸水稀釋可以消除基質(zhì)影響。添加回收實(shí)驗(yàn)表明,自來水、海河水、白芷、胡蘿卜和蝦蛄中汞的回收率均在70%-120%,變異系數(shù)在20%以內(nèi)。使用電感耦合等離子體質(zhì)譜(ICP-MS)法驗(yàn)證方法的準(zhǔn)確性,儀器檢測結(jié)果和建立的ELISA分析方法具有很好的相關(guān)性(白芷樣品檢測判定系數(shù)為0.988),表明建立的方法具有很好的準(zhǔn)確性。 本實(shí)驗(yàn)建立的用于檢測食品和藥品中的重金屬汞殘留的酶聯(lián)免疫檢測方法,具有靈敏度高、使用簡便、不需要昂貴的儀器等優(yōu)點(diǎn),可以作為批量樣品的篩選方法和現(xiàn)場檢測的便捷工具,對(duì)于應(yīng)對(duì)突發(fā)食品或者藥品汞安全事故提供了更好的選擇
[Abstract]:An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) method for the determination of heavy metal mercury ions was established. Because the mercury ion has no immunogenicity, the mercury ion and keyhole hemocyanin (KLH) were ligated with (ITCBE) as the bifunctional chelator to prepare the immunogen, and the immunogen was prepared by linking the mercury ion with the keyhole hemocyanin (KLH) as the bifunctional chelator. On the basis of this, the specific antibody was obtained by immunizing New Zealand white rabbits, and the titer of antibody was 1? 700 000. The indirect competitive enzyme-linked immunosorbent assay (Elisa) for mercury was established by using the obtained antibodies. On the basis of optimizing the amount of coated antigen, the dilution multiple of antibody, the ionic strength of diluent, the pH value of diluent and the blocking solution, the standard curve of inhibition rate for indirect competition of ELISA by heavy metal mercury was established. The detection limit was 0.45 渭 g / L and the sensitivity was 4.10 渭 g / L. the detection limit of IC-ELISA was 0.45 渭 g / L and the sensitivity was 4.10 渭 g / L. The specific test showed that the antibody had some cross-reaction to nickel and copper, the cross-reaction rates were 10.8% and 13.5%, respectively, and there was no obvious cross-reaction between the antibody and other metals. In this experiment, tap water, Haihe water, Angelica dahurica, carrots and prawns were selected as samples. The method of sample pretreatment was determined by matrix effect elimination experiment, and the experiment of adding and recovering was carried out at the same time. After water film of 0.22 渭 m in tap water and water in Haihe River, the effect of substrate can be eliminated by dilution with double evaporated water. Angelica dahurica, carrots and oratosquilla were nitrified by nitrification system with the volume ratio of nitric acid and perchloric acid being 5:1. Then the effect of matrix can be eliminated by diluting with double evaporated water. The recovery rate of mercury in tap water, Haihe water, Angelica dahurica, carrots and Oratosquilla was 70% and 120% respectively, and the coefficient of variation was less than 20%. Inductively coupled plasma mass spectrometry (ICP-MS) was used to verify the accuracy of the method. The results of the instrument had good correlation with the established ELISA analysis method (the determination coefficient of the samples of Angelica dahurica was 0.988). It is shown that the established method has good accuracy. The enzyme-linked immunosorbent assay established in this experiment for the determination of heavy metal mercury residues in food and medicine has the advantages of high sensitivity, simple use and no need of expensive instruments, etc. It can be used as a screening method for batch samples and a convenient tool for on-the-spot detection, which provides a better choice for dealing with sudden mercury accidents in food or drugs.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R155.51
本文編號(hào)：2433868
[Abstract]:An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) method for the determination of heavy metal mercury ions was established. Because the mercury ion has no immunogenicity, the mercury ion and keyhole hemocyanin (KLH) were ligated with (ITCBE) as the bifunctional chelator to prepare the immunogen, and the immunogen was prepared by linking the mercury ion with the keyhole hemocyanin (KLH) as the bifunctional chelator. On the basis of this, the specific antibody was obtained by immunizing New Zealand white rabbits, and the titer of antibody was 1? 700 000. The indirect competitive enzyme-linked immunosorbent assay (Elisa) for mercury was established by using the obtained antibodies. On the basis of optimizing the amount of coated antigen, the dilution multiple of antibody, the ionic strength of diluent, the pH value of diluent and the blocking solution, the standard curve of inhibition rate for indirect competition of ELISA by heavy metal mercury was established. The detection limit was 0.45 渭 g / L and the sensitivity was 4.10 渭 g / L. the detection limit of IC-ELISA was 0.45 渭 g / L and the sensitivity was 4.10 渭 g / L. The specific test showed that the antibody had some cross-reaction to nickel and copper, the cross-reaction rates were 10.8% and 13.5%, respectively, and there was no obvious cross-reaction between the antibody and other metals. In this experiment, tap water, Haihe water, Angelica dahurica, carrots and prawns were selected as samples. The method of sample pretreatment was determined by matrix effect elimination experiment, and the experiment of adding and recovering was carried out at the same time. After water film of 0.22 渭 m in tap water and water in Haihe River, the effect of substrate can be eliminated by dilution with double evaporated water. Angelica dahurica, carrots and oratosquilla were nitrified by nitrification system with the volume ratio of nitric acid and perchloric acid being 5:1. Then the effect of matrix can be eliminated by diluting with double evaporated water. The recovery rate of mercury in tap water, Haihe water, Angelica dahurica, carrots and Oratosquilla was 70% and 120% respectively, and the coefficient of variation was less than 20%. Inductively coupled plasma mass spectrometry (ICP-MS) was used to verify the accuracy of the method. The results of the instrument had good correlation with the established ELISA analysis method (the determination coefficient of the samples of Angelica dahurica was 0.988). It is shown that the established method has good accuracy. The enzyme-linked immunosorbent assay established in this experiment for the determination of heavy metal mercury residues in food and medicine has the advantages of high sensitivity, simple use and no need of expensive instruments, etc. It can be used as a screening method for batch samples and a convenient tool for on-the-spot detection, which provides a better choice for dealing with sudden mercury accidents in food or drugs.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R155.51
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