Development of an indirect ELISA for Acid Red 73 in Shrimps
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摘要: 本文采用活性酯法和N,N-羰基二咪唑法分别合成了酸性红73的人工抗原,方阵滴定实验表明活性酯法所得兔抗酸性红73的Ig G更适合用于后继实验,以此为基础建立了一种虾仁中酸性红73的间接ELISA检测方法并对反应条件进行了优化。优化后,该方法的半数抑制浓度（IC50）为45.39μg/L,检测限（LOD）为5.64μg/L。交叉反应实验结果表明除苏丹红3号（0.15%）外与番红花红、苯酚红、刚果红、碱性品红和胭脂红等其他竞争物无交叉反应。在虾仁中的空白添加回收率范围为71.3%84.4%,变异系数（RSD）<9.55%。此法可用于虾仁中酸性红73的残留检测,并为进一步制备单克隆抗体和研制快速检测试剂盒奠定了基础。
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关键词:
- 虾仁 /
- 酸性红73 /
- 活性酯法 /
- N,N-羰基二咪唑法 /
- ELISA
Abstract: Acid red 73( AR73) was coupled to bovine serum albumin( BSA) and ovalbumin( OVA) by the use of N- hydroxysuccinimide active method and the CDI method to prepare the immunogen and coating antigen,respectively.The anti- AR73 antibody were obtained and purified by the caprylic acid- ammonium sulfate method.Based on the Ig G,an indirect competitive ELISA method and optimum reaction conditions for detection of AR73 was developed.The half of inhibition concentration( IC50) was 45.39 μg /L,the limit of detection for acid red 73 was5.64 μg / L,and the cross- reactivity study showed that the polyclonal antiserum were highly specific to AR73,and no cross- reactivity was detected between the obtained polyclonal antiserum and the other competitors( Congo Red,Phenol Red,Safranine,Basic Fuchsin and Carmine) except to Sudan red Ⅲ( 0.15%).The recoveries from the standards fortified blank samples were in the range of 71.3% 84.4% with Coefficient of Variance lower than 9.55%.This developed method can be used to determine the acid red 73 residue in shrimps,and can help to prepare the monoclonal antibody and develop the rapid test kits for acid red 73.-
Keywords:
- Shrimps /
- Acid Red 73 /
- Active ester method /
- CDI method /
- ELISA
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[1] Stiborova M,Martinek V,Rydlova H,et al.Sudan I is a potential carcinogen for humans:evidence for its metabolic activation and detoxication by human recombinant cytochrome P450 1A1 and liver microsomes[J].Cancer Research,2002,62(20):5678-5684.
[2] Stiborova M,Martinek V,Rydlova H,et al.Expression of cytochrome P450 1A1 and its contribution to oxidation of a potential human carcinogen 1-phenylazo-2-naphthol(Sudan I)in human livers[J].Cancer Letters,2005,220(2):145-154.
[3] Dawkar V V,Jadhav U U,Kagalkar A N,et al.Govindwar Decolorization and detoxification of sulphonated azo dye Red HE7B by Bacillus sp.VUS[J].World J.Microbiol.Biotechnol,2010,26(5):909-916.
[4] Guo Y M,Yue Q Y,Gao B Y,et al.Probing the molecular mechanism of C.I.Acid red 73 binding to human serum albumin[J].Environ.Toxicol.Phar,2010,30(1):45-51.
[5] Guo Y M,Yue Q Y,Gao B Y,et al.Molecular docking study investigating the possible mode of binding of C.I.Acid Red 73 with DNA[J].Int.J.Biol.Macromol,2011,49(1):55-61.
[6] 宁尚勇,高洁,许志强,等.反相液相色谱法同时检测虾仁中合成色素的研究[J].分析实验室,2008,27(4):73-75. [7] 常向彩,杨晓农,宋定州,等.酸性红73多克隆抗体的制备及应用[J].分析化学,2012,40(10):1593-1597. [8] 李冰,李剑勇,周绪正,等.动物性食品中兽药残留分析检测技术研究进展[J].畜牧与兽医,2012,44(5):82-86. [9] 宋姗姗.食品中罗丹明B的残留免疫检测方法的研究[D].无锡:江南大学,2011. [10] Zhao D,He L,Pu C,et al.A highly sensitive and specific polyclonal antibody based enzyme-linked immunosorbent assay for detection of antibiotic olaquidox in animal feed samples[J].Anal Bi OAnal Chem,2008,391(7):2653-2661.
[11] 常向彩,杨晓农,朱子凤,等.两种纯化兔抗酸性红73免疫球蛋白G方法的比较研究[J].中国畜牧兽医,2013,40(7):124-126. [12] Chang X C,HU X Z,Li Y Q,et al.Multi-determination of para red and sudan dyes in egg by a broad-specific antibody based enzyme linked immunosorbent assay[J].Food Control,2011,22(11):1770-1775.
[13] Goodrow M H,Hammock B D.Hapten design for compound2selective antibodies:ELISA for environmentally deleterious small molecules[J].Analy tica Chimica Acta,1998,376(1):83-91.
[14] Li J,Liu J,Zhang H,et al.Broad-specificity indirect competitive ELISA for determination of nitrofurans in animal feeds[J].Analytica Chimica Acta,2010,678(1):1-6.
[15] Galve R,Sanchez-Baeza F,Camps F,et al.Indirect competitive immun OAssay for trichlorophenol determination:Rational evaluation of the competitor heterology effect[J].Analytica Chimica Acta,2002,452(2):191-206.
[16] 谢桂勉,孙远明,徐振林,等.新型含硫半抗原的晶体结构、构象分析及其电性研究[J].高等学校化学学报,2009,30(11):2193-2198. [17] Franek M,Diblikova I,Cernoch I,et al.Br OAd-specificity immun OAssays for sulfonamide detection:immunochemical strategy of generic antibodies and competitors[J].Analytical Chemistry,2006,78(5):1559-1567.
[18] Schneider P,Hammock B D.Influence of the ELISA format and the hapten-enzyme conjugate on the sensitivity of an immun OAssay for Striazine herbicides using monoclonal antibodies[J].J Agric Food Chem,1992,40(3):525-530.
[19] Parker C W.生物活性化合物的放射免疫测定法[M].北京:科学出版社,1981:165. [20] Freia J.Detection of pesticides residues by enzymeimmun OAssay[J].Pestic Sci,1989(26):303-307.
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