虾仁中酸性红73间接ELISA检测方法的建立

Development of an indirect ELISA for Acid Red 73 in Shrimps

本文采用活性酯法和N，N-羰基二味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IgG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检测方法井对反应条件进行了优化。优化后，该方法的半敬抑制浓度（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制盒奠定了基础。

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-ART3 antibody were obtained and purified by the caprylic acid-ammonium sulfate method. Based on the IgG,an indirect competitive ELISA method and optimum reaction conditions for detection of AR73 was developed.The half of inhibition concentration（ ICso）was 45.39 μg/L,the limit of detection for acid red 73 was 5.64 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.