摘要
为了检测海产品贝毒大田软海绵酸,保障其食用安全,利用活泼酯法将小分子OA与载体蛋白偶联,免疫BALB/c小鼠,细胞融合技术建立分泌抗OA的杂交瘤细胞株,并对其各种特性进行分析;小鼠腹水法大量生产抗体,纯化后建立ELISA方法,同时建立OA的高效液相色谱-串联质谱(HPLC-MS)检测法,并对部分市售海产品进行了实际检测,ELISA方法标准曲线为y=-34.212x+83.49,相关系数为0.9784,线性范围0.4～25μg/L,灵敏度0.18μg/L;HPLC-MS/MS检测方法标准曲线y=193.07x-780.6(Q1/Q3:m/z827.4～m/z723.5)和y=83.021x-335.6(Q1/Q3:m/z827.4～m/z809.5),R2均为0.9991,线性范围10～800μg/L,灵敏度小于2μg/L,平均RSD为4.34%。在检测的实际样品中两种样品ELISA呈阳性反应,其中一种经过了HPLC-MS/MS的验证。所建立的ELISA及HPLC-MS检测方法均可用于海产品腹泻性贝毒OA限量标准检测,为进出口海产品OA标准方法的建立提供实验基础。
The aim of this experiment was to establish rapid detection methods of okadaic acid （OA） in shellfish in order to ensure edible safety. The OA was coupled with human IgG as immunity antigen and with BSA as detection antigen by active ester method. The Balb/c mice were inoculated with the prepared antigen. The hybridoma secreting monoclonal antibody against OA was constructed by the cell-fusion technology and the specialities of the monoclonai antibody were analyzed. The ELISA method was established for detecting OA after ascites was prepared and purified. At the same time, the HPLC-MS/MS method was also set up. Some shellfish samples were detected by the two methods. The linear regression equation of ELISA method is y =- 34.212x ＋ 83.49 （y is optical density at 490-nm wavelength, and x is OA concentration,μg/L） with the determination coefficient of 0.9784, which shows good linearity over the concentration range of 0.4 to 25 μg/L, and the detection limit to OA is 0.18μg/L. The linear regression equations of HPLC-MS/MS method are y = 193.07x - 780.6 （Q1/Q3： m/z 827.4-m/z 723.5） and y = 83.021x - 335.6 （Q1/Q3： m/z 827.4-m/z 809.5）, where y is peak height, and x is OA concentration, μg/L. Both the equations have the same determination coefficients of 0.9991, show good linearity over the concentration range of 10 to 800 μg/L. The detection limit to OA is less than 2 μg/L and the stand average RSD is 4.34%. Two shellfish samples were the positive result by ELISA method, and one of them was verifed by HPLC-MS/MS. The two methods both can be used for detecting OA and provide experimental foundation to establish standard detection method for OA in shellfish.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2009年第8期158-162,共5页
Food Science
基金
国家自然科学基金项目(30671762)
国家质检总局项目(2003IK044
2008IK003)
