Abstract:

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 monoclonal 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 25μg/L, and the detection limit to OA is 0.18 μg/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.

Key words: okadaic acid (OA), ELISA, HPLC-MS/MS, shellfish