基于虾类原肌球蛋白共同表位肽抗体的过敏原检测方法

Rapid detection of shrimp allergens based against the polyclonal antibodies of shrimp tropomyosin common peptide epitopes

目的建立一种能够同时检测多种虾原肌球蛋白的方法。方法通过合成不同虾类的共同表位肽,制备能够识别多种虾类原肌球蛋白的共同表位肽多克隆抗体,建立快速灵敏的虾类原肌球蛋白酶联免疫检测方法。结果该检测方法在4.79～1400 ng/mL的浓度范围内线性关系良好,其线性回归方程为Y=-19.083X+98.303(R^2=0.9813),最低检出限(IC_(10))为1.85 ng/mL;样品加标回收率在94.37%～103.45%之间;该检测方法与软体动物的原肌球蛋白有交叉反应,与鱼肉蛋白、牛奶、花生蛋白等无交叉反应;批内变异系数为3.4%～9.1%,板间变异系数为12.9%～19.6%,贮藏试验显示该酶联免疫试剂盒可以在4℃下保存6个月以上,能够应用于食品中虾类过敏原的检测。结论采用共同表位抗体,成功建立了基于酶联免疫的过敏原检测方法。

Objective To establish a method for simultaneous determination of various shrimp tropomyosins. Methods By synthesis of common peptide epitopes from different kinds of shrimp, polyclonal antibodies were produced which could identify a variety of shrimp tropomyosins. A rapid and sensitive ELISA method was established for the detection of shrimp tropomyosins. Results The method had a good linear relationship in the range of 4.79～1400 ng/mL, and the linear regression equation was Y=-19.083 X＋98.303（R～2=0.9813）. The limit of detection was 1.85 ng/mL（IC（10））, while the recovery rates were 94.37%～103.45%. The detection method was cross-responsive with invertebrate tropomyosin and had no cross reaction with fish protein, milk, and peanut protein. The coefficients of variation of the batch were 3.4%～9.1%, and the coefficients of variation of the plates were 12.9%～19.6%. The storage test showed that the ELISA kit could be stored more than 6 months at 4 ℃, which could be applied for the detection of shrimp allergens in food. Conclusion A simultaneous detection method of a variety of shrimp tropomyosins is establised successfully based on ELISA using common peptide epitopes.