直接竞争ELISA法快速检测动物源性食品中磺胺嘧啶残留

Competitive Direct ELISA for Rapid Detection of Sulfadiazine Residues in Edible Animal Products

采用重氮化-偶联法将磺胺嘧啶（SD）与人血清白蛋白（HSA）相连制备了免疫抗原SD—HSA，与卵清白蛋白（OVA）相连制备了包被抗原SD—OVA，用过碘酸钠法将SD—OVA与辣根过氧化物酶（HRP）连接制备了酶标抗原（SD—OVA—HRP）。用SD—HSA免疫BALB／c小鼠，经融合、筛选和克隆化制备了单克隆抗体，利用单抗建立了直接竞争ELISA方法。该方法具有好的特异性，在1—729ng／mL浓度范围标准曲线方程Y=12．05x＋2．2538，R^2=0．995，IC50为41．7ng／mL，在猪肉、鸡肉、鸡肝、鸡蛋、牛奶中的检测限分别为20、20、20、5、5μg/kg。在20μg／kg和100μg／kg水平猪肉、鸡肉、鸡肝、牛奶、鸡蛋样品中添加，回收率为82．78％-104．6％。与高效液相色谱方法比较，二者的阳性符合率为81．3％，阴性符合率为83．3％，总符合率为88．9％；与同类英国RANDOX试剂盒比较，二者符合率为100％。

Immunizing antigen （SD -HSA） and coating antigen （SD -OVA） were synthesized by the method of diazotization and coupling. HRP labeled antigen （ SD - OVA - HRP） was synthesized by periodate method. After McAb specific to SD was prepared and competitive direct ELISA was established. This method had good specificity with sulfadiazine. The equation of calibration curve was y -- 12. 05x ＋ 2.2538 with the RE of 0. 995 and IC50 of 41.7 ng/mL in the range from 1 ng/mL to 729 ng/mL. The limits of detection of ELISA were 20, 20, 20, 5, 5 μg/kg in pork, chicken, chick liver, egg, milk, respectively. Recoveries of sulfadiazine in spiked samples between 20 and 100 ng/mL ranged from 82.78% - 104.6%. This method was compared with HPLC. The positive agreement rates was 81. 3%, the negative agreement rates was 83. 3%, and the total agreement rates was 88.9% ; This method was compared with RANDOX Kit,with an agreement rate of 100%.