牛奶中三聚氰胺的ELISA检测研究

Analysis of Melamine in milk by ELISA

戊二醛法将三聚氰胺(Melamine)与载体蛋白进行偶联制备免疫抗原Melamine-BSA及包被抗原Melamine-OVA。对人工合成抗原进行紫外扫描,其紫外扫描谱图与载体蛋白及三聚氰胺的图谱有明显的差异,初步显示载体蛋白与三聚氰胺偶联成功。进一步分析Melamine-BSA中Melamine与BSA的摩尔比,复合物中三聚氰胺与BSA的摩尔比为8.56∶1。用免疫抗原Melamine-BSA腹腔免疫BALB/c小白鼠,获得抗三聚氰胺的多克隆抗体,间接非竞争ELISA分析所得抗血清的效价达10000以上。间接竞争ELISA显示所制备的抗血清能有效识别三聚氰胺,与氰脲酸、三聚氰酸酰胺、三聚氰酸二酰胺无交叉反应。利用自制抗体建立牛奶中三聚氰胺的间接竞争ELISA检测方法,对奶样作0.1μg/mL,1μg/mL,10μg/mL,100μg/mL,500μg/mL和1000μg/mL六个加标浓度的回收率在70.30%113.59%。

GA method was used to conjugate melamine to carrier protein to form immunogen(Melamine-BSA) and coated antigen(Melamine-OVA). Then the artificial antigen,melamine and carrier protein were each taken UV spectrum scan. The UV spectrum of artificial antigen had big difference compared to BSA and melamine,indicating the artificial antigen was primarily synthesized successfully. The molar ratio of melamine to BSA in artificial antigen was 8.56 ∶1. BALB/c small mice were immunized with immunogen (Melamine-BSA) through intraperitoneal,the titer of the anti-melamine antibodies were detected by indirect ELISA. The highest titer of the antiserum was more than 1 ∶10000. Indirect competitive ELISA showed that the antiserum could detect melamine specifically. There was no cross reaction among melamine and cyanuric acid,ammeline and cyanurodiamide. The recovery rate of milk on six levels of 0.1μg/mL,1μg/mL,10μg/mL,100μg/mL,500μg/mL and 1000μg/mL was among 70.30%113.59% by ELISA assay.