转Bt抗虫基因植物中杀虫蛋白的酶联免疫检测技术研究 Ⅲ.酶联免疫(ELISA)间接法检测Bt杀虫蛋白研究

采用ELISA间接检测法,研究了2种酶标羊抗兔抗体(辣根过氧化物酶与碱性磷酸酶)和2种检测载体(聚苯乙烯多孔板和硝酸纤维膜)对检测灵敏度和样本检测效果的影响。结果表明,在采用纯的杀虫晶体蛋白进行灵敏度检测时,2种酶标抗体的测定结果非常相近,无明显差别,灵敏度可达7·8~15·6ng。但在检测植物样本时,不同酶标抗体则存在差异。表现为碱性磷酸酶标记的抗体优于辣根过氧化物酶标记的抗体。这主要是由于植物样本中有内源过氧化物酶所致。用硝酸纤维素膜和聚苯乙烯多孔板作载体进行检测纯的Bt杀虫蛋白的效果二者相当。但检测植物样本时,植物中的叶绿素对Dot-ELISA的干扰很大,准确性差。本研究所制备的Bt杀虫蛋白抗体具有高的特异性。

Studies on the Enzyme-linked Immunosorbent Assay for Detection of Bacillus thuringiensis Insecticidal Protein Expressed in Transgenic Plants Ⅲ.Detection of Bt Insecticidal Protein by Indirect Enzyme-linked Immunosorbent Assay

The effects of second antibody conjugated with two enzymes (horseradish peroxidase and alkaline phosphatase) and two solid supports (polystyrene micro-well plate and nitrocellulose membrane) on sensitivity and detection results were investigated. The results show that when the pure Bt insecticidal protein is assayed by a indirect ELISA, there are no significant differences in sensitivity. The sensitivity of second antibody conjugated with alkaline phosphatase is equal to that of the second antibody conjugated with horseradish peroxidase. However, when the Bt insecticidal protein in cotton leaves is assayed on polystyrene plate, the detection value of horseradish peroxidase is higher than that of alkaline phosphatase. The reason is endogenous peroxidase activity in plant. There are some problems caused by chlorophyll in plant extract from leaf tissue when Dot-ELISA on nitrocellulose membrane is used to detect insecticidal protein in plant specimen. The antibody of Bt insecticidal protein prepared in this study is high specific.