草甘膦残留的酶联免疫分析方法的建立

Establishment of the Enzyme-linked Immunosorbent Assay for Glyphosate Residues

通过碳化二亚胺法将草甘膦(Glyphosate,GLY)分别与卵清蛋白(OVA)和牛血清白蛋白(BSA)偶联制备免疫原和包被原。基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)检测表明,草甘膦与牛血清白蛋白和卵清蛋白的偶联比分别为11∶1和9∶1。合成的免疫原作用新西兰大白兔制备出草甘膦多克隆抗体,ELISA检测抗体溶液效价为1∶1×107,该抗体不与草甘膦的结构类似物增甘膦和双甘膦起交叉反应。以此抗体建立了草甘膦间接竞争ELISA检测方法(ciELISA),其线性范围为0.78～100μg/mL,线性回归方程为y=15.851 0 lnx+7.780 5,R2=0.993 8。草甘膦的检测限为IC10=1.15μg/mL,IC50=14.35μg/mL。在添加回收试验中,草甘膦添加值5和10μg/g时,玉米粉中的草甘膦回收率为104.12%和81.37%,小麦粉中的回收率则分别为118.94%和为109.39%。结果表明,采用所制备的多克隆抗体而建立的草甘膦ciELISA方法可有效地应用于玉米粉和小麦粉中草甘膦的残留检测。

The diimine carbonization methods were used to synthesize immunogen and coating antigen by glyphosate being conjugated to ovalbumin （OVA） and bovine serum albumin （BSA）, respectively. The results showed the ratios of hapten to BSA and OVA were 11：1 and 9：1 by matrix-assisted laser desorption ionization-time of flight mass spectrometry （MALDI- TOF-MS）. The polyclonal antibodies against glyphosate were produced through immunizing New Zealand white rabbits with the immunogen. The titer of antibody against glyphosate was 1：1 ×107 by the test of the enzyme-linked immunosorbent assay （ELISA）. The glyphosate polyclonal antibodies did not cross-react with glyphosine and N-（Phosphonomethyl） iminodiaeetic. An indirect competitive ELISA （ciELISA） was developed for glyphosate detection, which has a detection limit （ICto） of 1.15 txg/mL and a linear working range of 0.78-100 p,g /mL with an IC5o value of 14.35μg/mL. The regression equation was y= 15.851 01nx＋7.780 5,R^2 was 0.993 8. When 5 and 10 μg/g glyphosate was added in the recovery exeperiments, the recoveries of glyphosate were 104.12% and 81.37% in corn meal samples, while 118.94% and 109.39% in wheat flour blank samples, respectively. The experiment results revealed that the eiELISA on the basis of the prepared antibodies against glyphosate was able to effectively detect the glyphosate residues in corn meal and wheat flour samples.