Establishment of an ELISA Method for Quantitative Detection of PAT/pat in GM Crops

Abstract

The phosphinothricin N-acetyltransferase gene (pat) is widely used to confer resistance to the herbicide phosphinothricin for genetically modified (GM) crops. A quantitative sandwich enzyme-linked immunosorbent assay (ELISA) is developed to detect PAT/pat in GM crops. Two anti-PAT/pat monoclonal antibodies (mAbs), 1F5-1F2 and 1B6-2D3, with titers of 1:1,024,000 and 1:896,000, respectively, against overexpressed His-PAT/pat, were screened out, raised, and characterized. An ELISA method was established with the 1F5-2F2 mAb for capture and the biotin-labeled 1B6-2D3 mAb for detection of PAT/pat. The linear detection range of the method was approximately 1.5625–12.5 ng/mL, with a sensitivity of 0.085 ng/mL and a coefficient of variation (CV) less than 5.0%. No cross-reactivity was found with other herbicide resistance proteins, especially PAT/bar. The established sensitive and specific ELISA was successfully applied in the detection of PAT/pat expression in GM crops.