A selective enzyme-linked immunosorbent assay (ELISA) was developed and validated to quantify double mutant 5-enolpyruvylshikimate-3-phosphate synthase (2mEPSPS) protein expression in soybean tissues. The 2mEPSPS protein is a modified maize EPSPS protein that when expressed in soybean, confers tolerance to glyphosate herbicides. The ELISA method developed to quantify this protein uses a sequential sandwich format in which soybean sample extracts are incubated in the wells of an immobilized anti-2mEPSPS polyclonal antibody coated plate. An enzyme-conjugated anti-2mEPSPS protein monoclonal antibody is sequentially added to the wells, which will bind to the immobilized polyclonal antibody-protein pair on the plate. The resulting assay has a quantitative range of 8 - 200 ng/mg dry tissue weight, with a lower limit of detection of 4 ng/mg dry tissue weight. The method showed no cross-reactivity to other herbicide tolerance transgenic proteins including CP4 EPSPS protein, and no false positive or false negative results were observed. Protein extraction efficiency was above 84% using a phosphate-buffered saline solution supplemented with Tween-20 and casein. Average protein recovery of fortified samples was above 76%. Method precision and ruggedness was assessed through inter-assay variability, which did not exceed 8% (%CV). Matrix effects were observed at lower sample dilution levels, requiring samples to be diluted at a 1:10 minimum dilution for accurate quantitation. All validation results confirmed that the 2mEPSPS ELISA method is suitable for its intended use. Application of the validated method in soybean tissue samples will also be discussed.

Development and validation of a quantitative enzyme-linked immunosorbent assay for the detection of 2mEPSPS protein in soybean tissues

246th National Meeting (2013)

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