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Multiplexed Sandwich Assay for Quantitation of Transgenic Proteins in Genetically Modified Plants Using the Luminex® FLEXMAP 3D® System
Brad Mire, Harold N. Baker, Thomas Copa, Michaela R. Hoffmeyer
Luminex Corporation, Austin, TX
Sponsored by Luminex B.V
Published in December 2010 2010 (p.3) DOI:  10.2144/000113596 Sponsored,vendor-submitted protocol
Methods for testing transgene copy number and protein expression are necessary for development of genetically modified crops (GMO). Most transgenes impart herbicide resistance or protection from crop damaging insects and are proprietary. With the recent increase in stacking of multiple traits in GMO crop species, the demand for a high throughput, sensitive, and multiplexed testing method is growing. Current ELISA-based testing methods prove problematic with the complex plant homogenate matrix, resulting in poor reproducibility, dynamic range, and sensitivity. We have developed a magnetic microsphere-based quantitative multiplex assay on the FLEXMAP 3D® system to detect proprietary GMO proteins in several crop species. Antibodies against the GMO targets were screened and the best performers were coupled to MagPlex® microspheres. Sample plant homogenates from cotton, corn, and soybean were diluted 1:100, and added to the coupled microspheres, followed by addition of biotinylated detection antibody and reporter. Reactions were analyzed on the Luminex® FLEXMAP 3D system at the high reporter gain setting. The assay, which takes < 2 hours, exhibits excellent reproducibility (CVs of ≤8% across all targets and species) and dynamic range of 4.5 to 5 logs.
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