1. Determination of phosphinothricin acetyltransferase in genetically transformed canola seed by a two-antibody sandwich enzyme immunoassay
- Author
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Brigitte Bauer-Weston, Arno Schulz, Michael M. Oelck, and Raymond J. A. Deschamps
- Subjects
food.ingredient ,Chromatography ,medicine.diagnostic_test ,biology ,Coefficient of variation ,Plant Science ,Absorbance ,Standard curve ,food ,Biochemistry ,Immunoassay ,Phosphinothricin acetyltransferase ,medicine ,biology.protein ,Antibody ,Canola ,Molecular Biology ,Selectable marker - Abstract
We report a quantitative, two-antibody sandwich immunoassay for phosphinothricin acetyltransferase (PAT), the selectable marker protein. The method yielded a standard curve with a working range of 0 to 5 ng PAT per mL extract. Replicate absorbance values for standards within a single assay showed a coefficient of variation typically less than 5 percent. Over three separate assays, the coefficient of variation for the slope and y-intercept of the standard curve was 3.8 and 3.7 percent, respectively. TransgenicBrassica napus L. seed was used to demonstrate the utility of the assay. Non-transgenic seed extracts did not show a positive immunoassay signal. Determinations of the PAT enzyme conducted on spiked non-transgenic seed extracts repeated in three separate assays fell in the acceptable range of 80 to 120 percent recovery. Transgenic canola seed, analyzed in three separate assays, showed a mean PAT enzyme content of 403 ng/g with a standard error of 19 ng/g and a coefficient of variation of 8.1 percent. The method has also been applied to several other tissues and processed products of canola, maize, and tobacco.
- Published
- 1996
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