Evaluation of an ELISA assay for rapid detection and quantification of neomycin phosphotransferase II in transgenic plants

摘要

Neomycin phosphotransferase II (neo) is a selectable marker gene used extensively in plant transformation experiments. Here we evaluate immunological detection of its gene product (NPTII) as an alternative to widely used radioactive assays. We have taken a commercially available non-radioactive NPTII Enzyme linked-Immunosorbant Assay (ELISA) kit, modified the protocol for application to plant tissues, and used it to quantify levels of NPTII protein in transformed plants. The ELISA proved safe, economical and convenient to reliably screen and quantify NPTII protein in large numbers of plant samples. The sensitivity of the ELISA for NPTII detection in tobacco plants is at least an order of magnitude greater than a widely used radioactive gel assay. Using three replicates per sample, standard errors are low and the assay is highly reproducibleover time for tissue-cultured tobacco. However, background readings varied with plant species, and also with plant age for untransformed glasshouse-grown tobacco. It is therefore essential to ensure that untransformed controls are closely matched to test plant.