Quantitative analysis of phosphinothricin-N-acetyltransferase in genetically modified herbicide tolerant pepper by an enzyme-linked immunosorbent assay.
An immunoassay method was developed to quantitatively detect phosphinothricin-N-acetyltransferase (PAT) encoded by the Bialaphos resistance (bar) gene in genetically modified (GM) pepper. The histidine-tagged PAT was overexpressed in Escherichia coli M15 (pQE31-bar) and efficiently purified by Ni2+ affinity chromatography. A developed sandwich enzyme-linked immunosorbent assay (S-ELISA) method (detection limit: 0.01 microg/ml) was 100-fold more sensitive than a competitive indirect ELISA (CI-ELISA) method or Western blot analysis in detecting the recombinant PAT. In real sample tests, PAT in genetically modified herbicide-tolerant (GMHT) peppers was successfully quantified [4.9 +/- 0.4 microg/g of sample (n = 6)] by the S-ELISA method. The S-ELISA method developed here could be applied to other GMHT crops and vegetables producing PAT.
Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, Saskatchewan S7N OX2, Canada.
Youn-Young
YY
Shim
Weon-Sun
WS
Shin
Gi-Seong
GS
Moon
Kyung-Hwan
KH
Kim
D000123:Acetyltransferases / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D000818:Animals; D002212:Capsicum / Q000201:enzymology / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006540:Herbicides / Q000494:pharmacology*; D030821:Plants, Genetically Modified; D011817:Rabbits
D006540:Herbicides; D000123:Acetyltransferases; C056243:phosphinothricin N-acetyltransferase