Application of immunoaffinity column as cleanup tool for an enzyme linked immunosorbent assay of phosphinothricin-N-acetyltransferase detection in genetically modified maize and rape.
We have developed a new immunoassay method to detect genetically modified (GM) maize and rape containing phosphinothricin-N-acetyltransferase (PAT). PAT encoded by Bialaphos resistance gene (bar) was highly expressed in soluble form in Escherichia coli BL21(DE3) and purified to homogeneity by Ni2+ affinity chromatography. A simple and efficient extraction and purification procedure of PAT from GM maize and rape was developed by means of the immunoaffinity column (IAC) as a cleanup tool. Purified polyclonal antibodies against PAT was produced and coupled covalently to CNBr-activated Sepharose 4B. Both the binding conditions and elution protocols were optimized. The IAC was successfully employed to isolate and purify the PAT from the various tissues of GM maize (Bt11 and Bt176) and rapes (MS1/RF1 and MS8/RF3). Enzyme linked immunosorbent assay (ELISA) procedures were established further on to measure the PAT protein. GM maize cannot be differentiated from non-GM maize by ELISA. But IAC-ELISA allowed 0.5% GMOs to be detected in MS1/RF1 and MS8/RF3 and 10% GMOs to be detected in Bt11 and Bt176, which makes this method an acceptable method to access PAT protein in GM rapes and maize.
Xu
Wentao
W
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
Huang
Kunlun
K
Zhao
Heng
H
Luo
Yunbo
Y
D000123:Acetyltransferases / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001483:Base Sequence; D029741:Brassica rapa / Q000201:enzymology / Q000235:genetics*; D002846:Chromatography, Affinity / Q000379:methods; D018744:DNA, Plant / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015870:Gene Expression; D007118:Immunoassay / Q000379:methods*; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000201:enzymology*; D003313:Zea mays / Q000201:enzymology / Q000235:genetics*
D018744:DNA, Plant; D000123:Acetyltransferases; C056243:phosphinothricin N-acetyltransferase