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Enzyme linked immunosorbent assay for PAT protein detection in genetically modified rape

Published online by Cambridge University Press:  20 March 2007

Xu Wen-Tao ,
Huang Kun-Lun ,
Deng Ai-Ke  and
Luo Yun-Bo
Xu Wen-Tao
Affiliation:
College of Food Science and Nutrition Engineering, China Agricultural University, Beijing 100083, China
Huang Kun-Lun
Affiliation:
College of Food Science and Nutrition Engineering, China Agricultural University, Beijing 100083, China
Deng Ai-Ke
Affiliation:
College of Food Science and Nutrition Engineering, China Agricultural University, Beijing 100083, China
Luo Yun-Bo*
Affiliation:
College of Food Science and Nutrition Engineering, China Agricultural University, Beijing 100083, China
*
*Corresponding author. E-mail: [email protected]
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Abstract

We have developed and applied an immunoassay method to detect genetically modified (GM) rape containing phosphinothricin acetyltransferase (PAT). The purified PAT was identified by Western blotting and enzymic activity analysis. The polyclonal antibody against purified PAT protein was obtained and purified by both a saturated ammonium sulphate method and protein A-Sepharose 4B. The sensitivity and cross-reactivity of the polyclonal antibody has been demonstrated in an enzyme-linked immunosorbent assay (ELISA). The result of the ELISA for antiserum sensitivity was about 2×10−5 mg/ml and the cross-reactivity determined experimentally showed a high degree of specificity for the antiserum used, because values were all less than 0.1%. Detection of transgenic plants was evaluated using two transgenic rape lines (MS1/RF1 and MS8/RF3) which could be easily distinguished by ELISA.

Keywords

ELISAgenetically modified organism detectionphosphinothricin acetyltransferase
Type
Research Article
Information
Chinese Journal of Agricultural Biotechnology , Volume 3 , Issue 3 , December 2006 , pp. 177 - 181
Copyright
China Agricultural University and Cambridge University Press 2006

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References

Anklam, E, Gadani, F, Heinze, P, Pijnenburg, H and Van Den Eede, G (2002) Analytical methods for detection and determination of genetically modified organisms in agricultural crops and plant-derived food products. European Food Research and Technology 214: 3–26.CrossRefGoogle Scholar
Anzai, H, Ishii, Y, Shichinohe, M, et al. (1996) Transformation of Phalaenopsis by particle bombardment. Plant Tissue Culture Letter 13: 265–272.CrossRefGoogle Scholar
Ba, D (1998) Contemporary Immunological Technology and Application. Beijing, China: Joint Press of Beijing Medical Sciences University and China Union Medical College, p. 312.Google Scholar
Bradford, MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72: 248–254.CrossRefGoogle ScholarPubMed
Brodmann, PD, Ilg, EC, Berthoud, H and Herrmann, A (2002) Real-time quantitative polymerase chain reaction methods for four genetically modified maize varieties and maize DNA. Journal of AOAC International 85(3): 646–653.CrossRefGoogle ScholarPubMed
Chen, C, Ratcliffe, NA and Rowley, AF (1993) Detection, isolation and characterization of multiple lectins from the haemolymph of the cockroach, Blaberus discoidalis. Journal of Biochemistry 294: 181–191.CrossRefGoogle ScholarPubMed
Deblaere, R, Reynaerts, A, Hoefte, H, Hernalsteens, JP, Leemans, J and Van, MM (1987) Vectors for cloning in plant cells. Methods in Enzymology 153: 277–292.CrossRefGoogle Scholar
DeBlock, M, DeBrouwer, D and Tenning, P (1989) Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bar and neo genes in the transgenic plants. Plant Physiology 91: 694–701.CrossRefGoogle Scholar
D'Halluin, K, Bossut, M, Bonne, E, Mazur, B, Leemans, J and Botterman, J (1992) Transformation of sugarbeet (Beta vulgaris L.) and evaluation of herbicide resistance in transgenic plants. Biotechnology 10: 309–314.Google Scholar
James, C (2003) Global hectarage of GM crops in 2002. ISAAA (International Science for the Acquisition of Agri-biotech Applications) Briefs 3(1): 2–5.Google Scholar
James, D, Schmidt, AM, Wall, E, Green, M and Masri, S (2003) Reliable detection and identification of genetically modified maize, soybean and canola by mutiplex PCR analysis. Journal of Agriculture and Food Chemistry 51: 5829–5834.CrossRefGoogle Scholar
Laemmli, UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680–685.CrossRefGoogle ScholarPubMed
Lipp, M, Anklam, E and Stave, JW (2000) Validation of an immunoassay for detection and quantitation of a genetically modified soybean in food and food fractions using reference materials: interlaboratory study. Journal of AOAC International 83(4): 919–927.CrossRefGoogle ScholarPubMed
McKenzie, MJ, Mett, V and Jameson, PE (2000) Modified ELISA for the detection of neomycin phosphotransferase II in transformed plant species. Plant Cell Reports 9: 286–289.CrossRefGoogle Scholar
Meyer, R (1999) Development and application of DNA analytical methods for the detection of GMOs in food. Food Control 10: 391–399.CrossRefGoogle Scholar
Petit, L, Baraige, F, Balois, AB, Bertheau, Y and Fach, P (2003) Screening of genetically modified organisms and specific detection of Bt176 maize in flours and starches by PCR-enzyme linked immunosorbent assay. European Food Research and Technology 217: 83–89.CrossRefGoogle Scholar
Rathore, KS, Chowdhury, VK and Hodges, TK (1993) Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts. Journal of Plant Molecular Biology 21(5): 871–884.CrossRefGoogle Scholar
Stave, JW (2002) Protein immunoassay methods for detection of biotech crops: applications, limits, and practical considerations. Journal of AOAC International 85(3): 780–786.CrossRefGoogle ScholarPubMed
Studer, E, Rhyner, C, Luthy, J and Hubner, P (1998) Quantitative competitive PCR for the detection of genetically modified soybean and maize. Zeitschrift fuer Lebensmittel-Untersuchyng und Forschung A 207: 207–213.CrossRefGoogle Scholar
Thompson, CJ, Movva, RN, Crameri, R, Davies, JE, Lauwereys, M and Botterman, J (1987) Characterisation of the herbicide gene bar from Streptomyces hygroscopicus. EMBO Journal 9: 2519–2523.CrossRefGoogle Scholar
Xu, WT, Huang, KL and Luo, YB (2004) High expression of bar gene in Escherichia coli and isolation and purification of Its expressed protein. Journal of Agricultural Biotechnology 12(5): 583–588 (in Chinese with English abstract).Google Scholar
Yamazaki, M, Son, L, Hayashi, T, et al. (1996) Transgenic fertile Scoparia dulcis L., a folk medicinal plant, conferred with a herbicide-resistant trait using an Ri binary vector. Plant Cell Reports 15: 317–321.CrossRefGoogle Scholar
Zhao, MP, Liu, Y, Li, YZ, Zhang, XX and Chang, WB (2003) Development and characterization of an immunoaffinity column for the selective extraction of bisphenol A from serum samples. Journal of Chromatography B 783: 401–410.CrossRefGoogle ScholarPubMed
Zimmermann, A, Hemmer, W, Liniger, M, Luthy, J and Pauli, U (1998) A sensitive detection method for genetically modified Maisgard™ corn using a nested PCR-system. Lebensmittel-Wiss. u-Technology 31: 664–667.CrossRefGoogle Scholar