Immunoblotting Assay for Glyphosate-tolerant Genetically Modified Soybean in Soybean Products

식품 중 제초제내성 유전자재조합 콩의 immunoblotting 분석

  • Published : 2004.06.30

Abstract

Genetically modified (GM) soybean Roundup Ready carries Agrobacterium sp. CP4 gene, which expresses 5-enolpyruvylshikimate-3-phosphate synthase (CP4EPSPS). CP4EPSPS in GM soybeans and soybean curds was screened using CP4EPSPS-specific polyclonal and monoclonal antibodies (pab and mab, respectively) by immunoblotting. Isolated recombinant CP4EPSPS was detected at detection limits of $0.006\;and\;0.0006{\mu}g$, whereas those of CP4EPSPS expressed in GM soybean were $0.001\;and\;0.0001{\mu}g$g, using mab and pab, respectively. From nine screened soybean curds, two had positive results with pab Immunoblotting method with pab and mab developed in this study could be applied to screen glyphosate-tolerant GM soybeans in soybean products.

Keywords

immunoblotting;glyphosate-tolerant soybeans;CP4EPSPS;monoclonal antibody;polyclonal antibody

References

  1. James C. Global Status of Commercialized Transgenic Crops: 2002. ISAAA, New York, NY, USA (2002)
  2. Malik J, Barry G, Kishore G. The herbicide glyphosate. Biofactors 2: 17-25 (1989)
  3. Kwak BY, Ko SH, Park CW, Son DY, Shon DH. Development of enzyme-linked immunosorbent assay for glyphosate-tolerant soybeans. Korean J. Food Sci. Technol. 35: 366-372 (2003)
  4. Hupfer C, Hotzel H, Sachse K, Engel KH. Detection of the genetic modification in heat-treated products of Bt maize by polymerase chain reaction. Z. Lebensm. Unters. Forsch. 206: 203-207 (1998) https://doi.org/10.1007/s002170050243
  5. Studer E, Rhyner C, Luethy J, Huebner P. Quantitative competitive PCR for the detection of genetically modified soybean and maize. Z. Lebensm. Unters. Forsch. 207: 207-213 (1998) https://doi.org/10.1007/s002170050320
  6. Van Hoef AMA, Kok EJ, Bouw E, Kuiper HA, Keijer J. Development and application of a selective detection method for genetically modified soy and soy-derived products. Food Addit. Contam. 15: 767-774 (1998) https://doi.org/10.1080/02652039809374708
  7. Son DY, Lee SI. Detection of genetically modified glyphosate-tolerant soybeans. Food Sci. Biotechnol. 11: 105-109 (2002)
  8. Son DY, Ahn KM, Lee SI. Sequencing, cloning and expression of CP4EPSPS Roundup Ready soybean insert. Food Sci. Technol. 12: 133-136(2003)
  9. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 (1976) https://doi.org/10.1016/0003-2697(76)90527-3
  10. Rogan GJ, Dudin YA, Lee TC, Magin KM, Astwood JD, Bhakta NS, Leach JN, Sanders PR, Fuchs RL. Immunodiagnostic methods for detection of 5-enolpyruvylshikimate-3-phosphate synthase in Roundup Ready? sobybeans. Food Cont. 10: 407-414 (1999) https://doi.org/10.1016/S0956-7135(99)00083-3
  11. Awazuhara H, Kawai H, Maruchi N. Major allergens in soybean and clinical significance of IgG4 antibodies investigated by IgE- and IgG4-immunob1otting with sera from soybean-sensitive patients. Clin. Exp. Allergy 27: 325-332 (1997) https://doi.org/10.1111/j.1365-2222.1997.tb00711.x
  12. Padgette SR, Taylor NB, Nida D, Bailey MR, MacDonald J, Holden LR, Fuchs RL. Safety assessment of glyphosate-tolerant soybeans: The composition of the seeds is equivalent to conventional soybeans. J. Nutr. 126: 702-716 (1996)
  13. Steinruken HC, Amrhein N. The herbicide glyphosate is a potent inhibitor of 5-enolpyruvyl shikimic acid-3-phosphate synthase. Biochem. Biophis. Res. Commun. 94: 1207-1212 (1980) https://doi.org/10.1016/0006-291X(80)90547-1
  14. Rolf M. Development and application of DNA analytical methods for the detection of GMOs in foods. Food Cont. 10: 391-399 (1999) https://doi.org/10.1016/S0956-7135(99)00081-X
  15. Vollenhofer S, Burg K, Schmidt J, Kroath H. Genetically modified organisms in food-screening and specific detection by polymerase chain reaction. J. Agric. Food Chem. 47: 5038-5043 (1999) https://doi.org/10.1021/jf990353l
  16. Coly PV, Wyatt D, McCaughey C, O'Neill HJ. A simple standardised protocol for the production of monoclonal antibodies against viral and bacterial antigens. J. Immunol. Meth. 153: 81-84 (1992) https://doi.org/10.1016/0022-1759(92)90308-G
  17. Lipp M, Brodmann P, Pietsch K, Pauwels J, Anklam E. IUPAC collaborative trial study of a method to detect genetically modified soy beans and maize in dried powder. J. AOAC Int. 82: 923-928 (1999)
  18. Padgette SR, Kolacz KH, Delannay X, Re DB, Lavallee BJ, Tinius CN, Rhodes WK, Otero YI, Barry GF, Eichholtz DA, Peschke VM, Nida DL, Taylor NB, Kishore GM. Development, identification, and characterization of a glyphosate-tolerant soybean line. Crop Sci. 35: 1451-1461 (1996) https://doi.org/10.2135/cropsci1995.0011183X003500050032x
  19. Haslam E. Shikimic acid. In: Metabolism and Metabolites. John Wiley and Sons, Chichester, England (1993)
  20. Brett GM, Chambers SJ, Huang L, Morgan MRA. Design and development of immunoassays for detection of proteins. Food Cont. 10: 401-406 (1999) https://doi.org/10.1016/S0956-7135(99)00082-1
  21. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 (1970) https://doi.org/10.1038/227680a0
  22. Baird DD. Introduction of a new broad spectrum postemergence herbicide class with utility for herbaceous perennial weed control. North Central Weed Control Conf. 26: 64-68 (1971)
  23. Barry G, Kishore G, Padgette S, Taylor M, Kolacz K, Weldon M, Re D, Eichholtz D, Fincher K, Hallas L. Inhibitors of amino acid biosynthesis: Strategies for imparting glyphosate-tolerance to crop plants. In: Biosysnthesis and Molecular Regulation of Amino Acids in Plants. Singh BK, Flores HE, Shannon JC (eds). American Society of Plant Physiologists, Rockville, MD, USA (1992)
  24. Wurz A, Bluth A, Zeltz P, Pfeifer C, Willmund R. Quantitative analysis of genetically modified organisms (GMO) in processed food by PCR-based methods. Food Cont. 10: 385-389 (1999) https://doi.org/10.1016/S0956-7135(99)00080-8