Analytical chemistry council

METHODS AND OBJECTS OF CHEMICAL ANALYSIS

(Metody i Ob''ekty Himicheskogo Analiza)

An international journal devoted to all aspects of Analytical chemistry

ISSN 2413-6166 (Online), ISSN 1991-0290 (Print)

Kiev University
 

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Development of ELISA Kit for Detection of Glyphosate-Resistant Genetically Modified Soybean

Y.V. Khomenko, L.M. Ishchenko*, V.D. Ishchenko, S.V. Midyk, D.Y. Rybalchenko, V.O. Ushkalov, V.G. Spyrydonov

* Corresponding authors

National University of Life and Environmental Sciences of Ukraine, 15, Heroiv Oborony St., Kyiv, 03041;
*e-mail: ischenko_lm@ukr.net

Methods Objects Chem. Anal., 2019, 14(1), p. 21-29

https://doi.org/10.17721/moca.2019.21-29

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Abstract

The goal of research was development of competitive ELISA kit for detection of genetically modified plants resistant to glyphosate. As the source for gene isolation we have used leaves of GM soybean RoundUp Ready, line 40-3-2 (Monsanto). Using polymerase chain reaction the gene encoding the enzyme 5-enolpiruvilshykimat-3-phosphate synthase (CP4 EPSPS) was isolated, which provides resistance to the herbicide glyphosate. Isolated gene has been cloned into plasmid vector pET24(a), creating genetic construct pET-CP4EPSPS. E.coli (BL 21(DE3)) has been transformed by genetic construct and over expressed after induction by 0.5 mM IPTG. Recombinant enzyme CP4 EPSPS with molecular weight 45 kD was purified as inclusion bodies and used as antigen for 96-well polystyrene plates immobilization. Monoclonal antibodies CP4 EPSPS mAb3 (Artron BioResearch Inc., Canada) was used for synthesis of horseradish peroxidase conjugate and receiving diagnostic reagent. We have shown that in the competitive ELISA we can detect at least 0.1% of GM plants resistant to glyphosate, on the example of soybean (line RR 40-3-2), moreover we can also detect rapeseed (line GT73) and sugar beet (line H7-1).

Keywords: genetically modified plants, enzyme immunoassay, 5-enolpyruvylshikimate-3-phosphate synthase, monoclonal antibody

Article language: En


Publisher:Taras Shevchenko National University, Kiev Ukraine

(Analytical chemistry department at Taras Shevchenko National University, 64 Vladimirskaya STR., Kiev, 01601 UKRAINE)
analysis@univ.kiev.ua, www.achem.univ.kiev.ua, +380-44-2393345

Papers published in Methods and Objects of Chemical Analysis are considered as publications suitable for PhD thesis

The journal website: www.moca.net.ua