DEVELOPMENT OF NEW ANALYTICAL TOOLS FOR GMO DETECTION: OBTENTION OF ANTIBODIES FOR AN ELECTROCHEMICAL IMMUNOSENSOR

M. FARIAS ; S. STAGNOLI ; M. A. LUNA; M. M. MARANI; A. NIEBYLSKI; N. M. CORREA; P. MOLINA

Rio Cuarto

Workshop; III Workshop "Protein-based vs DNA Strategies for GMO detection"; 2015

Grupo de Sistemas Organizados (UNRC)

In recent years, agricultural enterprises in different countries have developed new plant varieties by adopting modern biotechnology, including genetic transformation. The use of genetically modified organisms (GMO) has been a topic of extensive discussion. The potential risks related to immediate and/or delayed adverse effects on human health environment and farmer´s economy are negative issues regarding GMO that cannot be ruled out.The soybean GTS 40-3-2 event is commercially known as Roundup ReadyTM (RR) soybean since it was especially developed to be tolerant to the herbicide with the same name. This event contains an introduced construction with a gene of cp4 epsps (aroA:CP4) from the Agrobacterium tumefaciens strain CP4 expressing the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) that decreases the binding affinity to glyphosate, conferring increased tolerance to the glyphosate herbicide. Many other transgenic constructions also include the referred gene expressing the CP4-EPSPS protein, which can be a potential marker for GMOscreening. Using bioinformatics, the CP4-EPSPS protein was in silico cleaved with trypsin/chymotrypsin and the resultant peptides were extensively analyzed for further selection of the best candidatepeptides for antibody production.In this work we propose to use a set of sintetic peptides that could function as potential immunogens for the production of novel antibodies for the CP4-EPSPS biorecognition. The first step of the process is to combine the selected peptides with bovine serum albumine (BSA) as carrier protein, to enhance recognition of the antigen by the immune system. The reaction is carried out with one single peptide and, on the other hand, a mixture (two or three) of them. Conjugates are administered to three groups of adult mice Balb-C by subcutaneous injection according to immunization protocol, each group with a different dose of antigen (10, 50 y 100 µg/mL). Simultaneously, others routes of inoculation and different animal species are used to optimize the antibodies production.The production of polyclonal antibodies is evaluated by Outcherlony test on serum samples taken at 25 and 35 days after de first injection. After a positive result, the purification is carried out using an affinity column with the tested peptide/s.Once obtained the antibodies will be used for the construction of an electrochemical inmunosensor for GMO detection. This detector will enable a quantitative analysis with very low limit of detection and high specificity for transgenic protein CP4-EPSPS, with the advantage of a faster and less expensive method compared with others analytical techniques.