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Implementation of procedures for extraction and quantification of proteins in genetically modified plants by ELISA (Enzyme Linked Immunono Sorbent Assay)

Abstract

Tecam Laboratories is a national company, which carries out a broad scope of analyses in the areas of physical chemistry, microbiology, molecular biology, toxicology and ecotoxicology. The laboratory is accredited by INMETRO in quality standards according to Good Laboratory Practice (GLP) and ABNT NBR ISO/IEC 17025, is also Certificate in Biosafety (CQB) to work with Genetically Modified Organism (GMO). The molecular biology laboratory began its activities in 2001, supported by FAPESP-PIPE, offering analysis of GMOs in soybean and corn by the technique of polymerase chain reaction (PCR) to the food market. Since 2006, the physical chemistry laboratory performs chemical composition analysis for comparative plant studies between GM and conventional. In addition, companies that develop GMOs utilize the services in the areas of Toxicology, ecotoxicology and mutagenicity. The arrival of genetically modified food in the market arose the need to carry out safety assessment of biotechnology-derived organisms to human and animals’ health, as well as the environment. For conventional foods, obtained by classical breeding techniques, the history of use has always been regarded as sufficient to consider them safe. The requirements for risk assessment of foods derived from genetically modified organisms vary from country to country. The divergences between different countries should not be used as a commercial barrier, in 2003 the Codex Alimentarius Commission, responsible for establishing international standards for foods, published the principles for human health risk assessment of GMOs to prevent this kind of problem. Regarding the safety assessment of food derived from biotechnology, the Codex establishes relevant factors about: a) expressed substances (except nucleic acids); b) compositional analysis; c) analysis of metabolites; d) food processing and d) nutritional modifications. Currently, there is a consensus regarding the adoption of the Principle of Substantial Equivalence in safety assessment of GMOs, which consists in the comparison of the characteristics between plants modified and conventional. The National Technical Biosafety Commission (CTNBio), responsible for the approval of GMO foods in Brazil, also adopts the principle of substantial equivalence for biosafety assessment of GMOs. Tecam Laboratories carries out composition analysis such as macro and micronutrients, anti-nuticionais factors, metabolites, however, does not perform the quantitation of proteins expressed in transgenic plant. Typically, the companies producing GMO carry out these analyses in laboratories of its research centers or laboratories overseas service providers. There is demand but not provider to perform transgenic protein quantification in Brazil, due to lack of laboratory training in this specialty. The project aims to empower Tecam Laboratories offering studies using the ELISA method to perform quantification of protein expressed by genetically modified organisms according with Good Laboratory Practice. The same sample received by Tecam Laboratories to perform GMO compositional analysis could have the quantification of GMO protein analyzed. In this way Tecam could offer a more fully service to regular customers as well as prospect new customers as a consequence of this differential. In phase 1, we plan to run a pilot experiment based on construction of a standard curve for 2 m-EPSPS protein (which confers tolerance to the glyphosate herbicide on cotton). Once standardized curve, a sample of conventional cotton will be prepared and fortified with different amounts of 2 m-EPSPS protein. The fortified samples shall be submitted for protein extraction and quantified by ELISA test method to verify the recoverability of 2 m-EPSPS protein after extraction procedures. (AU)