بهينه سازي آزمايشات ELISA براي تشخيص و شناسايي پروتئين پسته در مواد غذايي

Optimisation of ELISA experiments for detection and quantification of pistachio protein in foods

Like other tree nuts, despite its high nutritional quality, pistachio carries specific proteins that elicit food-induced allergy symptoms in ccrtain individuals. In contrast to other tree nuts, little is known about the way of detecting pistachio allergenic proteins in foods. New labelling regulations require precise detection techniques to trace a specified list of allergenic food residues to the ppm level. In the present study pistachios were extracted using three extraction media i.e., (i) saline 10%, (ii) phosphate- buffered saline, and (iii) Tris buffer at pH 1.5 to find the best solution in order to optimise the extraction condition for a particular protein. In the second part, 2S proteins were isolated and purified from pistachios. Protein purification was carried out through a multiple stage procedure starting with an ammonium sulfate precipitation in which the concentration of the salt was increased step-by-step from 20% to 90%. The semi-purified protein fraction obtained was further purified using gel filtration chromatography. In the third part, antigens in the form of the purified 2S proteins from pistachio nut were used to produce antibodies in rabbits. Sensitive, specific ELISAs were set up and applied to 12 selected food samples extracted by 4 different methods of extraction (PBS, urea, PBS followed by acetone and urea followed by acetone) and 3 levels of antibody dilutions. The results showed that the higher total protein yield was not always an indication of an efficient extraction method for a particular protein. The results of determination of pistachio protein residues also proved that immunochemical techniques can reliably detect traces amounts of pistachios in food samples.