|Isleib, Thomas -|
Submitted to: International Journal of Food Science and Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 21, 2011
Publication Date: May 31, 2011
Citation: Hurlburt, B.K., Schmitt, D.A., Isleib, T.G., Cheng, H., Garvey, C.E., Koenig, R.L., Maleki, S.J. 2011. Production of pure protein antibodies and development of immunoassays to detect Ara h 3 levels in peanut varieties. International Journal of Food Science and Technology. 46:1477-1484. Interpretive Summary: Peanuts are one of the most allergenic foods, and are widespread in western food products. There is a need for the development of hypoallergenic products as well as diagnostic tools; therefore, there has been intense research into the allergic nature of the proteins involved. Three dominant proteins have been identified as the major allergens. In this work, one of those proteins, Ara h 3, was studied. Towards developing better diagnostic tools, a recombinant version of Ara h 3 was expressed and purified. The pure protein was used to generate antibodies that can be used to detect Ara h 3 in food products; furthermore, the antibodies have been used to screen hundreds of varieties of peanuts some of which have reduced Ara h 3 levels.
Technical Abstract: Peanuts are one of the most allergenic foods, and are widespread in western food products; therefore, there has been intense research into the allergic nature of the proteins involved. Ara h 3 is one of three immunodominant allergenic proteins responsible. It is a 60 kDa protein which forms following cleavage of the preprotein, and subsequent association of the resultant 40 and 20 kDa subunits. The large subunit has been shown to harbor most of the reactive epitopes, and has the protein fold likely responsible for its trypsin inhibitor activity. In this work, we have developed a method for the high-level expression and purification of recombinant Ara h 3, 40 kDa subunit. Specific antibodies have been produced and applied to the secondary and tertiary screens of hundreds of peanut cultivars. Several of these cultivars were identified that have significantly reduced accumulation of Ara h 3.