Application of mouse monoclonal antibody (mAb) 4C10-based enzyme-linked immunosorbent assay (ELISA) for amandin detection in almond (Prunus dulcis L.) genotypes and hybrids Mengna Su a, 1 , Mahesh Venkatachalam a, 2 , Thomas M. Gradziel b , Changqi Liu a , Ying Zhang a , Kenneth H. Roux c , Shridhar K. Sathe a, * a Department of Nutrition, Food & Exercise Sciences, Florida State University, Tallahassee, FL 32306, USA b Pomology Department, University of California, Davis, CA 95616, USA c Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA article info Article history: Received 3 September 2013 Received in revised form 29 June 2014 Accepted 31 August 2014 Available online 6 September 2014 Keywords: Almond Amandin ELISA Genotypes/hybrids Immunoassay abstract Amandin presence in 108 almond genotypes/hybrids and 80 almond marketing varieties grown in different locations was determined using murine monoclonal antibody 4C10-based sandwich ELISA. The results indicated that amandin was present in all the tested samples. The ELISA immunoreactivity var- iations were up to 8 fold among genotypes/hybrids and 2.5 fold among the almond marketing varieties. Amandin content variations were also confirmed using Western blot and dot blot. No correlation was observed between almond seed size and total soluble protein or amandin content. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Almonds are globally popular edible tree nut seeds and are consumed in a variety of forms. Among tree nuts, almonds rank number one in global consumption and the US is the global leader in almond production and exports (Almond Almanac, 2013). Valued for their sensory and nutritional qualities almond seeds are safely enjoyed by most. However, sensitive individuals, those who suffer from Type I allergies caused by the almond seed proteins, need to avoid unwarranted exposure to almond seeds. Type I allergies are immunoglobulin E (IgE)- mediated food allergies caused by some food proteins that serve as both the original sensitizing agent and, subsequently, as the symptom-eliciting agent. To prevent the unwarranted exposure to almond seeds by allergic individuals, the presence of almond seeds in a food and/ or a food ingredient must be known. For the purpose of almond seed detection, amandin, also known as almond major protein (AMP), prunin, 11S globulin, and Pru du 6, is a good target protein for several reasons including: a) comprising ~ 65 g/100 g of the water-soluble almond seed proteins (Wolf & Sathe, 1998); b) a recognized major allergen (Roux, Teuber, Robotham, & Sathe, 2001); c) representing a major seed storage protein in marketed almond varieties (Sathe, 1993) and almond genotypes and hybrids (Sathe, Teuber, Gradziel, & Roux, 2001); d) demonstrated stability towards processing (Su, Venkatachalam, Teuber, Roux, & Sathe, 2004; Venkatachalam, Teuber, Roux, & Sathe, 2002); e) biochemically well characterized (Sathe et al., 2002); and f) extensive epitope (portion of molecule recog- nized by the almond allergic patient sera IgE) mapping (Willison et al., 2013). Earlier, we reported a non-competitive, sensitive (1e 10 ng/mL amandin), anti-amandin rabbit polyclonal antibody (pAb)-based enzyme-linked-immunosorbent assay (ELISA) for almond detec- tion (Acosta, Roux, Teuber, & Sathe, 1999). The rabbit pAb-based ELISA is sensitive and is a useful screening tool to detect the pres- ence of almond seeds. Further testing of the assay (Tiwari et al., 2010) revealed that although the assay is a useful screening tool, cross-reactivity of the anti-amandin rabbit pAbs may interfere with * Corresponding author. Department of Nutrition, Food & Exercise Sciences, The Florida State University, Tallahassee, FL 32306-1493, USA. Tel.: þ1 (850) 644 5837; fax: þ1 (850) 645 5000. E-mail address: ssathe@fsu.edu (S.K. Sathe). 1 Current address: Fujian Hwanan Women's College, No. 66 XueYuan Rd, Uni- versity City, Fuzhou, Fujian 350108, PR China. 2 Current address: Pfizer Inc.,1211 Sherwood Avenue, Richmond, VA 23220, USA. Contents lists available at ScienceDirect LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt http://dx.doi.org/10.1016/j.lwt.2014.08.042 0023-6438/© 2014 Elsevier Ltd. All rights reserved. LWT - Food Science and Technology 60 (2015) 535e543