Electrophoretic and Immunological Analyses of Almond (Prunus dulcis L.) Genotypes and Hybrids

Abstract

Aqueous extracts from sixty almond samples representing various genotypes and interspecies hybrids of almond, including almond-peach, were analyzed for protein and peptide content using electrophoresis, Western immunoblotting, and enzyme-linked immunosorbent assay (ELISA). Nondenaturing nondissociating polyacrylamide gel electrophoresis (NDND-PAGE) of the aqueous extracts indicated that a single major storage protein (almond major protein -- AMP or amandin) dominated the total soluble protein composition. Denaturing SDS--PAGE analyses of the aqueous extracts revealed that the AMP was mainly composed of two sets of polypeptides with estimated molecular masses in the ranges of 38--41 kDa and 20--22 kDa, regardless of the source; however, distinct variations in the intensity and electrophoretic mobility of some bands were noted between samples. In addition to AMP, several minor polypeptides were also present in all the genotypes, and variations were seen in these as well. Regardless of the genotype, AMP was recognized in Western blots by rabbit polyclonal anti-AMP antibodies, mouse monoclonal anti-AMP antibodies (mAbs), and serum IgE from patients displaying strong serum anti-almond IgE reactivity. As with protein staining results, antibody reactivity also revealed common patterns but displayed some variation between samples. An anti-AMP inhibition ELISA was used to quantify and compare aqueous extracts for various samples. All samples (n = 60) reacted in this assay with a mean +/- standard deviation (sigma n) = 0.82 +/- 0.18 when compared to reference aqueous extract from Nonpareil designated as 1.0.

Tree Nut Allergens

“…As a tree nut allergen, it ranks third (15% reactive) behind walnut and cashew nut in the FAAN self reporting survey of tree nut allergies [45]and can be found in many foods. Despite these statistics and despite the fact that the proteins in almonds have been biochemically characterized [60, 61, 62, 63, 64, 65, 66, 67], relatively little has been reported regarding the identity of the allergens involved in almond sensitivity.…”

“…Sathe et al [60], Osborne and Campbell [64]and Acosta et al [65]have reported the purification and biochemical characterization of the major soluble seed storage protein in almond, a 14S globulin (legumin) known as almond major protein (AMP) or, from early (1897) descriptions, as amandin. Amandin is a complex nonglycosylated storage protein with a molecular weight estimated to be in the 375- to 475-kD range and constitutes 65–70% of the extractable proteins in almond [66]. SDS-PAGE under reducing conditions separated two major pairs of polypeptides in the 42- to 64-kD range and 20- to 27-kD range that appeared to be the respective disulfide-linked acidic and basic polypeptides of amandin, consistent with the classical structural model of legumin proteins.…”

“…The various peptides recognized by human IgE appear to be inherently immunogenic in that rabbit and goat polyclonal and mouse monoclonal IgG Abs recognize most of the same bands as does human IgE on immunoblots [60, 66, 67], and unreported data]. The amandin polypeptides are highly resistant to the wet and dry heat used during food processing and have been shown to be excellent marker proteins for immunoassays to detect almond contamination in foods [67, 69].…”

Cloning, Expression and Patient IgE Reactivity of Recombinant Pru du 6, an 11S Globulin from Almond

“…Almond protein extract has been extensively studied by Sathe et al [7] and Wolf and Sathe [11], who showed that prunin is a major component of the nut, accounting for up to 65% of the protein in a soluble aqueous extract. Immunoblotting assays using rabbit polyclonal and murine monoclonal anti-prunin antibodies reveal that a wide variety of commonly consumed almond cultivars have similarly high levels of prunin [12]. Immunoblot assays have also been performed using a pool of sera from almond-allergic patients to demonstrate high levels of IgE binding to presumptive prunin bands in aqueous almond extract [12,13].…”

Review of genetic diversity studies in almond (Prunus dulcis)

“…Isoenzymes may be encoded by genes at different loci or different alleles at the same locus (known as alloenzymes or allozymes). The detection of isoenzymes was the first molecular marker strategy to be used, because of their co-dominant expression and good reproducibility (Arulsekar et al, 1986;Hauagge et al, 1987a, b;Cerezo et al, 1989;Foolad et al, 1995;Vezvaei et al, 1995;Sathe et al, 2001). Isozyme studies have detected high levels of genetic variability in almond and allowed the individual identification of most genotypes studied.…”