Effect of temperature on structural configuration and immunoreactivity of pH-stressed soybean (Glycine max) agglutinin.

Soybean agglutinin (SBA) was purified using ammonium sulfate precipitation and liquid chromatography. Purified SBA was used to produce monoclonal antibodies through hybridoma technology. SBA secondary structure was studied using circular dichroism. pH-stressed (pHs 3.0, 7.2, 8.5, and 9.6) SBA physical properties (particle size, ζ-potential, and aggregation temperature) were investigated. Gel electrophoresis (non-native and native) was used to study heat-induced structural configuration changes in SBA. The effect of pH and temperature on the immunoreactivity of SBA was analyzed using enzyme-linked immunosorbent assay and immunoblots probed with two anti-SBA monoclonal antibodies with either linear or conformational epitopes. The hemagglutinating activity of heated SBA was measured by hemagglutination assay. Our results indicated that SBA had the least thermostability at pH 3.0 and the highest at pH 8.5. Temperature-induced structural configuration change on pH-stressed SBA led to immunoreactivity change. Heat-induced (70 and 80 °C) soluble SBA aggregation was proportionally related to hemagglutinating activity reduction.

Effect of deglycosylation on immunoreactivity and in vitro pepsin digestibility of major cashew (Anacardium occidentale L.) allergen, Ana o 1.

Major cashew allergen, Ana o 1, was purified in its native form from cashew seeds and subjected to enzymatic deglycosylation using PNGase F to assess the potential role of N-glycans in immunoreactivity. Western and dot blotting with pooled human plasma containing anticashew IgE revealed that deglycosylation increased IgE-binding of Ana o 1. Removal of N-glycans may have exposed previously masked Ana o 1 epitopes. Purified glycosylated and deglycosylated Ana o 1 were also subjected to in vitro pepsin digestion at pH 3.0 for 2 hr. Both glycosylated and deglycosylated Ana o 1 remained stable and reactive with IgE antibodies following digestion. PRACTICAL APPLICATION: Understanding the role of glycosylation in Ana o 1 immunoreactivity may provide insight into the potential development of hypoallergenic cashews/cashew products for sensitive individuals in the future.

Quality of a Chickpea-Based High Protein Snack.

A chickpea-based high protein, ready-to-eat snack was prepared using six chickpea types. The chickpea seeds and their corresponding snack products were analyzed for proximate composition, antinutrients, and select quality parameters. Chickpea types had: lipid (4.25% to 6.98%), moisture (6.63% to 9.15%), protein (23.33% to 30.95%), and carbohydrate (54.60% to 60.40%) contents exhibiting significant (P ≤ 0.05) differences. Ash content (1.94% to 2.41%) did not register significant differences. Chickpea types did not show variability in either polypeptide profile or in vitro protein digestibility. In the tested seeds, hemagglutinins and α-amylase inhibitors were not detected, while trypsin (12.73 to 19.58 units/mg sample) and chymotrypsin inhibitors (62.91 to 84.91 units/mg sample) activity varied significantly (P ≤ 0.05). The chickpea-based snack product had intermediate-moisture (23.31% to 27.81%), was low in lipids (5.09% to 5.84%), free of antinutrients, and was a good source of proteins (12.45% to 14.10%), carbohydrates (51.86% to54.96%), and minerals (1.53% to 2.43%). The L* , a* , and b* values of the products ranged from 75.97 to 79.38, 3.46 to 4.75, and 27.65 to 34.65, respectively. The hardness, springiness, and fracturability of the product were 700.89 to 955.23 g, 43.38% to 47.14%, and 5.26 to 5.90 mm, respectively. PRACTICAL APPLICATION: Development of new bean-based products, such as a chickpea-based snack with an overall good nutrition and taste, may play an important role in increasing the consumption of underutilized dry beans in the United States, shown to promote better health and wellness.

Functional Properties of Select Dry Bean Seeds and Flours.

Select functional properties of 21 dry beans and soybean seeds and their corresponding flours were evaluated. Among the tested seeds, dark-red and light-red kidney beans had larger length (L)/breadth (B) ratios than the rest. Lentil seeds were the smallest (L/B = 0.98), thinnest (2.23 mm), and lightest (density 1.14 g/cm3 ) among the tested seeds. Garbanzo seeds were the thickest (thickness 7.05 mm). Highest bulk density (g/cm3 ), porosity (%), specific volume (cm3 /g), and bulk volume (cm3 /g) were registered, respectively, by moth bean (0.86 g/cm3 ), pink bean (66.53%), lentil (0.88 cm3 /g), and soybean (1.46 cm3 /g) seeds. Water uptake during soaking peaked in ≤12 hr by all bean seeds. Soybean and black bean flours, respectively, exhibited the highest water-holding (3.14 g/g) and oil-holding (2.15 g/g) capacities. Great Northern bean flour exhibited higher foaming volume (30.8% increase) than all the other tested seed flours. Stability of all bean flour foams were ≤36 hr. Black gram flour formed a gel at the lowest concentration (4% w/v) among all the tested seed flours. Garbanzo, soybean, tepary, and val bean flours failed to form a gel at ≤ 20% (w/v). The results indicate that the tested dry bean flours have great potential as functional ingredients for food formulations. PRACTICAL APPLICATION: Understanding the functional properties of the dry bean seeds and flours is important for designing handling, transportation, storage, and processing methods for these beans and in guiding the selection of appropriate dry bean flours as food ingredients.

Food Allergen Epitope Mapping.

With the increased global awareness and rise in food allergies, a multifold interest in food allergens is evident. The presence of undeclared food allergens results in expensive food recalls and increased risks of anaphylaxis for the sensitive individuals. Regardless of the allergenic food, the immunogen needs to be identified and detected before making any efforts to inactivate/eliminate it. In type I food allergies, protein immunogen cross-links immunoglobulin E, leading to basophil/mast cell degranulation, resulting in the symptoms that range from mild irritation to anaphylaxis. A portion/part of the protein, known as the epitope, can interact with either antibodies to elicit allergic reactions or T-cell receptors to initiate allergic sensitization. Antibody-recognized epitopes can be either a linear sequence of amino acids (linear epitope) or a three-dimensional motif (conformational epitope), while T-cell-receptor-recognized epitopes are exclusively linear peptides. Identifying and characterizing human-allergy-relevant epitopes are important for allergy diagnosis/prognosis, immunotherapy, and developing food processing methods that can reduce/eliminate immunogencity/immunoreactivity of the allergen.

Immunoreactivity of Biochemically Purified Amandin from Thermally Processed Almonds (Prunus dulcis L.).

Almond seeds were subjected to select thermal processing and amandin was purified from processed and unprocessed (control) seeds using cryoprecipitation. Amandin immunoreactivity was assessed using two murine monoclonal antibodies (mAbs)-4C10 and 4F10 detecting human IgE-relevant conformational and linear epitopes, respectively. Overall amandin immunoreactivity following thermal treatment ranged from 64.9% to 277.8% (4C10) and 81.3% to 270.3% (4F10). Except for autoclaving (121 °C, 15 psi, 30 min) and roasting (160 °C, 30 min), the tested processing conditions resulted in increased immunoreactivity as determined by mAbs 4C10 and 4F10-based enzyme-linked immunosorbent assays (ELISAs). A significant, yet not complete, reduction in immunoreactivity was caused by autoclaving (121 °C, 15 psi, 30 min) and roasting (160 °C, 30 min). Western- and dot-blot immunoassays corroborated the ELISA results, confirming amandin thermal stability. PRACTICAL APPLICATION: The tested immunoassays indicated amandin to be stable, regardless of the targeted epitope and the processing method that whole almond seeds were subjected to.

Effects of processing and storage on almond (Prunus dulcis L.) amandin immunoreactivity.

A murine monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) was used to assess amandin immunoreactivity in processed and long-term stored almonds. The results demonstrated that amandin immunoreactivity is stable in variously processed almond seeds. Using the ELISA, amandin immunoreactivity could be detected in commercial whole raw and processed (blanched, sliced, dry roasted, and indicated combinations thereof) almond seeds stored for eleven years and eight months, defatted almond seed flours from several almond varieties/hybrids and their borate saline buffer-solubilized protein extracts stored for ten years and seven months, and several almond varieties grown in different California counties (full fat flours and their defatted flour counterparts). Roasting Nonpareil whole full fat almond seeds, full fat flour, and defatted flour at 170°C for 20min each with 2, 5, 10, and 20% w/w corn syrup or sucrose did not prevent amandin detection by ELISA. Similarly, amandin detection in select food matrices spiked with Nonpareil almond protein extract was not inhibited. In conclusion, amandin is a stable target protein for almond detection under the tested processing and storage conditions.

Comparison of Laboratory-Developed and Commercial Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assays for Almond (Prunus dulcis) Detection and Quantification.

A commercially available monoclonal antibody (mAb)-based direct sandwich enzyme-linked immunosorbent assay (ELISA) kit (BioFront Technologies, Tallahassee, Fla., U.S.A.) was compared with an in-house developed mAb 4C10-based ELISA for almond detection. The assays were comparable in sensitivity (limit of detection < 1 ppm full fat almond, limit of quantification < 5 ppm full fat almond), specificity (no cross-reactivity with 156 tested foods at a concentration of 100000 ppm whole sample), and reproducibility (intra- and interassay variability < 15% CV). The target antigens were stable and detectable in whole almond seeds subjected to autoclaving, blanching, frying, microwaving, and dry roasting. The almond recovery ranges for spiked food matrices were 84.3% to 124.6% for 4C10 ELISA and 81.2% to 127.4% for MonoTrace ELISA. The almond recovery ranges for commercial and laboratory prepared foods with declared/known almond amount were 30.9% to 161.2% for 4C10 ELISA and 38.1% to 207.6% for MonoTrace ELISA. Neither assay registered any false-positive or negative results among the tested commercial and laboratory prepared samples. PRACTICAL APPLICATION: Ability to detect and quantify trace amounts of almonds is important for improving safety of almond sensitive consumers. Two monoclonal antibody-based ELISAs were compared for almond detection. The information is useful to food industry, regulatory agencies, scientific community, and almond consumers.

Effects of the Maillard Reaction on the Immunoreactivity of Amandin in Food Matrices.

Amandin is the major storage protein and allergen in almond seeds. Foods, containing almonds, subjected to thermal processing typically experience Maillard browning reaction. The resulting destruction of amino groups, protein glycation, and/or denaturation may alter amandin immunoreactivity. Amandin immunoreactivity of variously processed almond containing foods was therefore the focus of the current investigation. Commercial and laboratory prepared foods, including those likely to have been subjected to Maillard browning, were objectively assessed by determining Hunter L* , a* , b* values. The L* values for the tested samples were in the range of 31.75 to 85.28 consistent with Maillard browning. Three murine monoclonal antibodies, 4C10, 4F10, and 2A3, were used to determine the immunoreactivity of the targeted samples using immunoassays (ELISA, Western blot, dot blot). The tested foods did not exhibit cross-reactivity indicating that the immunoassays were amandin specific. For sandwich ELISAs, ratio (R) of sample immunoreactivity to reference immunoreactivity was calculated. The ranges of R values were 0.67 to 15.19 (4C10), 1.00 to 11.83 (4F10), and 0.77 to 23.30 (2A3). The results of dot blot and Western blot were consistent with those of ELISAs. Results of these investigations demonstrate that amandin is a stable marker protein for almond detection regardless of the degree of amandin denaturation and/or destruction as a consequence of Maillard reaction encountered under the tested processing conditions. PRACTICAL APPLICATION: Foods containing almond are often subjected to processing prior to consumption. Amandin, the major allergen in almond, may experience Maillard reaction. Understanding the change in amandin immunoreactivity as a result of Maillard reaction is important for amandin detection and production of hypoallergenic food products.

A Cherry Seed-Derived Spice, Mahleb, is Recognized by Anti-Almond Antibodies Including Almond-Allergic Patient IgE.

There are a number of examples of immunologic cross-reactivity elicited by pollens, fruits, seeds, and nuts of closely related plant species. Such cross-reactivity is of particular concern for patients with food allergies. In this report, we investigated a spice (mahleb) that is prepared from the kernel of the St. Lucie cherry, Prunus mahaleb, for cross-reactivity with almond (Prunus dulcis), using enzyme-linked immunosorbent assay (ELISA) and Western blot. Almond and mahleb are members of the same genus. Cross-reactivity between the mahleb and almond was demonstrated by reaction of cherry and almond kernel protein extracts with antibodies raised against almond proteins. Almond-specific murine monoclonal IgG, rabbit polyclonal IgG, and almond-allergic serum IgE each exhibited cross-reactivity with cherry kernel protein. Because of the demonstrated cross-reactivity between almond and mahleb, these findings should be of special concern to almond-allergic patients and attending medical personnel.

Recombinant Allergen Production in E. coli.

Recombinant protein allergens have been used in allergy studies, allergy diagnosis, and epitope mapping. Messenger RNAs (mRNAs) are isolated from tissues of interest for complementary DNA (cDNA) library construction. Subsequently, the allergen gene is amplified by polymerase chain reaction (PCR) and sequenced. The amplified gene is then cloned into an expression vector, expressed in Escherichia coli cells, and purified from the cell lysate. This chapter describes the protocols for recombinant allergen production.

Food Allergy.

Food allergy is receiving increased attention in recent years. Because there is currently no known cure for food allergy, avoiding the offending food is the best defense for sensitive individuals. Type I food allergy is mediated by food proteins, and thus, theoretically, any food protein is a potential allergen. Variability of an individual's immune system further complicates attempts to understand allergen-antibody interaction. In this article, we briefly review food allergy occurrence, prevalence, mechanisms, and detection. Efforts aimed at reducing/eliminating allergens through food processing are discussed. Future research needs are addressed.

Pistachio (Pistacia vera L.) Detection and Quantification Using a Murine Monoclonal Antibody-Based Direct Sandwich Enzyme-Linked Immunosorbent Assay.

A commercially available direct sandwich enzyme-linked immunosorbent assay (ELISA) (BioFront Technologies, Tallahassee, FL, USA) using murine anti-pistachio monoclonal antibodies (mAbs) as capture and detection antibodies was evaluated. The assay was sensitive (limit of detection = 0.09 ± 0.02 ppm full fat pistachio, linear detection range = 0.5-36 ppm, 50% maximum signal concentration = 7.9 ± 0.7 ppm), reproducible (intra- and inter-assay variability < 24% CV), and rapid (post-extraction testing time ∼ 1.5 h). The target antigen was stable and detectable in whole pistachio seeds subjected to autoclaving (121 °C, 15 psi, 15, 30 min), blanching (100 °C, 5, 10 min), frying (191 °C, 1 min), microwaving (500, 1000 W, 3 min), and dry roasting (140 °C, 30 min; 168 °C, 12 min). No cross-reactivity was observed in 156 food matrices, each tested at 100,000 ppm, suggesting the ELISA to be pistachio specific. The pistachio recovery ranges for spiked (10 ppm) and incurred (10-50000 ppm) food matrices were 93.1-125.6% and 35.7-112.2%, respectively. The assay did not register any false-positive or -negative results among the tested commercial and laboratory prepared samples.

Epitope mapping of 7S cashew antigen in complex with antibody by solution-phase H/D exchange monitored by FT-ICR mass spectrometry.

The potential epitope of a recombinant food allergen protein, cashew Ana o 1, reactive to monoclonal antibody, mAb 2G4, has been mapped by solution-phase amide backbone H/D exchange (HDX) monitored by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Purified mAb 2G4 was incubated with recombinant Ana o 1 (rAna o 1) to form antigen:monoclonal antibody (Ag:mAb) complexes. Complexed and uncomplexed (free) rAna o 1 were then subjected to HDX-MS analysis. Five regions protected from H/D exchange upon mAb binding are identified as potential conformational epitope-contributing segments.

Production and analysis of recombinant tree nut allergens.

Allergic reactions to tree nuts are a growing global concern as the number of affected individuals continues to rise. Unlike some food allergies, tree nuts can cause severe reactions that persist throughout life. The tree nuts discussed in this review include those most commonly responsible for allergic reactions: cashew, almond, hazelnut, walnut, pecan, Brazil nut, pistachio, and chestnut. The native allergenic proteins derived from tree nuts are frequently difficult to isolate and purify and may not be adequately represented in aqueous nut protein extracts. Consequently, defined recombinant allergens have become useful reagents in a variety of immunoassays aimed at the diagnosis of tree nut allergy, assessing cross-reactivity between various nuts and other seeds, mapping of IgE binding epitopes, and analyzing the effects of the food matrix, food processing, and gastric digestion on allergenicity. This review describes the approaches that can be used for the production of recombinant tree nut allergens and addresses key issues associated with their production and downstream applications.

A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection.

A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (<15% CV) and reproducible (intra- and inter assay variability <15% CV). The assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

Rapid screening for potential epitopes reactive with a polycolonal antibody by solution-phase H/D exchange monitored by FT-ICR mass spectrometry.

The potential epitopes of a recombinant food allergen protein, cashew Ana o 2, reactive to polyclonal antibodies, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ana o 2 polyclonal antibodies were purified in the serum from a goat immunized with cashew nut extract. Antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen:polyclonal antibody (Ag:pAb) complexes. Complexed and uncomplexed (free) rAna o 2 were then subjected to HDX-MS analysis. Four regions protected from H/D exchange upon pAb binding are identified as potential epitopes and mapped onto a homologous model.

Conformational epitope mapping of Pru du 6, a major allergen from almond nut.

Tree nuts are a widely consumed food. Although enjoyed safely by most individuals, allergic reactions to tree nuts, including almond, are not uncommon. Almond prunin (Pru du 6), an 11S globulin (legumin), is an abundant nut seed protein and a major allergen. Conformational epitope mapping studies of prunin have been performed with a murine monoclonal antibody (mAb) 4C10. This mAb reacts with non-reduced but not reduced prunin in immunoblotting assays, indicating the recognition of a conformational epitope. 4C10 competes with patient IgE, as assessed by ELISA, indicating clinical significance of the epitope. To characterize the 4C10 epitope, hydrogen/deuterium exchange (HDX) monitored by 14.5 T Fourier transform ion cyclotron resonance mass spectrometry (MS) was performed on the native prunin-4C10 complex and on uncomplexed native prunin. Several epitope candidate peptides that differ in deuterium uptake between the complexed and uncomplexed forms were identified. The epitope was further mapped by analyzing chimeric molecules incorporating segments of the homologous soybean allergen, Gly m 6, in immunoassays. These data indicate that the 4C10 epitope overlaps with a subset of patient IgE binding epitopes on almond prunin and further supports HDX-MS as a valid technique for mapping conformational epitopes.

Solubilization, fractionation, and electrophoretic characterization of Inca peanut (Plukenetia volubilis L.) proteins.

Effects of different solvents, ionic strength, and pH on Inca peanut seed protein solubility were assessed by quantitatively analyzing solubilized proteins using Lowry and Bradford methods. Soluble proteins were fractionated using Osborne procedure and the polypeptide composition of solubilized proteins was determined by one dimensional 25 % monomer acrylamide linear gradient SDS-PAGE. Osborne protein fractions were analyzed by the 2D gel electrophoresis. Total seed proteins were efficiently solubilized by 2 M NaCl among the tested solvents. The soluble seed proteins registered a minimum solubility at pH ~4.0. Osborne protein fractions, albumins, globulins, prolamins, and glutelins accounted for 43.7, 27.3, 3.0, and 31.9 %, respectively, of the total aqueous soluble proteins. Soluble seed flour proteins are mainly composed of polypeptides in the MW range of 6-70 kDa of which the predominant polypeptides were in the 20-40 kDa range. Prolamin fraction was mainly composed of four polypeptides (MW < 15 kDa). Glycoprotein staining indicated 32-35 and <14 kDa peptides to be positive.