Recalls Associated with Food Allergens and Gluten in FDA-Regulated Foods from Fiscal Years 2013 to 2019.

Allergens are one of the leading causes of food recalls in the US. The Food and Drug Administration (FDA) enforces requirements relating to major food allergens (MFAs) and gluten-free labeling to ensure food safety for allergic and celiac patients, respectively. Violative foods are subject to recalls. In this study, recall data for FDA-regulated foods were analyzed for fiscal years (FYs) 2013-2019 to identify trends and root causes associated with 1471 food allergen and gluten recalls. Of the 1471 recalls, 1415 recalls were due to MFAs, 34 recalls were due to gluten-free labeling violation and 23 recalls involved other allergens. Recalls due to MFAs overall increased during the study period with a peak incidence in FY 2017. MFA recall health hazard classifications were assessed as Class I (51.2%), Class II (45.5%), and Class III (3.3%). A majority of MFA recalls involved one allergen (78.8%). Milk was the most common MFA involved in MFA recalls (37.5%), followed by soy (22.5%) and tree nuts (21.6%). Almond, anchovy, and shrimp were the most common allergens recalled within the MFA groups of tree nuts, fish, and Crustacean shellfish, respectively. About 97% of MFA recalls involved one product category and among them, the category of 'bakery products, dough, bakery mixes and icings' ranked first (367 recalls), followed by the category of 'chocolate and cocoa products' (120 recalls). Labeling-associated errors accounted for 71.1% of MFA recalls with known root causes (914 out of 1286). It is important for the industry to develop and implement appropriate allergen controls to reduce the number of MFA recalls.

Effects of Emulsifiers on an In vitro Model of Intestinal Epithelial Tight Junctions and the Transport of Food Allergens.

SCOPE: Certain food emulsifiers may interfere with gut barrier function in ways correlating to increased exposure to allergens. Understanding the consequences of interactions between these food ingredients and the intestinal epithelium is important for evaluating allergen dose exposure characteristics. METHODS AND RESULTS: This study challenged Caco-2 cell monolayers, an in vitro model of human intestinal epithelial tight junctions with synthetic polysorbate-80 or natural lecithin alone, or in combination with known allergens (egg proteins: ovalbumin, ovomucoid, and ovotransferrin; and a synthetic form of galactose-alpha-1,3-galactose [alpha-gal], an allergen of increasing concern). For most doses of individual emulsifiers and allergens, >90% cell viability and <15% cytotoxicity are observed; however, toxicity increased at a 0.5% concentration of emulsifiers. At low cytotoxic concentration (0.2%), only polysorbate-80 treatment reduced monolayer integrity (≈20%) with increased lucifer yellow passage. Dose-related differences in expression of tight junction-associated genes and occludin protein are observed with emulsifier treatments. The transport of all tested allergens across the cell monolayers, excluding ovotransferrin, nearly doubled in the presence of 0.2% polysorbate-80 compared to lecithin and untreated control. CONCLUSION: By modulating paracellular permeability, polysorbate-80 may enhance absorption of allergens in a size-dependent manner.

Enhanced quantitation of egg allergen in foods using incurred standards and antibodies against processed egg in a model ELISA.

Underestimation of egg allergen from processed foods prompted the evaluation of critical Enzyme-Linked Immunosorbent Assay (ELISA) parameters: (1) extraction of egg proteins from a processed matrix; (2) use of anti-heat processed egg antibodies (Abs) on detectability of modified proteins, and (3) utilization of incurred material as standards. The relative affinity of two combinations of raw (R), boiled (B) and fried (F) Abs to unprocessed/processed egg proteins with or without matrix was determined from antibody (Ab) binding curves. In ELISAs using RBF-Abs and BF-Abs, denaturing buffer, and incurred standards, the Limit of Detection (LOD) and Limit of Quantitation (LOQ) were 0.47 and 0.25; and 1.58 and 0.85, respectively, and the linear range was 0-24 µg g-1 egg protein. The recoveries of egg protein from cookies, cereal bar, and muffin (incurred levels 4.8-48 µg g-1) with the developed ELISAs were in an acceptable range (50-130%). These ELISAs consistently detected more declared/undeclared egg proteins in market samples compared to assays using PBS for extraction. Overall, better assay performance was observed using BF-Abs. An ELISA combining anti-processed egg Abs, denaturing buffer, and incurred standards promises improved quantitation of egg proteins in processed foods.

Extraction Conditions Affect the Immunoreactivity of Peanut Allergens.

Peanut allergic consumers rely on food package labels to avoid foods containing peanut. The inadvertent presence of peanut in foods due to cross-contact can be fatal if ingestion of such food leads to an allergic reaction. Analytical methods are available to detect undeclared peanut in foods. However, depending on the type of food matrix and food processing parameters, method performance can be adversely affected due to reduction in the extraction efficiency of peanut proteins. Temperature and probe sonication were used as a preincubation treatment for peanut flour slurries to assess their effect on the total peanut protein solubility from raw, light-roasted, and dark-roasted peanut flours. The effect of these treatments on the immunoreactivity of peanut allergens (Ara h 1, Ara h 2, Ara h 3, and Ara h 6) was determined by an indirect enzyme-linked immunosorbent assay using antibodies raised against these individual peanut proteins. Preincubation at 50 °C did not significantly improve the peanut protein solubility, whereas an increase in protein solubility was observed when light- and dark-roasted peanut flour slurries were preincubated at 90 °C or sonicated. The immunoreactivity of peanut allergens varied depending on the degree of peanut flour roasting and type of preincubation treatment. Overall, the immunoreactivity of peanut allergens from most peanut flour slurries was unaffected when preincubated at 50 °C for up to 60 min or sonicated with a probe for up to 5 min, whereas preincubation at 90 °C resulted in a time-dependent reduction in immunoreactivity of peanut allergens. Sonication treatment may improve peanut protein extraction without markedly affecting their immunoreactivity. PRACTICAL APPLICATION: Extraction of peanut proteins is vital for developed analytical methods to estimate peanut allergens in foods. The manuscript describes the effect of two different temperatures (50 and 90 °C) and probe-type sonication on peanut protein solubility. The findings suggest sonication can improve peanut protein solubility without markedly affecting their immunoreactivity.

In vivo and in vitro evaluation of tissue colonization and survival capacity of Salmonella Oranienburg in laying hens.

Salmonella enterica serovar Oranienburg (SO) was linked to a human salmonellosis outbreak in the Midwest in 2015 and 2016 from consumption of eggs. However, unlike Salmonella enterica serovar Enteritidis (SE), little is known regarding the potential of SO to colonize in laying hens and contaminate eggs. We used in vivo and in vitro models to evaluate tissue colonization and survival capacity of SO. Twenty eight-week-old laying hens were each challenged with an oral dose of approximately 107 (n = 92) or 109 (n = 96) colony-forming units (CFU) in 1 mL saline and evaluated after 1, 2, and 4 wk. Standard microbiological methods with pre-enrichment and enrichment in selective media were used for detection of SO in tissues, egg shell wash, internal egg contents, and excreta. Peak colonization of spleen (86.9%), ovaries (31.6%), upper oviduct (15.8%), and lower oviduct (34.3%) was detected between 1 and 2 wk post-infection (pi), while at 4 wk SO was only recovered from spleens (25%). Salmonella enterica serovar Oranienburg was not recovered from internal egg contents. However, the presence of SO on egg shells was seen when there were traces of excreta. Shedding in excreta was found in 92 and 100% birds gavaged with 107 and 109 CFU at 2 wk pi, respectively. The invasion and proliferation of SO in ovarian granulosa cells (GC) was compared to that of SE, and while the invasion of SO into GC was comparable to SE, proliferation of SO was significantly lower (P < 0.05). The infective potential of SO was also assessed by enumerating survival in egg white over 4 wk under refrigerated conditions, resulting in 65% survival at 4 wk. Overall, our data suggested that SO infection in layers did not result in egg contamination via vertical transmission, and colonization of egg-forming tissues was limited to 2 wk pi. Survival within GC and egg white demonstrates the ability of SO to withstand antibacterial factors and the potential of SO to penetrate the yolk.

Undeclared Food Allergens and Gluten in Commercial Food Products Analyzed by ELISA.

Undeclared allergen(s) in commercial food products are responsible for many food recalls, as reported by regulatory agencies in various countries, including the United States. Correct allergen labeling practices are essential for the safety of food-allergic consumers. However, this practice may be hindered by the introduction of allergens all along the food supply chain, including unintentionally through cross-contact. To understand the pervasiveness of undeclared allergen(s) in commercial food products, the objective of this review is to summarize the prevalence of undeclared milk, egg, hazelnut, peanut, soy, and gluten as detected by ELISA from previously published surveys. The prevalence of undeclared allergen(s) in products with or without an advisory statement was also summarized and compared. As compiled by this review, there are some food categories that may be at higher risk for containing undeclared allergen(s). However, the data on prevalence and amount of allergen present may vary widely within any particular allergen or food category. Factors, such as food survey product selection, geography, awareness of allergen/gluten issues, and/or the choice of ELISA method, may be responsible for such differences.

Survey of undeclared soy allergen levels in the most frequently recalled food categories with or without precautionary labelling.

A comprehensive study was designed to determine the frequency and levels of soy allergen in packaged bakery and snack food products. A representative sample of products with no soy allergen disclosed on the label was analysed using two widely used enzyme-linked immunosorbent assay (ELISA) methods. Samples were chosen that either had no soy identified on the product label or which had a soy precautionary statement. Among 558 bakery and snack products, soy protein was detected in 17% of the products using the Neogen (NE) kit and 11% of the products using the Elisa Systems (ES) kit. The disagreement rates between kits were 8.8% for bakery products and 3.3% for snack products. Overall soy protein was detected at higher frequency in bakery products than in snack foods. Among 284 bakery samples, soy protein was detected in 25% of the samples with no precautionary statement and 19% of the samples which had a precautionary statement. Among 274 snack samples, soy protein was detected in 11% of the samples with no precautionary statement and 9% of the samples which had a precautionary statement. The sample repeatability was at an acceptable level (< 9%) for each method and food commodity. The reproducibility between kits was 23% for bakery foods and 36% for snack foods. None of the bakery (21) and snack (6) products without precautionary labelling (measured level > 5 ppm) had a higher level of soy protein per serving compared with the eliciting dose10 (ED10) of 10.6 mg for soy allergic patients. But the level of soy protein per serving may be clinically relevant to a subpopulation of soy allergic patients if a more stringent eliciting dose is applied. These findings emphasise that suitable detection methodologies and references doses are crucial for labelling accuracy and the safety of soy allergic consumers.

Survey of undeclared egg allergen levels in the most frequently recalled food types (including products bearing precautionary labelling).

Since the number of recalls involving undeclared allergens is commonly associated with bakery and snack foods, we aimed to determine the frequency of egg allergens in a large number of these products using two commercial enzyme-linked immunosorbent assay (ELISA) methods. Samples were chosen that either had no egg identified on the product label or which had an egg precautionary statement. Among all samples, egg protein was detected in 5% of products using a Morinaga (MO) kit and 1% of products using a R-Biopharm (RB) kit. For bakery samples, egg protein was detected in 6% of 363 samples with no precautionary labelling (6% by MO and 1% by RB kit) and 12% of 80 samples which had precautionary labelling. For snack samples, egg protein was detected in 2% of 371 samples with no precautionary labelling (2% by MO and < 1% by RB kit) and 5% of 21 samples which had precautionary labelling. The disagreement rates between two methods were 5.2% for bakery products and 2.6% for snack products. The sample repeatability was at an acceptable level for bakery (< 12.5%) and snack foods (< 7.5%) for each method. The relative standard deviation between test kits was high (103.1%) for bakery foods. Four bakery products without precautionary labelling had a higher level of egg protein per serving compared with the eliciting dose (ED10 of 3.7 mg protein) for egg allergic patients. These results highlight the fact that detection methodology plays a vital role for accurate labelling control and mitigation of risk for egg allergic consumers.

Effects of grain species and cultivar, thermal processing, and enzymatic hydrolysis on gluten quantitation.

Gluten from wheat, rye, and barley can trigger IgE-mediated allergy or Celiac disease in sensitive individuals. Gluten-free labeled foods are available as a safe alternative. Immunoassays such as the enzyme-linked immunosorbent assay (ELISA) are commonly used to quantify gluten in foods. However, various non-assay related factors can affect gluten quantitation. The effect of gluten-containing grain cultivars, thermal processing, and enzymatic hydrolysis on gluten quantitation by various ELISA kits was evaluated. The ELISA kits exhibited variations in gluten quantitation depending on the gluten-containing grain and their cultivars. Acceptable gluten recoveries were obtained in 200mg/kg wheat, rye, and barley-spiked corn flour thermally processed at various conditions. However, depending on the enzyme, gluten grain source, and ELISA kit used, measured gluten content was significantly reduced in corn flour spiked with 200mg/kg hydrolyzed wheat, rye, and barley flour. Thus, the gluten grain source and processing conditions should be considered for accurate gluten analysis.

Characterization of Antibodies for Grain-Specific Gluten Detection.

Gluten ingestion causes immunoglobulin E (IgE)-mediated allergy or celiac disease in sensitive individuals, and a strict gluten-free diet greatly limits food choices. Immunoassays such as enzyme-linked immunosorbent assay (ELISA) are used to quantify gluten to ensure labeling compliance of gluten-free foods. Anti-gluten antibodies may not exhibit equal affinity to gluten from wheat, rye, and barley. Moreover, because wheat gluten is commonly used as a calibrator in ELISA, accurate gluten quantitation from rye and barley contaminated foods may be compromised. Immunoassays utilizing grain-specific antibodies and calibrators may help improve gluten quantitation. In this study, polyclonal antibodies raised against gluten-containing grain-specific peptides were characterized for their immunoreactivity to gluten from different grain sources. Strong immunoreactivity to multiple gluten polypeptides from wheat, rye, and barley was observed in the range 34 to 43 kDa with anti-gliadin, 11 to 15 and 72 to 95 kDa with anti-secalin, and 30 to 43 kDa with anti-hordein peptide antibodies, respectively. Minimal or no cross-reactivity with gluten from other grains was observed among these antibodies. The anti-consensus peptide antibody raised against a repetitive amino acid sequence of proline and glutamine exhibited immunoreactivity to gluten from wheat, rye, barley, and oat. The antibodies exhibited similar immunoreactivity with most of the corresponding grain cultivars by ELISA. The high specificity and minimal cross-reactivity of grain-specific antibodies suggest their potential use in immunoassays for accurate gluten quantitation.

Immunological characterization of the gluten fractions and their hydrolysates from wheat, rye and barley.

Gluten proteins in wheat, rye and barley cause celiac disease, an autoimmune disorder of the small intestine, which affects approximately 1% of the world population. Gluten is comprised of prolamin and glutelin. Since avoidance of dietary gluten is the only option for celiac patients, a sensitive gluten detection and quantitation method is warranted. Most regulatory agencies have set a threshold of 20 ppm gluten in foods labeled gluten-free, based on the currently available ELISA methods. However, these methods may exhibit differences in gluten quantitation from different gluten-containing grains. In this study, prolamin and glutelin fractions were isolated from wheat, rye, barley, oats and corn. Intact and pepsin-trypsin (PT)-digested prolamin and glutelin fractions were used to assess their immunoreactivity and gluten recovery by three sandwich and two competitive ELISA kits. The Western blots revealed varied affinity of ELISA antibodies to gluten-containing grain proteins and no reactivity to oat and corn proteins. ELISA results showed considerable variation in gluten recoveries from both intact and PT-digested gluten fractions among different kits. Prolamin fractions showed higher gluten recovery compared to their respective glutelin fractions. Among prolamins, barley exhibited higher recovery compared to wheat and rye with most of the ELISA kits used. Hydrolysis resulted in reduced gluten recovery of most gluten fractions. These results suggest that the suitability of ELISA for accurate gluten quantitation is dependent upon various factors, such as grain source, antibody specificity, gluten proteins and the level of their hydrolysis in foods.

Gluten detection in foods available in the United States - a market survey.

Many gluten-free (GF) food choices are now available in supermarkets. However, the unintentional presence of gluten in these foods poses a serious health risk to wheat-allergic and celiac patients. Different GF labelled foods (275) and non-GF labelled foods, without wheat/rye/barley on the ingredient label (186), were analysed for gluten content by two different enzyme linked immunosorbent assay (ELISA) kits. Considering the gluten threshold of 20ppm, GF labelled foods had 98.9% GF labelling compliance with 1.1% (3 out of 275) of foods being mislabelled/misbranded. Among the non-GF labelled foods, 19.4% (36 out of 186) of foods had >20ppm of gluten, as measured by at least one ELISA kit, of which 19 foods had >100ppm of gluten. The presence of oats in non-GF labelled foods was strongly correlated with a positive ELISA result. Gluten was also found in a significant number of foods with gluten/wheat-related advisory warnings.

Development of an incurred cornbread model for gluten detection by immunoassays.

Gluten that is present in food as a result of cross-contact or misbranding can cause severe health concerns to wheat-allergic and celiac patients. Immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and lateral flow device (LFD), are commonly used to detect gluten traces in foods. However, the performance of immunoassays can be affected by non-assay-related factors, such as food matrix and processing conditions. Gluten (0-500 ppm) and wheat flour (20-1000 ppm) incurred cornbread was prepared at different incurred levels and baking conditions (204.4 °C for 20, 27, and 34 min) to study the accuracy and precision of gluten measurement by seven immunoassay kits (three LFD and four ELISA kits). The stability and immunoreactivity of gluten proteins, as measured by western blot using three different antibodies, were not adversely affected by the baking conditions. However, the gluten recovery varied depending upon the ELISA kit and the gluten source used to make the incurred cornbread, affecting the accuracy of gluten quantification (BioKits, 9-77%; Morinaga, 91-137%; R-Biopharm, 61-108%; and Romer Labs, 113-190%). Gluten recovery was reduced with increased baking time for most ELISA kits analyzed. Both the sampling and analytical variance increased with an increase in the gluten incurred level. The predicted analytical coefficient of variation associated with all ELISA kits was below 12% for all incurred levels, indicative of good analytical precision.

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.

Immunoreactivity and detection of wheat proteins by commercial ELISA kits.

Wheat proteins are responsible for sensitivities, including baker's asthma, immunoglobulin E (IgE)-mediated allergic reaction, wheat-dependent, exercise-induced anaphylaxis, and celiac disease. The detection of gluten/wheat traces in foods is important to safeguard the health of wheat-sensitive individuals and comply with food labeling. Many immunoanalytical-based commercial kits are available for the quantification of gliadin/gluten/wheat proteins. We compared the immunoreactivity of wheat fractions with wheat-allergic human serum IgE and antibody conjugates used in six commercial immunoassay kits. Moreover, the performance of the kits was tested using corn flour spiked with gluten (5, 10, 25, and 50 ppm) and wheat flour (50, 100, 250, and 500 ppm). The albumin, globulin, gliadin, and glutenin fractions reacted with IgE from nine, eight, two, and eight patients' sera, respectively, out of nine wheat allergic patients tested. Among the antibodies from commercial kits, those from R-Biopharm, Morinaga, and Romer Labs reacted strongly with the gliadin fraction, whereas those from BioKits, ALLER-TEK, and ELISA Systems reacted strongly with the glutenin fraction. All kits showed minimal or no reactivity with albumin and globulin fractions. All kits detected the gluten and wheat flour in a corn flour matrix at the lowest spiked levels of 5 and 50 ppm, respectively. However, there was wide variation among the kits when comparing the recovery of gluten and wheat flour. The recovery was also dependent on the source material (gluten or wheat flour) used for spiking the corn flour matrix.

Cloning and characterization of an 11S legumin, Car i 4, a major allergen in pecan.

Among tree nut allergens, pecan allergens remain to be identified and characterized. The objective was to demonstrate the IgE-binding ability of pecan 11S legumin and characterize its sequential IgE-binding epitopes. The 11S legumin gene was amplified from a pecan cDNA library and expressed as a fusion protein in Escherichia coli. The native 11S legumin in pecan extract was identified by mass spectrometry/mass spectrometry (MS/MS). Sequential epitopes were determined by probing the overlapping peptides with three serum pools prepared from different patients' sera. A three-dimensional model was generated using almond legumin as a template and compared with known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot blot, 16 (57%) bound to 11S legumin, designated Car i 4. MS/MS sequencing of native 11S legumin identified 33 kDa acidic and 20-22 kDa basic subunits. Both pecan and walnut seed protein extracts inhibited IgE binding to recombinant Car i 4, suggesting cross-reactivity with Jug r 4. Sequential epitope mapping results of Car i 4 revealed weak, moderate, and strong reactivity of serum pools against 10, 5, and 4 peptides, respectively. Seven peptides were recognized by all three serum pools, of which two were strongly reactive. The strongly reactive peptides were located in three discrete regions of the Car i 4 acidic subunit sequence (residues 118-132, 208-219, and 238-249). Homology modeling of Car i 4 revealed significant overlapping regions shared in common with other tree nut legumins.

Cloning and characterization of 2S albumin, Car i 1, a major allergen in pecan.

Although pecans are associated with IgE-mediated food allergies, the allergens responsible remain to be identified and characterized. The 2S albumin gene was amplified from the pecan cDNA library. Dot-blots were used to screen the recombinant protein with pecan allergic patients' serum. The affinity purified native protein was analyzed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS) analysis. Cross-reactivity with walnut was determined by inhibition enzyme-linked immunosorbent assay (ELISA). Sequential epitopes were determined by probing the overlapping peptides with three different patients' serum pool. The 3-dimensional homology model was generated, and the locations of the pecan epitopes were compared with those of known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot-blot, 22 (79%) bound to 2S albumin, designated as Car i 1. Edman sequencing and the MS/MS sequencing of native 2S albumin confirmed the identity of recombinant (r) Car i 1. Both pecan and walnut protein extracts inhibited the IgE-binding to rCar i 1. Sequential epitope mapping indicated weak, moderate, and strong reactivity against 12, 7, and 5 peptides, respectively. Of the 11 peptides recognized by all serum pools, 5 peptides were strongly reactive and located in 3 discrete regions of the Car i 1 (amino acids 43-57, 67-78, and 106-120). Three-dimensional modeling revealed IgE-reactive epitopes to be solvent accessible and share significant homology with other tree nuts providing a possible basis for previously observed cross-reactivity.

Biochemical and spectroscopic characterization of almond and cashew nut seed 11S legumins, amandin and anacardein.

Native, undenatured amandin and anacardein secondary structures were estimated to be, respectively, 56.4 and 49% ß-sheet, 14 and 23.7% α-helix, and 29.6 and 27.4% random coil. Circular dichroic (CD) and fluorescence spectroscopy were used to assess structural changes in amandin and anacardein subjected to denaturing treatments that included heat (100 °C, 5 min), guanidium HCl (GuHCl), urea, sodium dodecyl sulfate (SDS), and reducing agent, 2% v/v ß-mercaptoethanol (ßME) + heat. Mouse monoclonal antibodies (mAbs) 4C10 and 4F10 directed against amandin and 1F5 and 4C3 directed against anacardein were used to assess the influence of denaturing treatments on the immunoreactivity of amandin and anacardein. Among the denaturing treatments investigated, SDS and ß-ME caused a significant reduction in the immunoreactivity of amandin and anacardein when probed with mAb 4C10 and 4C3, respectively.

Purification and biochemical characterization of Brazil nut (Bertholletia excelsa L.) seed storage proteins.

Brazil nut storage proteins, 2S albumin, 7S vicilin, and an 11S legumin, were purified using column chromatography. Analytical ultracentrifugation of the purified albumin, vicilin, and legumin proteins, respectively, registered sedimentation coefficients of 1.8, 7.1, and 11.8 S. Under reducing conditions, the major polypeptide bands in 2S albumin were observed at 6.4, 10-11, and 15.2 kDa. The 7S globulin was composed of one 12.6 kDa, two approximately 38-42 kDa, and two approximately 54-57 kDa polypeptides, whereas the 11S globulin contained two major classes of polypeptides: approximately 30-32 and approximately 20-21 kDa. The 7S globulin stained positive when reacted with Schiff reagent, indicating that it is a glycoprotein. The estimated molecular mass and Stokes radius for 2S albumin and 7S and 11S globulins were 19.2 kDa and 20.1 A, 114.8 kDa and 41.1 A, and 289.4 kDa and 56.6 A, respectively. Circular dichroism spectroscopic analysis indicated the secondary structure of the three proteins to be mainly beta-sheets and turns. Emission fluorescence spectra of the native proteins registered a lambda(max) at 337, 345, and 328 nm for 2S albumin and 7S and 11S globulins, respectively. When probed with anti-Brazil nut seed protein rabbit polyclonal antibodies, 7S globulin exhibited higher immunoreactivity than 2S albumin and 11S globulin.