Background: Buckwheat allergies are not common, however, it is considered to be a very potent allergen. Ingestion of small amounts has been found to produce anaphylactic reactions, particularly in children. Identification and characterization of the major allergen(s) in buckwheat are currently underway, however, there are some discrepancies in the findings. Methods: Identification of the major allergen(s) was determined through Western blotting using buckwheat-allergic patients’ sera. Once the allergenic proteins were identified, they were purified, their IgE-binding activity assessed through an indirect ELISA and the N-terminal amino acid sequence completed. To assess the stability of the IgE-binding epitopes, protein fractions were exposed to various treatments and assayed using an indirect ELISA. Lastly, the presence of anti-buckwheat IgG in the patients’ sera was analyzed through Western blotting and ELISA. Results: IgE binding was detected to proteins with molecular masses of approximately 14 and 18 kDa. N-terminal sequencing was completed and found to share some homology with rice proteins associated with rice allergies and cross-allergenicity with buckwheat proteins. When the water-soluble protein fraction was heated, exposed to acidic and alkaline conditions and fully denatured, IgE-binding activity was reduced. When the fraction was partially denatured through urea, IgE-binding activity increased. Furthermore, IgG-binding activity was detected with proteins only above the 20 kDa region. Conclusions: Proteins with molecular masses around 14 and 18 kDa were identified as the major allergenic proteins in the buckwheat-allergic patients’ sera tested in this study. Results also indicate that these two proteins possess IgE-binding capability.

A study was undertaken to examine the effects of N-linked glycosylation on the structure-function of porcine pepsin. The N-linked motif was incorporated into four sites (two on the N-terminal domain and two on the C-terminal domain), and the recombinant protein expressed using Pichia pastoris. All four N-linked recombinants exhibited similar secondary and tertiary structure to nonglycosylated pepsin, that is, wild type. Similar K(m) values were observed, but catalytic efficiencies were approximately one-third for all mutants compared with the wild type; however, substrate specificity was not altered. Activation of pepsinogen to pepsin occurred between pH 1.0 to 4.0 for wild-type pepsin, whereas the glycosylated recombinants activated over a wider range, pH 1.0 to 6.0. Glycosylation on the C-terminal domain exhibited similar pH activity profiles to nonglycosylated pepsin, and glycosylation on the N-domain resulted in a change in activity profile. Overall, glycosylation on the C-domain led to a more global stabilization of the structure, which translated into enzymatic stability, whereas on the N-domain, an increase in structural stability had little effect on enzymatic stability. Finally, glycosylation on the flexible loop region also appeared to increase the overall structural stability of the protein compared with wild type. It is postulated that the presence of the carbohydrate residues added rigidity to the protein structure by reducing conformational mobility of the protein, thereby increasing the structural stability of the protein.

Salmonella enterica serovar Enteritidis was detected in artificially inoculated eggs within 24 h through a rapid monoclonal antibody-based dot blot immunoassay. Detection in poultry and other products required 28 h. Samples were directly enriched in homogenized egg without the need for pre-or postenrichment steps. Serovar Enteritidis was detected in the presence of other bacteria when outcompeted 1:400.Conventional methods for detection of Salmonella in foods are labor-intensive, time-consuming, and expensive. It has also been found that some of the routinely used selective enrichment broths are inhibitory towards Salmonella enterica serovar Enteritidis (28). Rapid methods based on principles such as membrane technology (11), latex agglutination (27), immunoassays (6,18,30), and immunomagnetic separation (8,9,19) have been developed. Methods employing PCR in combination with preenrichment broths (24,31,32), immunomagnetic separation (25, 26), or centrifugation (21, 22) are currently being developed.Immunologically based methods specific for serovar Enteritidis suffer from the same drawbacks as the above methods: one or more enrichment broths, or in some cases, postenrichment broths, are required. Cross-reactivity has also been observed with most monoclonal antibodies produced against serovar Enteritidis (17,18,27). This report describes the development of a rapid dot blot immunoassay for the detection of serovar Enteritidis in eggs, poultry, and other products.Large grade A eggs were scrubbed with 70% ethanol and opened aseptically. Eggs were mixed for 30 s using a stomacher lab blender 400 (Seward Laboratory, London, England) and were inoculated with either serovar Enteritidis phage type 1, 4, 8, 13, or 13a. After incubation, 25-ml portions were placed into 50-ml polypropylene tubes, and 0.1 volume of 15% sodium cholate in phosphate-buffered saline (PBS) (pH 7.2) was added. After being mixed, tubes were placed in boiling water for 10 min and cooled for 30 min at 4°C. Through heating, the egg mixture was solidified, forming a solid egg gel. After cooling, the gel was removed from the tube, and a sterile core borer (10-mm diameter) was used to create small cylindrical gels. The cylindrical gels were then cut into disks of 2 mm in thickness.Nitrocellulose strips (8.5 by 2.5 cm) were prewetted with PBS prior to use. Egg disks were placed on the membrane for 5 min, removed, and washed for 2 min in PBS. Strips were blocked for 45 min in 5% skim milk powder in Tris-buffered saline (pH 7.5) and incubated with a murine monoclonal antibody solution (tissue culture supernatant) for 45 min, followed by 1 h of incubation in biotinylated goat anti-mouse immunoglobulin G. Strips were incubated with streptavidinalkaline phosphatase for 1 h and developed with a BCIP (5-bromo-4-chloro-3-indolylphosphate)-nitroblue tetrazolium chloride substrate solution. Membranes were washed twice with Tris-buffered saline containing 0.05% Tween 20 for 2 min between each step. All steps were performed at room temperature.Bacterial cultures were...

No abstract