Comparison of real-time PCR and ELISA for the detection of crustacean shellfish allergens.

Food allergies are a significant public health concern, and crustacean shellfish represent one of the major FDA regulated food allergens. Allergic individuals must avoid foods containing crustaceans, and this necessitates highly sensitive and accurate detection methods. Two of the major methods used are protein-based ELISA and DNA-based real-time PCR.

Multi-laboratory validation of the xMAP-Food Allergen Detection Assay: A multiplex, antibody-based assay for the simultaneous detection of food allergens.

The increasing prevalence of individuals with multiple food allergies and the need to distinguish between foods containing homologous, cross-reactive proteins have made the use of single-analyte antibody-based methods (e.g., ELISAs) sometimes insufficient.

Robustness Testing of the xMAP Food Allergen Detection Assay: A Multiplex Assay for the Simultaneous Detection of Food Allergens.

Abstract: The xMAP food allergen detection assay (xMAP FADA) can simultaneously detect 15 analytes (14 food allergens plus gluten) in one analysis. The xMAP FADA typically employs two antibody bead sets per analyte, providing built-in confirmation that is not available with other antibody-based assays. Before an analytical method can be used, its reliability must be assessed when conditions of the assay procedure are altered.

Single-Laboratory Validation of the Multiplex xMAP Food Allergen Detection Assay with Incurred Food Samples.

An xMAP Food Allergen Detection Assay (xMAP FADA) was developed to meet analytical needs when responding to complaints by individuals with multiple food allergies and to address potential ambiguities associated with cross-reactive proteins. A single-laboratory validation (SLV) was conducted to examine the reliability of the xMAP FADA to detect 15 analytes individually or as part of a mixture at more than six concentrations in four foods. The xMAP FADA reliably detected the analytes despite the incurred dark chocolate and incurred baked muffins displaying recoveries of 10-20% and <60%, respectively.

Analysis and Characterization of Proteins Associated with Outer Membrane Vesicles Secreted by spp.

Little is known about secretion of outer membrane vesicles (OMVs) by . In this study, OMVs isolated from , and were examined by electron microscopy (EM) and their associated outer membrane proteins (OMP) and genes were analyzed by SDS-PAGE, protein sequencing, BLAST, PCR, and DNA microarray. EM of stained cells revealed that the OMVs are secreted as pleomorphic micro-vesicles which cascade from the cell's surface.

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.

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.

Respiratory syncytial virus fusion protein-induced toll-like receptor 4 (TLR4) signaling is inhibited by the TLR4 antagonists Rhodobacter sphaeroides lipopolysaccharide and eritoran (E5564) and requires direct interaction with MD-2.

Unlabelled: Respiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Toll-like receptor 4 (TLR4), a signaling receptor for structurally diverse microbe-associated molecular patterns, is activated by the RSV fusion (F) protein and by bacterial lipopolysaccharide (LPS) in a CD14-dependent manner. TLR4 signaling by LPS also requires the presence of an additional protein, MD-2.

Gluten and celiac disease--an immunological perspective.

Gluten, a complex protein group in wheat, rye, and barley, causes celiac disease (CD), an autoimmune enteropathy of the small intestine, in genetically susceptible individuals. CD affects about 1% of the general population and causes significant health problems. Adverse inflammatory reactions to gluten are mediated by inappropriate T-cell activation leading to severe damage of the gastrointestinal mucosa, causing atrophy of absorptive surface villi.

Sialyl residues modulate LPS-mediated signaling through the Toll-like receptor 4 complex.

We previously reported that neuraminidase (NA) pretreatment of human PBMCs markedly increased their cytokine response to lipopolysaccharide (LPS). To study the mechanisms by which this occurs, we transfected HEK293T cells with plasmids encoding TLR4, CD14, and MD2 (three components of the LPS receptor complex), as well as a NFκB luciferase reporting system. Both TLR4 and MD2 encoded by the plasmids are α-2,6 sialylated.

Measuring TLR function in transfectants.

This unit summarizes a combination of methods that can be optimized for measuring toll-like receptor (TLR) function. TLRs serve as primary innate immune sensors and exhibit high specificity towards evolutionarily conserved microbial and viral structures. The unit focuses specifically on TLR4, the principal Gram-negative lipopolysaccharide (LPS) sensor.

Expression of functional D299G.T399I polymorphic variant of TLR4 depends more on coexpression of MD-2 than does wild-type TLR4.

Two missense variants (D299G and T399I) of TLR4 are cosegregated in individuals of European descent and, in a number of test systems, result in reduced responsiveness to endotoxin. How these changes within the ectodomain (ecd) of TLR4 affect TLR4 function is unclear. For both wild-type and D299G.

Vibrio cholerae flagellins induce Toll-like receptor 5-mediated interleukin-8 production through mitogen-activated protein kinase and NF-kappaB activation.

Vaccine reactogenicity has complicated the development of safe and effective live, oral cholera vaccines. Delta ctx Vibrio cholerae mutants have been shown to induce inflammatory diarrhea in volunteers and interleukin-8 (IL-8) production in cultured intestinal epithelial cells. Bacterial flagellins are known to induce IL-8 production through Toll-like receptor 5 (TLR5).

TLR4-mediated activation of dendritic cells by the heat shock protein DnaK from Francisella tularensis.

Francisella tularensis is the causative agent of tularemia, a severe, debilitating disease of humans and other mammals. As this microorganism is also classified as a "category-A pathogen" and a potential biowarfare agent, there is a need for an effective vaccine. Several antigens of F.

Analysis of proteinase-activated receptor 2 and TLR4 signal transduction: a novel paradigm for receptor cooperativity.

Proteinase-activated receptor 2 (PAR2), a seven-transmembrane G protein-coupled receptor, is activated at inflammatory sites by proteolytic cleavage of its extracellular N terminus by trypsin-like enzymes, exposing a tethered, receptor-activating ligand. Synthetic agonist peptides (AP) that share the tethered ligand sequence also activate PAR2, often measured by Ca2+ release. PAR2 contributes to inflammation through activation of NF-kappaB-regulated genes; however, the mechanism by which this occurs is unknown.

Gliadin induces an increase in intestinal permeability and zonulin release by binding to the chemokine receptor CXCR3.

Background & Aims: Celiac disease is an immune-mediated enteropathy triggered by gliadin, a component of the grain protein gluten. Gliadin induces an MyD88-dependent zonulin release that leads to increased intestinal permeability, a postulated early element in the pathogenesis of celiac disease. We aimed to establish the molecular basis of gliadin interaction with intestinal mucosa leading to intestinal barrier impairment.

Differential activation of human TLR4 by Escherichia coli and Shigella flexneri 2a lipopolysaccharide: combined effects of lipid A acylation state and TLR4 polymorphisms on signaling.

The lipid A of LPS activates TLR4 through an interaction with myeloid differentiation protein-2 (MD-2) and the degree of lipid A acylation affects TLR4 responsiveness. Two TLR4 single nucleotide polymorphisms (Asp299Gly and Thr399Ile) have been associated with LPS hyporesponsiveness. We hypothesized that the combination of hypoacylation and these single nucleotide polymorphisms would exhibit a compounded effect on TLR4 signaling.

Association of TLR4 polymorphisms with symptomatic respiratory syncytial virus infection in high-risk infants and young children.

Respiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Although anti-RSV Ab prophylaxis has greatly reduced infant mortality in the United States, there is currently no vaccine or effective antiviral therapy. RSV fusion (F) protein activates cells through TLR4.

Toll-like receptor 2-mediated signaling requirements for Francisella tularensis live vaccine strain infection of murine macrophages.

Francisella tularensis, an aerobic, non-spore-forming, gram-negative coccobacillus, is the causative agent of tularemia. We reported previously that F. tularensis live vaccine strain (LVS) elicited strong, dose-dependent NF-kappaB reporter activity in Toll-like receptor 2 (TLR2)-expressing HEK293T cells and proinflammatory gene expression in primary murine macrophages.

Analysis of TLR4 polymorphic variants: new insights into TLR4/MD-2/CD14 stoichiometry, structure, and signaling.

TLR4 is the signal-transducing receptor for structurally diverse microbial molecules such as bacterial LPS, respiratory syncytial virus fusion (F) protein, and chlamydial heat shock protein 60. Previous studies associated two polymorphic mutations in the extracellular domain of TLR4 (Asp(299)Gly and Thr(399)Ile) with decreased LPS responsiveness. To analyze the molecular basis for diminished responsiveness, site-specific mutations (singly or coexpressed) were introduced into untagged and epitope (Flag)-tagged wild-type (WT) TLR4 expression vectors to permit a direct comparison of WT and mutant signal transduction.

Immunologic consequences of Francisella tularensis live vaccine strain infection: role of the innate immune response in infection and immunity.

Francisella tularensis (Ft), a Gram-negative intracellular bacterium, is the etiologic agent of tularemia. Although attenuated for humans, i.p.

Mutations in TLR4 signaling that lead to increased susceptibility to infection in humans: an overview.

In this overview, we will present current information on known mutations in the TLR4 signaling pathway that have been associated with increased susceptibility to disease. To date, mutations in the extracellular domain of TLR4 itself, IRAK-4, NEMO (IKK gamma), and I kappa B alpha have been identified and profoundly affect the host response to infection.