Specific allergen profiles of peanut foods and diagnostic or therapeutic allergenic products.

BACKGROUND: Generic immunoassays for peanut cannot discriminate between allergen levels in peanut-derived food products or therapeutics. Clinical trials of oral immunotherapy (OIT) are strengthened by using standardized peanut preparations with defined doses of major allergens. OBJECTIVE: This article describes measurement of Ara h 1, Ara h 2, and Ara h 6 in peanut foods and in peanut flour extracts used for allergy diagnosis and OIT. METHODS: Monoclonal antibody-based enzyme immunoassays for Ara h 1, Ara h 2, and Ara h 6 were used to compare allergen levels in peanut (n = 16) and tree nut (n = 16) butter, peanut flour (n = 11), oils (n = 8), extracts used for diagnosis and OIT (n = 5), and the National Institute for Standards and Technology Peanut Butter Standard Reference Material 2387. RESULTS: Roasted peanut butters contained 991 to 21,406 µg/g Ara h 1 and exceeded Ara h 2 and Ara h 6 levels by 2- to 4-fold. Similarly, National Institute for Standards and Technology Peanut Butter Standard Reference Material 2387 contained 11,275 µg/g Ara h 1, 2,522 µg/g Ara h 2, and 2,036 µg/g Ara h 6. In contrast, peanut flours contained 787 to 14,631 µg/g Ara h 2 and exceeded Ara h 1 levels by 2- to 20-fold. Flour extracts used for OIT contained 394 to 505 µg/mL Ara h 1, 1,187 to 5,270 µg/mL Ara h 2, and 1,104 to 8,092 µg/mL Ara h 6. In most cases specific peanut allergens were not detected in tree nut butters or peanut oils. CONCLUSIONS: The results show marked differences in specific peanut allergen profiles in peanut butter and flour and peanut preparations for clinical use. Roasting can increase Ara h 1 levels in peanut butter. Variability in allergen levels could affect the outcome of clinical trials of peanut OIT, especially with respect to Ara h 1. Specific allergen measurements will improve standardization and provide accurate dosing of peanut preparations that are being used for OIT.

Biological activity of human IgE monoclonal antibodies targeting Der p 2, Fel d 1, Ara h 2 in basophil mediator release assays.

Background: Human Immunoglobulin E monoclonal antibodies (hIgE mAb) are unique tools for investigating IgE responses. Here, the biological activity of hIgE mAb, derived from immortalized B cells harvested from the blood of allergic donors, targeting three allergens (Der p 2, Fel d 1 and Ara h 2) was investigated. Methods: Three Der p 2-, three Fel d 1- and five Ara h 2-specific hIgE mAb produced by human B cell hybridomas, were combined in pairs and used to passively sensitize humanized rat basophilic leukemia cells and compared with sensitization using serum pools. Sensitized cells were stimulated with corresponding allergens (recombinant or purified), allergen extracts or structural homologs, having 40-88% sequence similarity, and compared for mediator (ß-hexosaminidase) release. Results: One, two and eight pairs of Der p 2-, Fel d 1- and Ara h 2-specific hIgE mAb, respectively, produced significant mediator release (>50%). A minimum hIgE mAb concentration of 15-30 kU/L and a minimum antigen concentration between 0.01-0.1 µg/mL were sufficient to induce a pronounced mediator release. Individual sensitization with one Ara h 2-specific hIgE mAb was able to induce crosslinking independently of a second specific hIgE mAb. Der p 2- and Ara h 2-specific mAb showed a high allergen specificity when compared to homologs. Mediator release from cells sensitized with hIgE mAb was comparable to serum sensitization. Conclusion: The biological activity of hIgE mAb reported here provides the foundation for novel methods of standardization and quality control of allergen products and for mechanistic studies of IgE-mediated allergic diseases, using hIgE mAb.

Unique allergen-specific human IgE monoclonal antibodies derived from patients with allergic disease.

Introduction: Allergic reactions are mediated by human IgE antibodies that bind to and cross-link allergen molecules. The sites on allergens that are recognized by IgE antibodies have been difficult to investigate because of the paucity of IgE antibodies in a human serum. Here, we report the production of unique human IgE monoclonal antibodies to major inhaled allergens and food allergens that can be produced at scale in perpetuity. Materials and methods: The IgE antibodies were derived from peripheral blood mononuclear cells of symptomatic allergic patients, mostly children aged 3-18 years, using hybridoma fusion technology. Total IgE and allergen-specific IgE was measured by ImmunoCAP. Their specificity was confirmed through ELISA and immunoblotting. Allergenic potency measurements were determined by ImmunoCAP inhibition. Biological activity was determined in vitro by comparing ß-hexosaminidase release from a humanized rat basophilic cell line. Results: Human IgE monoclonal antibodies (n = 33) were derived from 17 allergic patients with symptoms of allergic rhinitis, asthma, atopic dermatitis, food allergy, eosinophilic esophagitis, or red meat allergy. The antibodies were specific for five inhaled allergens, nine food allergens, and alpha-gal and had high levels of IgE (53,450-1,702,500 kU/L) with ratios of specific IgE to total IgE ranging from <0.01 to 1.39. Sigmoidal allergen binding curves were obtained through ELISA, with low limits of detection (<1 kU/L). Allergen specificity was confirmed through immunoblotting. Pairs of IgE monoclonal antibodies to Ara h 6 were identified that cross-linked after allergen stimulation and induced release of significant levels of ß-hexosaminidase (35%-80%) from a humanized rat basophilic cell line. Conclusions: Human IgE monoclonal antibodies are unique antibody molecules with potential applications in allergy diagnosis, allergen standardization, and identification of allergenic epitopes for the development of allergy therapeutics. The IgE antibody probes will enable the unequivocal localization and validation of allergenic epitopes.

Corrigendum: Biological activity of human IgE monoclonal antibodies targeting Der p 2, Fel d 1, Ara h 2 in basophil mediator release assays.

[This corrects the article DOI: 10.3389/fimmu.2023.1155613.].

Simultaneous quantification of specific food allergen proteins using a fluorescent multiplex array.

The aim was to develop a fluorescent multiplex array for simultaneously measuring regulated food allergens using specific allergen protein molecules from peanut, tree nut, cow's milk, egg, soy, fish, shellfish, sesame, mustard and celery. Microspheres coupled to specific monoclonal antibodies were used for allergen detection, with purified allergens as reference standards.Standard curves for 17 allergens covered a 5-log dynamic range. Intra- and inter-assay acceptance criteria were within 70-130% recovery and a CV of ≤15%. Food reference materials contained high levels of their respective major allergens (2000-175,000 µg/g), Similar high allergen levels were found in 10 selected foods analysed using a 9-plex array. Egg, milk, peanut, hazelnut and walnut allergens were detectable in chocolate bars with incurred allergens at 3, 10, 30, and 100 ppm. The multiplex array is an efficient tool for measuring specific food allergens, with applications for risk assessment and standardization of therapeutic products for food allergy.

T lymphocyte rolling and recruitment into peripheral lymph nodes is regulated by a saturable density of L-selectin (CD62L).

L-selectin mediates tethering and rolling of lymphocytes in high endothelial venules (HEV) of lymph nodes (LN) and of leukocytes at inflammatory sites. We used transgenic mice expressing varying levels of wild-type or a non-cleavable mutant form of L-selectin on T cells to determine the relationship between L-selectin density, tethering and rolling, and migration into LN. T cells expressing supraphysiological levels of either wild-type or non-cleavable L-selectin showed rolling parameters similar to C57BL/6 T cells in hydrodynamic flow assays and during rolling in Peyer's patch HEV. In contrast, PMA- or antigen-activated T cells and L-selectin(+/-) T cells expressing subphysiological levels of L-selectin showed reduced numbers of rolling cells with increased rolling velocity. Short-term homing studies showed that elevated expression of L-selectin above physiological levels had no effect on T cell migration to LN; however, low L-selectin expression resulted in reduced T cell homing to LN. Thus, T lymphocyte migration into LN is regulated by the density of cell surface L-selectin. In addition, there is a saturable density of L-selectin required for optimal homing to PLN in C57BL/6 mice, the L-selectin level on circulating naive T cells promotes optimal homing, and increased expression above saturating levels promotes no further increase in T cell recruitment.

Bioactive hydrogel substrates: probing leukocyte receptor-ligand interactions in parallel plate flow chamber studies.

The binding of activated integrins on the surface of leukocytes facilitates the adhesion of leukocytes to vascular endothelium during inflammation. Interactions between selectins and their ligands mediate rolling, and are believed to play an important role in leukocyte adhesion, though the minimal recognition motif required for physiologic interactions is not known. We have developed a novel system using poly(ethylene glycol) (PEG) hydrogels modified with either integrin-binding peptide sequences or the selectin ligand sialyl Lewis X (SLe(X)) within a parallel plate flow chamber to examine the dynamics of leukocyte adhesion to specific ligands. The adhesive peptide sequences arginine-glycine-aspartic acid-serine (RGDS) and leucine-aspartic acid-valine (LDV) as well as sialyl Lewis X were bound to the surface of photopolymerized PEG diacrylate hydrogels. Leukocytes perfused over these gels in a parallel plate flow chamber at physiological shear rates demonstrate both rolling and firm adhesion, depending on the identity and concentration of ligand bound to the hydrogel substrate. This new system provides a unique polymer-based model for the study of interactions between leukocytes and endothelium as well as a platform to develop improved scaffolds for cardiovascular tissue engineering.

Functional analysis of the combined role of the O-linked branching enzyme core 2 beta1-6-N-glucosaminyltransferase and dimerization of P-selectin glycoprotein ligand-1 in rolling on P-selectin.

Leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) is expressed as a homodimer and mediates leukocyte rolling through interactions with endothelial P-selectin. Previous studies have shown that PSGL-1 must be properly modified by specific glycosyltransferases including alpha1,3-fucosyltransferase-VII, core 2 beta1-6-N-glucosaminyltransferase (C2GlcNAcT-I), one or more alpha2,3-sialytransferases, and a tyrosulfotransferase. In addition, dimerization of PSGL-1 through its sole extracellular cysteine (Cys(320)) is essential for rolling on P-selectin under shear conditions. In this report, we measured the contributions of both C2GlcNAcT-I glycosylation and dimerization of PSGL-1 to adhesive bonds formed during tethering and rolling of transfected cell lines on purified P-selectin. Tethering to P-selectin under flow increased with dimerization compared with cells expressing monomeric PSGL-1 (referred to as C320A). The rolling defects (decreased cellular accumulation, PSGL-1/P-selectin bond strengths and tethering rates, and increased velocities and skip distance) demonstrated by transfectants expressing monomeric PSGL-1 could be overcome by increasing the substrate P-selectin site density and by overexpressing C2GlcNAcT-I in C320A transfectants. Two molecular weight variants of PSGL-1 were isolated from cell lines transfected with PSGL-1, C320A, and/or C2GlcNAcT-I cDNAs, and these differences in electrophoretic mobility appeared to correlate with C2GlcNAcT-I expression. C320A transfectants expressing low molecular weight PSGL-1 had lower C2GlcNAcT-I levels (measured by reactivity to core 2 specific linkage antibody, CHO-131) and compromised rolling on P-selectin (regardless of site density) compared with C320A cells with high levels of C2GlcNAcT-I and high molecular weight PSGL-1. Both C2GlcNAcT-I glycosylation and PSGL-1 dimerization increased the rate of tethering to P-selectin under flow, whereas C2GlcNAcT-I levels primarily influenced tether bond strength.