Pea (Pisum sativum) allergy in children: Pis s 1 is an immunodominant major pea allergen and presents IgE binding sites with potential diagnostic value.

BACKGROUND: Food allergy to pea (Pisum sativum) has been rarely studied in children at the clinical and molecular levels. OBJECTIVE: To elucidate the allergenic relevance and diagnostic value of pea 7S globulin Pis s 1, nsLTP, and 2S albumins PA1 and PA2 in children. METHODS: Children with pea-specific IgE ≥ 0.35 kUA /L and clinical evidence of pea allergy or tolerance were included in the study. IgE binding against pea total protein extract, recombinant (r) rPis s 1, rPA1, rPA2, and natural nsLTP was analysed using IgE immunoblot/inhibition. Mediator release potency was investigated in passively sensitized rat basophil leukaemia (RBL) 2H3-cells. IgE binding to synthetic overlapping peptides of Pis s 1 was detected on multipeptide microarrays. RESULTS: 19 pea-sensitized children were included, 14 with doctors' diagnosed allergy and 5 with tolerance to pea (median age 3.5 and 4.5 years, respectively). 11/14 (78%) pea-allergic and 1/5 (20%) tolerant children were sensitized to Pis s 1. Under the reducing conditions of immunoblot analysis, IgE binding to rPA1 was negligible, sensitization to rPA2 and nsLTP undetectable. Compared to pea total protein extract, rPis s 1 displayed on average 58% IgE binding capacity and a 20-fold higher mediator release potency. Selected Pis s 1-related peptides displayed IgE binding in pea-allergic but not in pea-tolerant children. CONCLUSIONS AND CLINICAL RELEVANCE: In this study group, Pis s 1 is a major immunodominant allergen in pea-allergic children. Evidence for sensitization to nsLTP and 2S albumins was low but requires further verification with regard to conformational epitopes. Recombinant Pis s 1 and related peptides which were exclusively recognized by pea-allergic children may improve in vitro diagnosis of pea allergy once verified in prospective studies with larger study groups.

Quality and potency profile of eight recombinant isoallergens, largely mimicking total Bet v 1-specific IgE binding of birch pollen.

BACKGROUND: To date, only limited information on structure, expression levels and IgE binding of Bet v 1 variants, which are simultaneously expressed in birch pollen, is available. OBJECTIVE: To analyse and compare structure and serum IgE/IgG binding of rBet v 1 variants to Bet v 1.0101. METHODS: Recombinant Bet v 1 variants were studied with sera of 20 subjects allergic to birch pollen. Folding, aggregation and solubility of the rBet v 1 variants were analysed to attribute diverging IgE binding to either allergen structure or methodological features. IgE/IgG binding was studied with rBet v 1 in solution or adsorbed to solid phases. Allergen-mediated cross-linking of FcεRI receptors was determined by mediator release of sensitized humanized rat basophil leukaemia cells. RESULTS: All variants, except for rBet v 1.0113, were monomeric and had Bet v 1-type conformation. Serum IgE binding to variants adsorbed to solid phase was reduced to 6.6%-36.5% compared with Bet v 1.0101. In contrast, inhibition of IgE binding to Bet v 1.0101 by rBet v 1 variants ranged from 62% to 83%. Similarly, mediator release ranged from 30.7% to 55.2% for all variants and was only clearly reduced for rBet v 1.0301 (10.4%). The IgE-binding potency of rBet v 1 variants representing their native quantities in birch pollen was only slightly lower compared to extract. IgG binding to variants was between 50.9% and 134.5% compared with rBet v 1.0101 (100%). CONCLUSION AND CLINICAL RELEVANCE: Bet v 1 variants previously classified as hypoallergenic can exhibit similar functional IgE binding as Bet v 1.0101. Eight rBet v 1 variants largely reproduce total Bet v 1-specific IgE binding of birch pollen extracts. Assay format-dependent variation in IgE-binding properties needs to be considered in the development of diagnostic or therapeutic products.

Control region length dynamics potentially drives amino acid evolution in tarsier mitochondrial genomes.

Patterns and processes of molecular evolution critically influence inferences in phylogeny and phylogeography. Within primates, a shift in evolutionary rates has been identified as the rationale for contrasting findings from mitochondrial and nuclear DNA studies as to the position of Tarsius. While the latter now seems settled, we sequenced complete mitochondrial genomes of three Sulawesi tarsiers (Tarsius dentatus, T. lariang, and T. wallacei) and analyzed substitution rates among tarsiers and other primates to infer driving processes of molecular evolution. We found substantial length polymorphism of the D-loop within tarsier individuals, but little variation of predominant lengths among them, regardless of species. Length variation was due to repetitive elements in the CSB domain-minisatellite motifs of 35 bp length and microsatellite motifs of 6 bp length. Amino acid evolutionary rates were second highest among major primate taxa relative to nucleotide substitution rates. We observed many radical possibly function-altering amino acid changes that were rarely driven by positive selection and thus potentially slightly deleterious or neutral. We hypothesize that the observed pattern of an increased amino acid evolutionary rate in tarsier mitochondrial genomes may be caused by hitchhiking of slightly deleterious mutations with favored D-loop length variants selected for maximizing replication success within the cell or the mitochondrion.

LAMP-LFD Based on Isothermal Amplification of Multicopy Gene ORF160b: Applicability for Highly Sensitive Low-Tech Screening of Allergenic Soybean (Glycine max) in Food.

Soybean (Glycine max) allergy can be life threatening. A lack of causative immunotherapy of soybean allergy makes soybean avoidance indispensable. Detection methods are essential to verify allergen labeling and unintentional allergen cross contact during food manufacture. Here, we aimed at evaluating our previously described primers for loop-mediated isothermal amplification (LAMP) of multicopy gene ORF160b, combined with a lateral flow dipstick (LFD)-like detection, for their performance of soybean detection in complex food matrices. The results were compared with those obtained using quantitative real-time Polymerase Chain Reaction (qPCR) as the current standard of DNA-based allergen detection, and antibody-based commercial lateral flow device (LFD) as the current reference of protein-based rapid allergen detection. LAMP-LFD allowed unequivocal and reproducible detection of 10 mg/kg soybean incurred in three representative matrices (boiled sausage, chocolate, instant tomato soup), while clear visibility of positive test lines of two commercial LFD tests was between 10 and 102 mg/kg and depending on the matrix. Sensitivity of soybean detection in incurred food matrices, commercial retail samples, as well as various processed soybean products was comparable between LAMP-LFD and qPCR. The DNA-based LAMP-LFD proved to be a simple and low-technology soybean detection tool, showing sensitivity and specificity that is comparable or superior to the investigated commercial protein-based LFD.

Folded or Not? Tracking Bet v 1 Conformation in Recombinant Allergen Preparations.

BACKGROUND: Recombinant Bet v 1a (rBet v 1a) has been used in allergy research for more than three decades, including clinical application of so-called hypoallergens. Quantitative IgE binding to rBet v 1a depends on its native protein conformation, which might be compromised upon heterologous expression, purification, or mutational engineering of rBet v 1a. OBJECTIVE: To correlate experimental/theoretical comparisons of IgE binding of defined molar ratios of folded/misfolded recombinant Bet v 1a variants and to determine accuracy and precision of immuno- and physicochemical assays routinely used to assess the quality of recombinant allergen preparations. METHODS: rBet v 1a and its misfolded variant rBet v 1aS112P/R145P were heterologously expressed and purified from Escherichia coli. Structural integrities and oligomerisation of the recombinant allergens were evaluated by 1H-nuclear magnetic resonance (1H-NMR), circular dichroism (CD) spectroscopy, and dynamic light scattering (DLS). IgE binding of defined combinations of rBet v 1a and rBet v 1aS112P/R145P was assessed using immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA) and mediator release (MR) of humanized rat basophilic leukemia cells sensitized with serum IgE of subjects allergic to birch pollen. Experimental and theoretically expected results of the analyses were compared. RESULTS: 1H-NMR spectra of rBet v 1a and rBet v 1aS112P/R145P demonstrate a native and highly disordered protein conformations, respectively. The CD spectra suggested typical alpha-helical and beta-sheet secondary structure content of rBet v 1a and random coil for rBet v 1aS112P/R145P. The hydrodynamic radii (RH) of 2.49 ± 0.39 nm (rBet v 1a) and 3.1 ± 0.56 nm (rBet v 1aS112P/R145P) showed monomeric dispersion of both allergens in solution. Serum IgE of birch pollen allergic subjects bound to 0.1% rBet v 1a in the presence of 99.9% of non-IgE binding rBet v 1aS112P/R145P. Immunoblot analysis overestimated, whereas ELISA and mediator release assay underestimated the actual quantity of IgE-reactive rBet v 1a in mixtures of rBet v 1a/rBet v 1aS112P/R145P with a molar ratio of rBet v 1a ≤ 10%. CONCLUSION: Valid conclusions on quantitative IgE binding of recombinant Bet v 1a preparations depend on the accuracy and precision of physico- and immunochemical assays with which natively folded allergen is detected.