Background: Consistency in composition and potency, particularly regarding major allergens, is crucial for the quality of extracts for allergen immunotherapy.Objective: To characterize the major allergen composition of house dust mite (HDM) extracts commercially available in the United States and the SQ HDM sublingual immunotherapy (SLIT) tablet, and to relate the composition to patient sensitization patterns.Methods: Der 1/Der 2 ratios were determined in 10,000- and 30,000-AU/mL HDM extracts from 5 US companies and the SQ HDM SLIT-tablet. Allergen content was analyzed by enzyme-linked immunosorbent assay and compared with an in-house reference. Sensitivity toward Der p 1, Der p 2, and Der p 10 was determined in serum from randomly selected subgroups of 220 individuals from North American and European SQ HDM SLIT-tablet trials.Results: Mean Der 1/Der 2 ratios in US HDM extracts ranged from 0.4 to 20.5. For the SQ HDM SLIT-tablet (20 batches), variability did not exceed 12% regarding content of Der f 1 (SD, 11.9%; 95% confidence interval [CI], 0.94-1.06), Der p 1 (SD, 6.1%; 95% CI, 0.97-1.03), and combined Der 2 allergen (SD, 6.4%; 95% CI, 0.97-1.03), indicating a consistent Der 1/Der 2 ratio. High allergen sensitivity frequencies toward Der p 1 and Der p 2 were observed regardless of geographic region. Efficacy of the SQ HDM SLIT-tablet has been demonstrated in 5 clinical trials.Conclusion: The SQ HDM SLIT-tablet has efficacy potential for a broad range of patients because it includes a consistent 1: 1 ratio of the 2 major HDM allergens to which individuals were most frequently sensitized across geographic regions. Efficacy has been demonstrated. (C) 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Consistency in composition and potency, particularly regarding major allergens, is crucial for allergen immunotherapy efficacy and safety. We compared allergen sensitivity profiles for house dust mite (HDM) in North American and European patients with the allergen content of commercially available US HDM extracts and an SQ-HDM sublingual immunotherapy tablet (SLIT-tablet; MK-8237; Merck/ALK). Sensitivity towards Der p 1, Der p 2, and Der p 10 allergens was determined in randomly selected subgroups of 220 subjects from European and North American SQ-HDM SLIT-tablet trials. Der 1/Der 2 ratios were determined in 10,000 and 30,000 AU/mL HDM extracts from 3 US companies. SQ-HDM SLIT-tablets are produced by mixing body and fecal fractions of Dermatophagoides farinae and Dermatophagoides pteronyssinus to achieve a 1:1:1:1 ratio between Der f 1/2 and Der p 1/2 allergens. Allergen content was analyzed by ELISA and compared with an in-house reference. Subject sensitization profiles indicated high reactivity frequencies towards Der p 1 and Der p 2 regardless of geographic region. The average Der 1/Der 2 ratio in US extracts ranged from 1:1 to 5:1. For SQ-HDM SLIT-tablet (20 batches), normalized mean (SD) content for Der f 1, Der p 1, and combined Der 2 allergens was 1.000 (0.119), 1.000 (0.061), and 1.000 (0.064) units, respectively. Other HDM allergens were also present. Der 1/Der 2 are major HDM allergens in North American and European populations. The described process enables content control of each major allergen and results in SQ-HDM SLIT-tablets containing both mites, and consistent and equivalent Der 1/Der 2 content.

Organic production benefits from fair competition and sustained consumer confidence. The latter can only be assured by paper trailing and verification assessments. Traditional analytical strategies for guaranteeing quality and uncovering adulteration have relied on the determination of the amount of a marker compound or compounds in a material and a subsequent comparison of the value(s) obtained with those established for equivalent material. Since it is unlikely to find a unique marker that allows discrimination between organic and conventional produce, selective fingerprinting (profiling) combined with chemometrics is a more promising approach. In this study, carotenoid High Performance Liquid Chromatography–Diode Array Detection profiling combined with k-nearest neighbour classification chemometrics was used to predict the production system (organic vs. non-organic) of chicken eggs. A proof-of-concept set (training set for the model) was composed of eggs from 24 organic farms, 12 free range, and 12 barn farms in the Netherlands. The identities of organic, free range, and barn eggs were predicted with success rates of 100%, 100%, and 84%, respectively. The identity of eggs determined using an additional market test set with eggs from 12 organic, 12 free range, and 12 barn farms originating from the Netherlands and New Zealand resulted in correct classifications for 35 of the 36 samples. The results of this study indicate that this fingerprint approach is a promising tool for analytical verification of the production system of organic eggs.

Natural and recombinant Bet v 1, the major birch pollen allergen, and homologous allergens, Api g 1 and Dau c 1, from celery and carrot, respectively, were studied by CD spectroscopy under conditions of varying denaturant concentration, pH and temperature to determine fundamental thermodynamic parameters for conformational stability. Thermodynamic studies increase basic knowledge regarding differences between birch pollen-related allergens and are of importance in choosing processing conditions. The conformational stability determined from guanidine hydrochloride denaturation curves was similar for rBet v 1.0101 and rApi g 1.0101. Conformational responses to chaotropic salt were different for recombinant allergens from different species, but were similar for the natural isoform mixtures. The conformational stabilities of nApi g 1 and nDau c 1, were shown to be similar to rBet v 1.2801 at pH>4.4 [Mogensen, J. E., Ipsen, H., Holm, J., & Otzen, D. E. (2004). Elimination of a misfolded folding intermediate by a single point mutation. Biochemistry, 43(12), 3357-3367], but nApi g and nDau c 1 were stable to heating at lower pH-values.

Birch pollen allergy is predominantly caused by the major allergen Bet v 1 and can lead to crossreactions with homologous proteins in food. Two major cross-reactive food allergens are Dau c 1 from carrot and Api g 1 from celery, which have never been purified from their natural source. Here, we describe a non-denaturing purification method for obtaining natural Bet v 1, Dau c 1 and Api g 1, comprising of ammonium sulfate precipitation, hydrophobic interaction chromatography and size exclusion chromatography. This method resulted in 98-99% pure isoform mixtures for each allergen. Characterization of these isoform mixtures with Q-TOF MS/MS clearly showed earlier reported isoforms of Bet v 1, Dau c 1 and Api g 1, but also new isoforms. The presence of secondary structure in the three purified allergens was demonstrated via circular dichroism and showed high similarity. The immune reactivity of the natural allergens was compared with recombinant proteins by Western blot and ELISA and showed similar reactivity.

The kinetics and spectroscopic properties of the single polypeptide and proteolytically cleaved form of recombinant Fe3+Fe2+ human purple acid phosphatase (recHPAP) exhibit significant differences, primarily due to a difference in pKes,1 (the value of an acid dissociation constant of the ES complex). These differences are due to the presence or absence, respectively, of an interaction between an aspartate residue in an exposed loop of the protein and one or more active site residues. To further explore the origin of these differences, the ferrous ion of recHPAP has been replaced by zinc. Analysis of the reconstituted Fe3+Zn2+recHPAP reveals an unexpected catalytic activity versus pH profile, in that the optimal pH is 6.3, similar to that of the proteolytically cleaved form (6.5). Moreover, replacement of the ferrous ion by zinc increases the turnover number more than 10-fold; the pKes values are also shifted as expected for the change in the divalent metal ion. Although the EPR spectra of both single polypeptide and proteolytically cleaved Fe3+Zn2+-recHPAP are independent of pH over the range 4.5–6.2, the visible spectrum of Fe3+Zn2+-recHPAP is pH dependent. These results suggest that the properties and environment of the divalent metal are important in determining the catalytic properties of mammalian PAPs, and in particular that a solvent molecule coordinated to the divalent metal ion may play a critical role in the catalytic cycle of these enzymes.