Major allergen content consistency of SQ house dust mite sublingual immunotherapy tablets and relevance across geographic regions.

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.

Purification and characterization of natural Bet v 1 from birch pollen and related allergens from carrot and celery.

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 Fe(III)Zn(II) form of recombinant human purple acid phosphatase is not activated by proteolysis.

The kinetics and spectroscopic properties of the single polypeptide and proteolytically cleaved form of recombinant Fe(3+)Fe(2+) human purple acid phosphatase (recHPAP) exhibit significant differences, primarily due to a difference in pK(es,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 Fe(3+)Zn(2+)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 pK(es) 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 Fe(3+)Zn(2+)-recHPAP are independent of pH over the range 4.5-6.2, the visible spectrum of Fe(3+)Zn(2+)-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.