Πέμπτη 31 Ιανουαρίου 2019

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

Abstract

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 analyze 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 was analyzed 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 crosslinking of FcεRI receptors was determined by mediator release of sensitized humanized rat basophil leukemia 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% ‐ 83%. Similarly, mediator release ranged from 30.7% ‐ 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% ‐ 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 of IgE binding properties needs to be considered in the development of diagnostic or therapeutic products.

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