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Licensed Unlicensed Requires Authentication Published by De Gruyter June 1, 2005

Engineering, Purification and Applications of His-Tagged Recombinant Antibody Fragments with Specificity for the Major Birch Pollen Allergen, Bet v1

  • Sabine Flicker , Sylvia Laffer , Peter Steinberger , Bashar Alhani , Ying Zhu , Marja-Leena Laukkanen , Kari Keinänen , Dietrich Kraft and Rudolf Valenta
From the journal Biological Chemistry

Abstract

Type I allergy, an immunodisorder affecting almost 20% of the population worldwide, is based on the production of IgE antibodies against per se harmless allergens. We report the expression of hexahistidine-tagged antibody fragments (Fabs) with specificity for Bet v1, the major birch pollen allergen, in Escherichia coli. The cDNA coding for the heavy chain fragment of a mouse monoclonal anti-Bet v1 antibody, Bip 1, was engineered by PCR to contain a hexahistidine-encoding 3′ end. The modified Bip 1 heavy chain cDNA was co-expressed in E. coli XL-1 Blue with the Bip 1 light chain cDNA using the combinatorial plasmid pComb3H. His-tagged recombinant (r) Bip1 Fabs were isolated by nickel affinity chromatography and rBip 1 Fabs without His-tag were purified via affinity to rBet v1. rBip 1 Fabs with and without His-tag bound specifically to rBet v 1 and, like Bet v1-specific human serum IgE and rabbit-anti rBet v1 antibodies, cross-reacted with Bet v1-related allergens in other plant-species (alder, oak, hazelnut). We demonstrate the usefulness of His-tagged rBip 1 Fabs (1) for the identification of pollen samples containing Bet v1 by particle blotting, (2) for the detection of Bet v1-specific IgE antibodies in human serum samples by sandwich ELISA and (3) for the quantification of Bet v1 in solution. Based on these examples we suggest to use rBip 1 Fabs for the detection of Bet v1 and Bet v1-related allergens in natural allergen sources for allergy prevention, as well as for the standardization of natural allergen extracts produced for diagnosis and immunotherapy of birch pollen allergy.

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Published Online: 2005-06-01
Published in Print: 2000-01-28

Copyright © 2000 by Walter de Gruyter GmbH & Co. KG

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