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, Rudolf Valenta

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13 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)39-47
Number of pages9
JournalBiological Chemistry
Volume381
Issue number1
DOIs
Publication statusPublished - 2000
MoE publication typeA1 Journal article-refereed

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Flicker, S., Laffer, S., Steinberger, P., Alhani, B., Zhu, Y., Laukkanen, M-L., Keinänen, K., Kraft, D., & Valenta, R. (2000). Engineering, purification and applications of his-tagged recombinant antibody fragments with specificity for the major birch pollen allergen, Bet v1. Biological Chemistry, 381(1), 39-47. https://doi.org/10.1515/BC.2000.006