TY - JOUR
T1 - Engineering, purification and applications of his-tagged recombinant antibody fragments with specificity for the major birch pollen allergen, Bet v1
AU - Flicker, Sabine
AU - Laffer, Sylvia
AU - Steinberger, Peter
AU - Alhani, Bashar
AU - Zhu, Ying
AU - Laukkanen, Marja-Leena
AU - Keinänen, Kari
AU - Kraft, Dietrich
AU - Valenta, Rudolf
PY - 2000
Y1 - 2000
N2 - 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.
AB - 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.
U2 - 10.1515/BC.2000.006
DO - 10.1515/BC.2000.006
M3 - Article
SN - 1431-6730
VL - 381
SP - 39
EP - 47
JO - Biological Chemistry
JF - Biological Chemistry
IS - 1
ER -