Engineering, purification and applications of His-tagged recombinant antibody fragments with specificity for the major birch pollen allergen, bet v1.
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 Bip1 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) Bip 1 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 v1 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 v 1 by particle blotting, (2) forthe 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.
Flicker
S
S
Institute of General and Experimental Pathology, AKH, University of Vienna, Austria.
Laffer
S
S
Steinberger
P
P
Alhani
B
B
Zhu
Y
Y
Laukkanen
M L
ML
Keinänen
K
K
Kraft
D
D
Valenta
R
R
D000485:Allergens; D000818:Animals; D000918:Antibody Specificity; D052179:Antigens, Plant; D001483:Base Sequence; D002846:Chromatography, Affinity; D003429:Cross Reactions; D018076:DNA, Complementary; D004797:Enzyme-Linked Immunosorbent Assay; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000276:immunology*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014197:Trees / Q000276:immunology
D000485:Allergens; D052179:Antigens, Plant; D018076:DNA, Complementary; D007128:Immunoglobulin Fragments; D010940:Plant Proteins; D011994:Recombinant Proteins; C061312:Bet v 1 allergen, Betula; D006639:Histidine