Purification and characterization of the major oak pollen allergen Que a 1 for component-resolved diagnostics using ImmunoCAP.
BACKGROUND
The aim of this study was to purify the major oak pollen allergen, Que a 1, to perform biochemical and immunological characterization of the allergen and to develop an experimental native (n) Que a 1 ImmunoCAP(R).


METHODS
Que a 1 was purified from oak pollen extract using affinity chromatography and characterized by SDS-PAGE, two-dimensional (2D) PAGE, mass spectrometry (MS), N-terminal sequencing and specific IgE inhibition on ImmunoCAP. Samples from 16 subjects sensitized to oak pollen were analyzed by ImmunoCAP for IgE reactivity to nQue a 1, and recombinant (r)Bet v 1 and 2 (profilin). They were also studied in IgE immunoblotting.


RESULTS
The purity of nQue a 1 was >95%, since a single band was observed on silver-stained SDS-PAGE. The identity was verified by MS analysis, and 2D-PAGE revealed several isoforms. The obtained N-terminal sequence of 50-amino-acid residues from nQue a 1 showed a 58-74% sequence identity with other pathogenesis-related class 10 allergens. Specific IgE inhibition verified a preserved immunoreactivity (70-92% inhibition). All subjects were sensitized to Que a 1 and Bet v 1, and two to profilin. The IgE antibody levels to nQue a 1 were generally lower than to rBet v 1. The obtained results correlated well with IgE immunoblotting.


CONCLUSIONS
We present a highly purified and extensively characterized preparation of nQue a 1. Que a 1 seems to be an allergen of equal importance in oak pollen as Bet v 1 in birch pollen. An nQue a 1 ImmunoCAP will be useful in component-resolved diagnostics.
Movérare
Robert
R
Phadia AB, Uppsala University, Uppsala, Sweden. robert.moverare@phadia.com
Everberg
Henrik
H
Carlsson
Raimo
R
Holtz
Anette
A
Thunberg
Roland
R
Olsson
Peter
P
Brostedt
Peter
P
Hogbom
Erik
E
D000485:Allergens / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology*; D008969:Molecular Sequence Data; D010865:Pilot Projects; D010940:Plant Proteins / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D011058:Pollen / Q000737:chemistry* / Q000276:immunology; D029963:Quercus / Q000737:chemistry / Q000276:immunology*; D006255:Rhinitis, Allergic, Seasonal / Q000097:blood / Q000276:immunology; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000485:Allergens; D052179:Antigens, Plant; D010940:Plant Proteins; C061312:Bet v 1 allergen, Betula; D007073:Immunoglobulin E