Affinity purification of egg-white allergens for improved component-resolved diagnostics.
BACKGROUND
Egg is a common cause of food-allergic reactions, especially among young children. Some egg-allergic patients do, however, tolerate heated egg products and component-resolved diagnostics (CRD) may facilitate prediction of different disease manifestations. Commercially available preparations of the egg-white allergens, ovomucoid, ovalbumin, conalbumin and lysozyme, have been reported to contain impurities which interfere with accurate CRD.


METHODS
Commercial preparations of the 4 egg-white allergens were characterized using allergen-specific monoclonal chimeric human/mouse IgE antibodies in experimental ImmunoCAP® tests. Further purification of commercial ovomucoid, ovalbumin and conalbumin preparations was performed by chromatography based on affinity to monoclonal antibodies. Purity was monitored by size exclusion chromatography, SDS-PAGE, Western blotting and experimental ImmunoCAP tests using allergen-specific chimeric IgE antibodies. IgE reactivity to the highly purified egg components was analyzed in 83 samples from egg white-sensitized individuals.


RESULTS
Preparations of commercially available ovomucoid, ovalbumin and conalbumin were found to contain other egg allergens which were removed by chromatographic purification. No impurities were detected in the commercial lysozyme preparation. Previously unknown complexes between the target allergens and contaminating allergens were detected and removed by affinity chromatography. IgE reactivity to ovalbumin was most common in the analyzed samples (87%), followed by ovomucoid (72%), conalbumin (69%) and lysozyme (58%).


CONCLUSIONS
In this study we demonstrate the advantage of using monoclonal antibodies for purification, and monoclonal chimeric IgE antibodies for characterization, of egg allergens intended for CRD. Our study also established that ovalbumin, ovomucoid, conalbumin and lysozyme are all major allergens.
Everberg
Henrik
H
Phadia AB, Uppsala, Sweden. henrik.everberg@phadia.com
Brostedt
Peter
P
Oman
Hans
H
Bohman
Svante
S
Movérare
Robert
R
D000485:Allergens / Q000276:immunology / Q000302:isolation & purification*; D000911:Antibodies, Monoclonal / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods*; D003207:Conalbumin / Q000276:immunology / Q000302:isolation & purification*; D021181:Egg Hypersensitivity / Q000175:diagnosis*; D004529:Egg White / Q000737:chemistry*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D007159:Immunologic Tests; D010047:Ovalbumin / Q000276:immunology / Q000302:isolation & purification*; D010059:Ovomucin / Q000276:immunology / Q000302:isolation & purification*
D000485:Allergens; D000911:Antibodies, Monoclonal; D003207:Conalbumin; D010059:Ovomucin; D007073:Immunoglobulin E; D010047:Ovalbumin