Optimization of critical factors affecting the performance of an allergen chip for the analysis of an allergen-specific human IgE in serum.
A sensitive and multiplexed assay of allergen-specific human immunoglobulin E (IgE) is of great significance in the precise diagnosis of allergies. We report on the optimization of critical factors for chip-based analysis of IgE in human serum with a high reliability. Extracts of two mite species were used as model allergens, and were spotted onto a glass slide for the construction of an allergen chip. Respective allergen-specific IgE in human serum was analyzed by using biotinylated anti-human IgE and a streptavidin-Cy3 conjugate. Factors affecting the performance of the allergen chip were investigated and optimized. Especially, the effect of additives, the concentrations of biotinylated anti-human IgE and the streptavidin-Cy3 conjugate, the serum dilution factor, and the concentration of allergen extract as a capturing agent were examined in detail. Under the optimized conditions, a chip-based analysis for sera from 43 patients revealed a reliable and reproducible diagnosis of respective allergies, showing a good correlation with a conventional CAP assay.
Han
Min-Kyu
MK
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Korea.
Seo
Moon-Hyeong
MH
Lee
Dohoon
D
Kim
Seung Hyun
SH
Park
Hae-Sim
HS
Kim
Hak-Sung
HS
D000485:Allergens / Q000276:immunology*; D000818:Animals; D000918:Antibody Specificity; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood* / Q000276:immunology*; D040081:Protein Array Analysis / Q000379:methods*; D039981:Pyroglyphidae / Q000276:immunology; D012680:Sensitivity and Specificity; D013045:Species Specificity
D000485:Allergens; D007073:Immunoglobulin E