Rapid method for arrayed investigation of IgE-reactivity profiles using natural and recombinant allergens.
BACKGROUND
The availability of increasing numbers of purified natural and recombinant allergens offer the possibility for component-resolved characterization of IgE binding. To make use of this potential, fast and simple methods with high capacity have to be developed.


METHODS
A laboratory multiscreen device was used in an innovative two-dimensional approach. In the first step, natural and recombinant allergens were immobilized onto the membrane using the sample chambers as application mask and, after blocking and rotating the membrane through 90 degrees, the same device was used to apply and incubate sera of allergic patients. Enzyme-linked immunoassay (ELISA) quantification of specific IgE was performed for purposes of comparison.


RESULTS
Proteins were most efficiently bound onto nitrocellulose in 20 mM sodium hydroxide (NaOH). Up to 45 proteins or extracts could be investigated with a maximum of 45 sera in a single application, resulting in a resolution of 2,025 spots on one membrane with a size comparable to a standard Western blot. A high correlation for IgE-binding between natural and recombinant allergens was observed. Development of the membrane resulted in very evenly distributed square patterns. The results corresponded with the conventional ELISA measurements of specific IgE.


CONCLUSIONS
The innovative usage of a standard incubation device for both application of proteins as well as screening of sera provides a simple high throughput method for the characterization of IgE binding to allergens. The results are important for component resolved diagnosis of allergy by means of fast monitoring of IgE- and IgG-reactivity spectra. Recombinant allergens may be used as targets for these purposes.
Suck
R
R
Allegropharma Joachim Ganzer KG, Reinbek, Germany.
Nandy
A
A
Weber
B
B
Stock
M
M
Fiebig
H
H
Cromwell
O
O
D000485:Allergens / Q000276:immunology*; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000175:diagnosis* / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology* / Q000378:metabolism; D007159:Immunologic Tests / Q000379:methods*; D008567:Membranes, Artificial; D011485:Protein Binding; D011994:Recombinant Proteins / Q000276:immunology
D000485:Allergens; D008567:Membranes, Artificial; D011994:Recombinant Proteins; D007073:Immunoglobulin E