A Multiplexed Immunoassay for Rapid Detection of Specific IgE in Allergy Diagnosis (Nystrand et.al.)
Specific IgE is an established marker of the in vitro diagnosis of allergy. As there is a need of screening a number of different allergens in order to establish or exclude the cause of the symptoms, an experimental protein microarray assay for detection of specific IgE antibodies has been developed.
The assay is based on microspots arrayed on a capillary flow membrane with an inherent 3-dimensional structure. A 3-dimensional structure has two major benefits. First, it has an enlarged inner surface area, which favours efficient detection of low-concentration analytes. Secondly, the structure enables a liquid flow driven by capillary force to take place, from the sample and reagent application site through the reaction zone, which allows a simple and robust instrument solution to be devised. In addition the enlarged inner surface area is necessary to secure the detection of a critical component of low content in natural allergen extracts.
|Experimental device: Nitrocellulose strips (5 x 55) mm laminated on a glass surface||Liquid transportation by capillary forces|
The microspots contain natural allergen extract, purified and recombinant allergens which are covalently immobilized. The allergen reagents are arrayed at density of up to 100 spots/cm2 on membrane, each spot being 250 µm in diameter. Starting with 30 µl serum sample applications, a sequential series of liquid flows and assay events are induced to take place including washing buffer and detecting antibody conjugated to a fluorophore, the total assay time requires less than 30 minutes.
|Typical results after assay performed|
|Using ImmunoCAP measurements as reference, the detection limit of the assay is less than 0.35 kUA/L of specific IgE and the average intra-assay precision was found in the range of 5-8%. No cross-reactivity to other immunoglobulins at normal serum levels is seen.|
Our results suggest that the inherent capability of the protein array assay method studied, regarding analytical sensitivity, background and dynamic range, comparable to the established reference assay. However, at the same time, our study highlights the critical importance of the antibody-capturing reagent attached to the solid phase, such that detection of specific IgE antibodies with adequate sensitivity can be achieved only if extraordinarily potent allergen reagents are used
Scanner: GenePix 4000B, Axon Instrument; S/W GenePix Pro 5.0
Arrayer: Nano-Plotter NP 2.0
Courtesy of: Nystrand M., Elfverson G., Lidholm J., Matsson P., Holmquist S.
Phadia AB , Uppsala, Sweden
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