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The Platelia Aspergillus EIA is a 1-stage immunoenzymatic sandwich microplate assay which detects galactomannan in human serum. The assay uses the rat monoclonal antibody EBA-2, which is directed against Aspergillus galactomannan. The monoclonal antibody is used 1) to coat the wells of the microplate and bind the antigen and 2) as the detector antibody in the conjugate reagent (peroxidase-linked monoclonal antibody).
Serum samples are heat-treated in the presence of EDTA in order to dissociate immune complexes and to precipitate serum proteins that could possibly interfere with the test. The treated serum samples and conjugate are added to the wells coated with the monoclonal antibody and incubated. A monoclonal antibody-galactomannan-monoclonal antibody/peroxidase complex is formed in the presence of Aspergillus antigen.
The strips are washed to remove any unbound material, and the substrate solution is added, which will react with the complex bound to the well to form a blue color reaction. The enzyme reaction is stopped by the addition of acid, which changes the blue color to yellow. The optical absorbance of specimens and controls is determined with a spectrophotometer set at 450 nm and 620/630 nm wavelengths.
Negative, cutoff (low-positive), and high-positive controls are analyzed each time the assay is performed. The presence or absence of Aspergillus galactomannan antigen in the test sample is determined by calculation of an index for the specimen. The index is the optical density (OD) value of the specimen divided by the mean OD of wells containing the cutoff control serum (low-positive control).(Package insert: Platelia Aspergillus EIA, Bio-Rad, Redmond, WA, 6/2003)
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