Preparation of Solid Tissue for Aspergillus Galactomannan |
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Aim Platelia Aspergillus (Biorad) is a 1-stage immunoenzymatic sandwich microplate kit, for the detection of antigen galactomannan. FDA information about Platelia Aspergillus EIA can be found here. The technique was designed for use on human serum. However, it may also be possible to perform this method on solid tissues and organic solutions. Viscous solution and tissue specimens need to be pre-treated to achieve the extraction of the Aspergillus antigen and to get a homogeneous sample in solution, using the following method:Equipment • safety cabinet• sterile Petri dish • sterile scalpel • sterile glass homogeniser • extra pure water (as recommended for use in assay) • 1.5ml sterile eppendorfs/microtubes • vortex • centrifuge • sterile pipette tips Ideally pyrogen free equipment should be used, although standard sterile equipment should be sufficient (following manufacturers advice). Health and Safety • perform procedure in a class II laminar flow safety cabinet• wear disposable gloves Procedure Preparation of the samples to be tested:• In a laminar flow safety cabinet (class II), fine cut the sample with a sterile scalpel in a Petri dish. It is important to cut the samples in all directions: length, width and diagonal to break the tissue fibres and to optimise the extraction of the antigen • Transfer the small pieces in the sterile glass homogeniser (1 - 5ml, depending on volume of specimen) and grind it to get an homogenate • Add extra pure water, of equal volume to the sample. This helps grinding and to get a liquid homogenate • Aliquot in one or several sterile eppendorf/microtubes of 1.5 ml capacity, 1 ml per aliquot. • Vortex the homogenate for 30 seconds • Centrifuge at 1000g for 5 minutes • Remove the supernatant in a tube with a sterile pipette tip • Analyse the supernatant with the same technique used for serum following the kits directions. Notes • Minimise contact of specimen with dust/air, due to the sensitivity of the Platelia test (1ng of galactomannan per ml). Any possibility of contamination during the various steps of the procedure must be prevented in order to avoid false positive results.• The manufacturer has only validated Platelia for serum, therefore, the result must be presented with careful interpretation. Written by: Laboratoire de Parasitologie-MycologieInstitut de Parasitologie et de Pathologie Tropicale Faculté de médecine Strasbourg Translated by: Dagmar BiegonAdapted by: Susan J. Howard,Mycology Lab, Hope Hospital, Manchester, UK |
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