DEVELOPMENT OF ALLERGOLOGY TEST IN MICROPLATE IMMUNOASSAY FORMAT BASED ON CHEMILUMINESCENCE

摘要

CARLA SYSTEM (Captur Assay RADIM Liquid Allergen) is a Capture Assay-ELISA system for quantitative detection of specific IgE in human serum. The samples are incubated with a biotinylated-allergen solution in a microplate coated with anti-human IgE. During the first incubation the sample IgE antibodies are bound to the solid phase and the biotinylated allergen are bound to the sample specific IgE. After removing the unbound material by an aspiration/washing cycle, the streptavidine HRP tracer is added to the wells to bind the biotinylated allergen. In order to quantify specific IgE, a standard curve with known amounts of total IgE antibodies is incubated in the same coated microplate with a biotinylated anti IgE antibody. The colorimetric reaction is evidenced by incubating the solid phase with the chromogen solution (tetramethylbenzidine, TMB) in a substrate buffer. Horseradish Peroxidase (HRP) in a commonly used reporter enzyme in enzyme linked immunoassay and TMB and Lumigen PS-1 has been reported for visualization of peroxidase activity. Chemiluminescent detection offers potential advantages in terms of sensitivity and specificity of the assay. In particular the possibility to reduce the sample volume is an important aspect for the allergological pediatric investigations, as a reduced sample volume could allow to increase the number of allergen tests with the same blood sample. Our aim was to investigate the contribute of antilgE and tracer concentration in order to increase the sensitivity on the Brio automatic System. Moreover a correlation with results obtained with an already assessed method commercially available has been studied.