Development of a two-site enzyme-linked immunosorbent assay for alpha-amylase from Aspergillus oryzae based on monoclonal antibodies.

A two-site monoclonal antibody ELISA was developed to quantify the allergen Asp o 2 (alpha-amylase from Aspergillus oryzae). Two mAbs recognizing distinct epitopes were selected, enriched by in vitro production in a modular minifermenter and affinity-purified. The first antibody was bound to microtiter plates which were then incubated with samples containing the allergen. Bound allergen was detected using a biotinylated second antibody and peroxidase-polymer-labelled streptavidin. The assay had a sensitivity of 0.6 ng/ml and did not react to high concentrations of wheat and rye flour or yeast proteins. The mAb ELISA will be useful in individual or epidemiological studies of baker's asthma to assess workplace allergen concentrations and the efficacy of allergen exposure prevention. It can be used as a standard assay for the quantification of alpha-amylase and the establishment and control of threshold limits in European bakeries.