Quantification of profilins by a monoclonal antibody-based sandwich ELISA

Abstract

Profilins are plant allergens responsible for cross-reactivities in pollen and fruit-allergic patients. A two-site enzyme-linked immunosorbent assay has been developed for the quantification of profilins and its suitability for quantifying profilin in different plant extracts has been evaluated. The assay is based on two profilin-specific monoclonal antibodies (mAbs) with different epitope specificities. These antibodies were immobilized on ELISA plates and incubated with samples containing profilin. Bound profilin was detected by a combination of biotinylated profilin-specific antiserum and peroxidase–streptavidin conjugate. The optimized ELISA measured profilin concentrations ranging from 4 to 250 ng/ml and could quantify profilins from plant species of a variety of different botanical families. No reactivity to mites, molds, or crustaceans was detected, suggesting that the immunoassay is plant-specific. The results indicate that this sensitive profilin-assay will be helpful both for quantifying the profilin content of allergenic extracts intended for clinical use and for studying cross-reactivities between pollen extracts.