Multiplex microbead measurements for the characterization of cat and ragweed allergen extracts

Abstract

Current assays for allergen extracts can measure either overall potency or the levels of individual allergens. To develop a multiplex allergen extract potency assay (MAEPA) for allergen extracts that can concurrently measure individual allergens and characterize the overall allergen levels in the mixture. Six anti-Fel d 1 and 6 anti-Amb a 1 recombinant antibodies were generated and were covalently bound to carboxy-labeled beads. Antibody-bound beads were then used to measure Fel d 1 and Amb a 1 levels in commercial cat hair and short ragweed pollen (SRP) extracts, respectively, using bead-based flow cytometry. These major allergen levels were compared with those obtained using a conventional antibody-based method. Allergen levels were calculated by comparing the half-maximal effective concentrations of dose-response curves analyzed using 4-parameter fits. Bead-antibody pairs were tested to determine whether the presence of additional bead-antibody pairs affected the apparent potency of the extract. Allergen contents of cat hair and SRP extracts determined using the MAEPA and anti-Fel d 1 and anti-Amb a 1 antibodies were comparable with potencies determined using conventional methods. Cross-interference from the concurrent use of multiple beads was minimal. Six lots of cat hair extract and 6 lots of SRP extract were tested. The MAEPA, a bead-based assay using recombinant antibodies, accurately determined Fel d 1 levels in cat hair allergenic extracts and Amb a 1 levels in SRP extracts. The results of this assay are reproducible and are consistent with data obtained using conventional methods.