BACKGROUND:Current assays for allergen extracts can measure either overall potency or the levels of individual allergens. OBJECTIVE: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. METHODS: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. RESULTS: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. CONCLUSIONS: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.

This chapter talks about natural or recombinant proteins or glycoproteins used in the diagnosis and/or treatment of immunoglobulin E (IgE)-mediated allergic disease. The purpose of allergen standardization is to ensure that the extracts are well characterized in terms of allergen content and that variation between lots is minimized even among different manufacturers. In the United States, the use of a biological model of allergen standardization has permitted the assignment of bioequivalent allergen units (BAU) for most standardized allergens. Although skin testing is an essential component of the allergen standardization program, it is not intended for routine use in the testing of manufactured lots of extracts prior to release. For the Hymenoptera venom allergens, the potency determination is also based on the content of the known principal allergens within the extract, hyaluronidase and phospholipase, which is determined by enzyme activity. Individual allergens may be measured and detected by various approaches using monospecific antisera or antibodies. Assay designs using these antisera include the radial immunodiffusion (RID) assay, crossed immunoelectrophoresis/ crossed radioimmunoelectrophoresis (CIE/CRIE), and enzyme-linked immunosorbent assay (ELISA) variants (direct, two-site, and competition). Each of the assays utilizes monospecific antisera or antibodies to detect and quantify the specified allergen. The identity of an allergen extract may be verified by visualizing the separated allergen proteins on the basis of their size and isoelectric points.