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.
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.
Authors: Geoffrey A Mueller; John A Ankney; Jill Glesner; Taruna Khurana; Lori L Edwards; Lars C Pedersen; Lalith Perera; Jay E Slater; Anna Pomés; Robert E London Journal: Mol Immunol Date: 2014-03-22 Impact factor: 4.407
Authors: Bartholomew N Ondigo; Gregory S Park; Severin O Gose; Benjamin M Ho; Lyticia A Ochola; George O Ayodo; Ayub V Ofulla; Chandy C John Journal: Malar J Date: 2012-12-21 Impact factor: 2.979