A human monoclonal antibody based immunoenzymetric assay to measure Fel d 1 concentrations in cat hair and pelt allergenic extracts.

In the United States, 19 allergen extracts of different specificities are standardized, which means that their potencies are determined in comparison to a US reference standard. For cat allergen extracts, potency is determined by measuring Fel d 1 content expressed in in Fel d 1 units, and with a unitage that correlates with skin test reactions (bioequivalent allergy units or BAU). Currently, Fel d 1 content is measured with a radial immunodiffusion (RID) assay that uses polyclonal sheep antisera to detect the allergenic protein by producing a white precipitin line in agar gel. However, the RID is considered cumbersome, and the polyclonal sera may qualitatively vary among animals and may recognize epitopes irrelevant to human allergic disease. In this report, we describe a quantitative two-site immunoenzymetric assay (IEMA) for Fel d 1 that uses immobilized capture and soluble biotin-labeled detection Fel d 1-specific human IgE monoclonal antibodies (mAb) that have been class-switched to IgG4. Together, they sandwich Fel d 1 molecules from extracts. Using purified natural Fel d 1 as a calibrator, the historically reported ∼4 micrograms Fel d 1/Fel d 1 unit assignment was directly measured in this mAb-based IEMA at 3.12 ± 0.24 micrograms of Fel d 1 per Fel d 1 unit. This IEMA appears to be equivalent to RID in the measurement of biological potencies of commercial cat hair and cat pelt extracts marketed in the United States.

Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts.

BACKGROUND: German cockroach (GCr) allergens induce IgE responses and may cause asthma. Commercial GCr allergen extracts are variable and existing assays may not be appropriate for determining extract composition and potency. OBJECTIVE: Our aim was to develop a multiplex antibody/bead-based assay for assessment of GCr allergen extracts. METHODS: Single chain fragment variable (scFv) antibodies against GCr were obtained by screening libraries derived from naïve human lymphocytes and hyperimmunized chicken splenocytes and bone marrow. Selected clones were sequenced and characterized by immunoblotting. Eighteen scFv antibodies (17 chicken, 1 human) coupled to polystyrene beads were used in this suspension assay; binding of targeted GCr allergens to antibody-coated beads was detected using rabbit antisera against GCr, and against specific allergens rBla g 1, rBla g 2, and rBla g 4. The assay was tested for specificity, accuracy, and precision. Extracts were also compared by IgE competition ELISA. RESULTS: Chicken scFv's generated eight different binding patterns to GCr proteins from 14 to 150 kDa molecular weight. Human scFv's recognized a 100 kDa GCr protein. The multiplex assay was found to be specific and reproducible with intra-assay coefficient of variation (CV) of 2.64% and inter-assay CV of 10.0%. Overall potencies of various GCr extracts were calculated using mean logEC50s for eight selected scFvs. Overall potency measures were also analyzed by assessing the contributions to potency of each target. CONCLUSIONS: An scFv antibody-based multiplex assay has been developed capable of simultaneously measuring different proteins in a complex mixture, and to determine the potencies and compositions of allergen extracts.

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

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.

Characterization of the N-glycans of recombinant bee venom hyaluronidase (Api m 2) expressed in insect cells.

Honeybee venom hyaluronidase (Api m 2) is a major glycoprotein allergen. Previous studies have indicated that recombinant Api m 2 expressed in insect cells has enzyme activity and IgE binding comparable with that of native Api m 2. In contrast, Api m 2 expressed in Escherichia coli does not. In this study, we characterized the carbohydrate side chains of Api m 2 expressed in insect cells, and compared our data with the established carbohydrate structure of native Api m 2. We assessed both the monosaccharide and the oligosaccharide content of recombinant Api m 2 using fluorophore-assisted carbohydrate electrophoresis and HPLC. To identify the amino acid residues at which glycosylation occurs, we digested recombinant Api m 2 with endoproteinase Glu-C and identified the fragments that contained carbohydrate by specific staining. Recombinant Api m 2 expressed in insect cells contains N-acetylglucosamine, mannose, and fucose, as well as trace amounts of glucose and galactose, and the oligosaccharide analysis is consistent with heterogeneous oligosaccharide chains consisting of two to seven monosaccharides. No sialic acid or N-acetylgalactosamine were detected. These results are similar to published data for native Api m 2, although some monosaccharide components appear to be absent in the recombinant protein. Analysis of proteolytic digests indicates that of the four candidate N-glycosylation sites, carbohydrate chains are attached at asparagines 115 and 263. Recombinant Api m 2 expressed in insect cells has enzymic activity and IgE binding comparable with the native protein, and its carbohydrate composition is very similar.