The FDA lot release protocol review and testing program insures that the potencies of standardized allergenic extracts distributed in the U.S. are within established limits. For cat and short ragweed pollen allergenic extracts, potency is defined according to the concentration of Fel d 1 and Amb a 1, respectively, and is measured with the radial immunodiffusion assay (RID). The RID is labor intensive and subjective, and dependent on devices that are no longer manufactured. We have therefore developed a sandwich ELISA to more accurately, precisely, and reproducibly measure the potency of these standardized allergenic extracts. Three candidate single chain variable fragment antibodies (scFv) against each allergen were purified by affinity chromatography and used as the coating (capture) antibody. Purified native Fel d 1 (Indoor Biotechnologies) and Amb a 1 were used as antigens. Revealing antibodies were polyclonal sheep anti-cat or anti-short ragweed pollen sera in combination with HRP-conjugated rabbit anti-sheep antibody (KPL). Data were analyzed using a 4-parameter logistic model. The assay was validated with CBER's current standardized cat and ragweed pollen reference standards, and with extracts purchased from the manufacturers. Each combination of capture scFv antibody and revealing polysera was sensitive, highly specific, and linear within a wide range of concentrations. Sandwich ELISAs for cat and short ragweed allergen extracts will enhance the FDA lot release protocol review and testing program's ability to measure and confirm the potency of standardized cat and short ragweed pollen allergenic extracts.

RATIONALE: Multiplex microbead flow cytometry has been used to diagnose allergy by binding allergens to beads and incubating these beads in the presence of patient IgE. We have shown that this technology may also be used to determine the potency of allergen extracts. In this study we examine the effect of the presence of multiple bead-bound antibodies on antibody binding. METHODS: Six anti-Fel d 1 recombinant antibodies were generated and were covalently bound to carboxy-labeled beads. Antibody-bound beads were then used to measure Fel d 1 in commercial cat hair extracts using bead-based flow cytometry (Bioplex, Biorad). These potencies were compared to those obtained by manufacturers and CBER/FDA using a conventional antibody-based method. Potencies were calculated by comparing dose response curves using Prism software (GraphPad). Bead-antibody pairs were tested to determine if the presence of additional bead-antibody pairs affected the apparent potency of the extract. RESULTS: Bead-based flow cytometry assay using anti-Fel d 1 recombinant antibodies yields potency numbers within the range of potencies determined using conventional methods. Minimal differences were detected in the apparent potencies of the extracts even when four additional bead-antibody pairs were added. Six lots of cat hair tested using six bead-antibody pairs had potencies (2, 6, 10, 4, 13, 4) similar to those obtained by a manufacturer (4, 6, 15, 6, 18, 7) (r2 > 0.95). CONCLUSIONS: We have developed a bead-based assay using recombinant antibodies that accurately determines Fel d 1 levels in cat allergenic extracts. This assay is reproducible and consistent with data obtained by other methods and testers.

Multiplex microbead flow cytometric technology has been used to identify allergies in individuals by binding recombinant allergens to bead surfaces and incubating these beads in the presence of patient IgE. Others have bound antibodies to the beads to detect a wide variety of proteins, such as signal transduction molecules, cytokines, and viral or bacterial surface antigens. In principal, this technology may be used to determine the potency and composition of complex protein mixtures, such as allergen extracts. Four anti-Fel d 1 recombinant antibodies were gerenareted by the phage display method, and were covalently bound to carboxy-labeled beads using EDC and sulfo-NHS. Antibody-bound beads were then used to detect levels of Fel d 1 in commercial cat hair extracts using a bead-based flow cytometry assay (Bioplex, Biorad). A bead-based flow cytometric potency assay using anti-Fel d 1 recombinant antibodies yields potency numbers within the range of potencies determined using a radial immunodiffusion assay by the manufacturers and CBER. Three lots of cat hair tested using this assay had potencies, as determined by the manufacturer, of 14.6, 15.4, and 14.4 Fel d 1 U/mL (95% CI = ± 25%). The potencies obtained by the microbead method were 11.6, 15.9, and 12.4 Fel d 1 U/mL (± 15%), respectively. We have developed a bead-based flow cytometric assay using recombinant antibodies that can accurately determine Fel d 1 levels in cat hair allergenic extracts.

RATIONALE: Protein microarrays have been used to identify specific IgE to solid phase allergens on the array surface. However, few papers have been published that utilize solid phase antibodies on the array surface to detect specific proteins in a complex mixture. Here we show that recombinant antibodies can be bound to the microarray surface and used to quantitavely detect allergens in allergen extracts. METHODS: Recombinant scFv antibodies that specifically recognize either nAmb a 1 or rFel d 1 (Clin Exp Allergy 2005;35:1040-1048) were spotted in 5 serial dilutions on nitrocellulose microarray slides using a pin and ring arrayer and blocked with ovalbumin. Three different ragweed or cat hair commercial extracts of predetermined potency (by radial immunodiffusion assay (RID)), as well as the existing CBER standards for cat and ragweed, were then incubated with the array and detected with secondary antibodies bound to HRP. Chemiluminescent signal was captured using a CCD camera and spot intensities were determined using Imagequant software. Relative potencies were determined by parallel line analysis of linear regression data. RESULTS: For the cat extracts, the potencies (in Fel d 1 U/mL) were 19.6 (95% CL = 13.6-28.4), 18.1 (13.9-23.4), and 12.5 (8.4-18.6). For the same extracts, the RID values were 20.5, 19.7 and 12.2, respectively. Ragweed results were similar. CONCLUSIONS: We have developed a microarray assay using recombinant antibodies that can detect Amb a I and Fel d 1 in allergenic extracts with accuracy similar to that of currently used standardized methods.

Background Monoclonal antibodies are a valuable tool in the study of allergens, but the technology used in their generation can be slow and labour-intensive. Therefore, we have examined recombinant antibody development by phage-display against single allergens and protein mixtures.Objective We used the avian immunoglobulin system (generated from single V-H and V-L genes) to provide a rapid method for generating highly specific recombinant antibody fragments from a minimal number of animals.Methods A single-chain antibody fragment (scFv) library was generated from a single chicken immunized with model allergens. ScFvs were isolated by phage-display and their properties investigated by ELISA and Western blot.Results Mono-specific scFvs were generated against recombinant Fel d 1 and native Amb a 1. Pannings against yellow jacket venom extracts only yielded clones that reacted with multiple proteins in the venom extract. The scFvs from each panning type were effectively expressed in Escherichia coli and readily purified. Highly specific and sensitive recognition of Fel d 1 and Amb a 1 was demonstrated in ELISA, with scFvs displaying antibody-concentration-dependent absorbance curves down to picogram levels of antibody. The specificity of selected antibodies for their cognate antigen was further confirmed in Western blot analysis, with scFvs directed to either Fel d 1 or Amb a 1 showing no reactivity for the other antigens used in immunization. Anti-Amb a 1 scFvs also mapped Amb a 1-isoform location in Western blot of ragweed extracts separated by 2D SDS-PAGE. DNA sequence analysis of scFvs showed that multiple different clones had been generated against Fel d 1 and Amb a 1. Using two anti-Fel d 1 scFv for ELISA analysis of Fel d 1 content in crude cat pelt extracts, we could produce data which were highly similar (P=0.33 and 0.89 by paired t-test analysis) to those obtained using conventional assays (radial immunodiffusion).Conclusion Phage-display technology may generate multiple allergen-specific recombinant antibody fragments from a single chicken, to allergens from mammalian, plant and insect sources. The resulting antibody fragments are of demonstrable use in allergen identification and quantification, in comparison with standard immunoassays.

RATIONALE: Cockroach allergens may be an important cause of inner city asthma. Although many allergens have been standardized, cockroach extract has not. We have shown (Clin. Exp. Allergy 2002; 32:721-727) that there is a wide disparity in potency and allergen content among commercially available cockroach extracts. As part of our efforts to standardize cockroach extracts, we have produced scFvs that recognize Bla g 1, Bla g 2, Bla g 4 and Bla g 5. METHODS: Three 6-month-old female white leghorn chickens were immunized with rBla g 1, rBla g 2, rBla g 4, and rBla g 5 (Indoor Biotechnologies) using Freund's adjuvants. The animals were sacrificed, and RNA was isolated from spleens and bone marrow. Libraries were constructed from amplified cDNA, and scFv recombinant antibodies were produced from the antibody repertoire of these chickens. Phage bearing these scFvs were panned against rBla g 1, rBla g 2, rBla g 4, and rBla g 5. Four clones from each library were identified as recognizing the specific recombinant Bla g. RESULTS: Anti-Bla g 1, anti-Bla g 2, anti-Bla g 4, and anti-Bla g 5 scFvs specifically recognize rBla g 1, rBla g 2, rBla g 4, and rBla g 5, respectively, by ELISA and Western blot analysis. The antibodies also recognize specific proteins in crude cockroach extracts. CONCLUSIONS: We have generated scFvs that specifically recognize each of the recombinant Bla g proteins. These recombinant antibodies will be useful in developing assays for the standardization of cockroach extracts.