Competition enzyme-linked immunosorbant assay (ELISA) can be a sensitive method for the specific detection of small quantities of allergen in a complex mixture.

RATIONALE: The competition ELISA assay is used to determine the potency of US standardized allergen extracts. We have been concerned that the competition ELISA is not sensitive to changes in individual allergen levels. This study was designed to determine the sensitivity of the competition ELISA to detect the specific loss of Bla g 1 and Bla g 2 in cockroach extracts. METHODS: German cockroach extract E3Cg was made from defatted German cockroaches. New Zealand White rabbits were immunized with rBla g 1 or rBla g 2. Optimal dilutions of anti-Bla g 1 and anti-Bla g 2 sera were established by ELISA. E3Cg was selectively depleted of Bla g 1 or Bla g 2 by immunoabsorption with anti-Bla g 1 or anti-Bla g 2 attached to Protein G agarose beads. Competition ELISA using pooled human sera, or mixed anti-Bla g 1 and anti-Bla g 2 serum, was performed on the depleted extracts, and on depleted extracts reconstituted with rBla g 1 or rBla g 2. RESULTS: Unlike pooled human-allergic IgE sera, anti-Bla g 1 and anti-Bla g 2 IgG -- in dilutions as low as 10(-6), could be used in the competition ELISA to measure the loss of allergen in depleted E3Cg. As little as 0.001 microg/mL of added rBla g 1 and 0.1 microg/mL of added rBla g 2, could be detected. CONCLUSION: The competition ELISA can be highly sensitive to compositional differences in complex allergen mixtures, even when the specific detecting antibody is present in relatively small amounts.

Exploiting the avian immunoglobulin system to simplify the generation of recombinant antibodies to allergenic proteins.

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.