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Background: The recombinant allergen of Blomia tropicalis, rBlo t 13, shows 11% of IgE reactivity to sera from allergic patients. This allergen belongs to the fatty acid-binding protein family and its natural equivalent remains to be isolated. Monoclonal antibodies (MAbs) are important tools for specific determination and isolation of natural allergens as well as for characterization of recombinant proteins. Methods: Mice were immunized with partially purified preparation of rBlo t 13 allergen expressed in the yeast Pichia pastoris. Spleen cells were fused with myeloma cells using polyethylene glycol. Hybridoma screening was performed using a direct ELISA with recombinant allergen. MAb specificity to rBlo t 13 was tested by immunoblotting. Topography of binding sites and binding of MAb to native allergen was studied by ELISA. Reactivity of MAb against allergenic extract of B. tropicalis and Dermatophagoides siboney was analyzed by ELISA inhibition. In addition, the reactivity of MAbs against rBlot 13 from Escherichia coli and P. pastoris expression was compared. Results: Two MAbs, 5G3 and 3G4 with IgG1 isotype, were generated. These MAbs specifically recognized the 16-kD band, which corresponds to the molecular weight shown by rBlo t 13 on SDS-PAGE. In ELISA, the binding of 5G3 MAb to B. tropicalis and D. siboney extracts was inhibited by rBlo t 13. Both MAbs showed the highest reactivity when the allergen was expressed in P. pastoris. Conclusion: Two MAbs specific for Blo t 13 were obtained. These MAbs recognized the same or close epitopes on the rBlo t 13 molecule. The occurrence of homologous allergens to Blo t 13 in D. siboney is suggested by the ELISA inhibition assay.

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Six stable clones secreting murine monoclonal antibodies (Mabs) against Der s 1 were obtained. The binding of Mabs showed cross-reactivity with Dermatophagoides farinae, as determined by enzyme-linked immunosorbent assay (ELISA). In a Western blot assay, antibodies reacted with a 24-kD protein considered to represent the major allergen Der s 1. The repertoire of antigenic sites on Der s 1 was studied using a panel of Mabs. Epitope specificity of the Mabs was determined by both competitive inhibition and sandwich ELISA assays. The results defined six different, non-overlapping and non-repeated antigenic sites on the allergen molecule. Der s 1 allergen from Dermatophagoides siboney extracts was purified by Mab affinity chromatography, this procedure gave 43% recovery of >90% pure allergen. The purified allergen had capacity to bind specific human IgE and demonstrated an allergenic activity of up to 77% of total D. siboney extract. An Mab-ELISA was developed using Mabs directed against different epitopes on Der s 1. This assay could detect up to 1 ng/ml of Der s 1 and Der f 1 in allergen preparations.