Monoclonal antibodies against Blo t 13, a recombinant allergen from Blomia tropicalis

Abstract

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.