title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Allergens, National Center of Bioproducts (BIOCEN), Havana, Cuba.
authors.forename: Mayrel
authors.initials: M
authors.lastname: Labrada
authors.forename: Keiko
authors.initials: K
authors.lastname: Uyema
authors.forename: Minerva
authors.initials: M
authors.lastname: Sewer
authors.forename: Alexis
authors.initials: A
authors.lastname: Labrada
authors.forename: Maritza
authors.initials: M
authors.lastname: González
authors.forename: Luis
authors.initials: L
authors.lastname: Caraballo
authors.forename: Leonardo
authors.initials: L
authors.lastname: Puerta
mesh_terms: D000485:Allergens / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D052179:Antigens, Plant; D001666:Binding Sites, Antibody / Q000276:immunology; D001667:Binding, Competitive / Q000276:immunology; D002352:Carrier Proteins / Q000276:immunology*; D003429:Cross Reactions / Q000276:immunology; D004306:Dose-Response Relationship, Immunologic; D004926:Escherichia coli / Q000276:immunology / Q000378:metabolism; D050556:Fatty Acid-Binding Proteins; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D023421:Models, Animal; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism
chemical_list: D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; C120818:Blo t 13 allergen; D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D011994:Recombinant Proteins