title: Generation of monoclonal antibodies against Blo t 3 using DNA immunization with in vivo electroporation.
abstract: BACKGROUND
  House dust mite allergy is closely associated with allergic diseases. Blomia tropicalis mite species is an important clinical species in the tropics. The cDNA clone encoding Blo t 3, a group 3 allergen from B. tropicalis, has been isolated in our laboratory.
  
  
  OBJECTIVE
  This study was designed to generate Blo t 3-specific monoclonal antibodies (mAbs) for the detection, characterization and purification of this allergen.
  
  
  METHODS
  Mice were immunized intramuscularly with naked plasmid DNA encoding Blo t 3 gene with in vivo electroporation. Hybridomas were generated by the fusion of the splenocytes to X63-Ag8.653 myeloma cells. Purified native Blo t 3 was obtained by mAb immuno-affinity purification and the allergenicity of native Blo t 3 was determined by human IgE enzyme-linked immunosorbent assay (ELISA).
  
  
  RESULTS
  A panel of class-switched and high-affinity mAb recognizing a wide spectrum of Blo t 3 epitopes have been generated. These mAbs are useful for western immunoblot assay, sandwich ELISA and affinity purification of native Blo t 3. Allergenicity of native Blo t 3 protein was examined with 44 mite-allergic sera and approximately 57% of the tested sera had positive serum IgE reactivity to the native Blo t 3.
  
  
  CONCLUSIONS
  These results demonstrated that intramuscular injection of naked DNA encoding Blo t 3 gene combined with in vivo electroporation is an effective and simple method to raise monoclonal antibodies that can be used for characterization and purification of Blo t 3.
authors.affiliation: Departments of Paediatrics and Otolaryngology and Bioprocessing Technology Centre, National University of Singapore, Republic of Singapore.
authors.forename: L
authors.initials: L
authors.lastname: Yang
authors.forename: N
authors.initials: N
authors.lastname: Cheong
authors.forename: D Y
authors.initials: DY
authors.lastname: Wang
authors.forename: B W
authors.initials: BW
authors.lastname: Lee
authors.forename: I C
authors.initials: IC
authors.lastname: Kuo
authors.forename: C H
authors.initials: CH
authors.lastname: Huang
authors.forename: K Y
authors.initials: KY
authors.lastname: Chua
mesh_terms: D000328:Adult; D000485:Allergens / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D052179:Antigens, Plant; D018274:Electroporation / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology; D007114:Immunization / Q000379:methods; D007073:Immunoglobulin E / Q000276:immunology; D007074:Immunoglobulin G / Q000096:biosynthesis; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008925:Mites / Q000276:immunology*; D019444:Vaccines, DNA / Q000276:immunology*
chemical_list: D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; C476862:Blo t 3 allergen, Blomia tropicalis; D007074:Immunoglobulin G; D019444:Vaccines, DNA; D007073:Immunoglobulin E