Generation of monoclonal antibodies against Blo t 3 using DNA immunization with in vivo electroporation

Summary 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.