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Generation of monoclonal antibodies against Blo t 3 using DNA immunization with in vivo electroporation
Author(s) -
Yang L.,
Cheong N.,
Wang D. Y.,
Lee B. W.,
Kuo I. C.,
Huang C. H.,
Chua K. Y.
Publication year - 2003
Publication title -
clinical and experimental allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.462
H-Index - 154
eISSN - 1365-2222
pISSN - 0954-7894
DOI - 10.1046/j.1365-2222.2003.01648.x
Subject(s) - biology , monoclonal antibody , microbiology and biotechnology , epitope , antibody , immunoglobulin e , house dust mite , virology , immunology
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.