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Updated: Sep 26, 2026
Generation of Murine Monoclonal Antibodies by Hybridoma Technology
Published on: January 2, 2017
Generation of monoclonal antibodies against Blo t 3 using DNA immunization with in vivo electroporation
1Departments of Paediatrics and Otolaryngology and Bioprocessing Technology Centre, National University of Singapore, Republic of Singapore.
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.
