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DNA immunization for the production of monoclonal antibodies to Blo t 11, a paramyosin homolog from Blomia tropicalis
Author(s) -
Ramos J. D. A.,
Teo A. S. M.,
Lee B. W.,
Cheong N.,
Chua K. Y.
Publication year - 2004
Publication title -
allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.363
H-Index - 173
eISSN - 1398-9995
pISSN - 0105-4538
DOI - 10.1046/j.1398-9995.2003.00409.x
Subject(s) - monoclonal antibody , recombinant dna , microbiology and biotechnology , biology , antibody , epitope , antigen , allergen , immunology , allergy , gene , biochemistry
Background: Blo t 11 is a high molecular weight allergen from Blomia tropicalis with significant immunoglobulin (Ig)E binding frequency. Native and recombinant Blo t 11 are susceptible to degradation and the isolation and expression of the allergen is problematic thus obtaining sufficient amounts of purified Blo t 11 for antibody production is limiting. DNA‐based immunization is an attractive alternative strategy that bypasses antigen purification for antibody production. Objectives: To use a DNA‐based immunization protocol for the production and characterization of Blo t 11 monoclonal antibodies (mAbs). Methods: The 2625 bp cDNA coding for Blo t 11 was cloned into a mammalian expression vector and immunized intramuscularly with electroporation into mice. Monoclonal antibodies to Blo t 11 were generated using a methylcellulose‐based hybridoma cloning kit. These mAbs were utilized for native Blo t 11 isolation and the development of sandwich enzyme‐linked immunosorbent assay (ELISA). Results: Six mAbs recognizing the native and recombinant Blo t 11 were generated and characterized. Native Blo t 11 was affinity purified from Bt extract and its identity was confirmed by matrix assisted laser desorption/ionization – time of flight mass spectrometry. The native Blo t 11 showed IgE reactivity with 67% of mite allergic sera. A two‐site ELISA developed showed a detection limit of 100 pg/ml of Blo t 11. Conclusion: A DNA‐based immunization protocol was successfully used to generate Blo t 11 mAbs with a spectrum of distinct epitopes located throughout the whole molecule, and they are useful for immunoaffinity purification and immunoassays.