Original article DNA immunization for the production of monoclonal antibodies to Blo t 11, a paramyosin homolog from Blomia tropicalis House dust mite (HDM) allergen exposure has been recognized as an important risk factor for the develop- ment of sensitization and allergic diseases (1–3). Blomia tropicalis (Bt) (4) is a clinically important HDM species in tropical and subtropical regions causing allergic sensitization among susceptible individuals (5–7). Several recombinant Bt allergens with complete cDNA sequences have been cloned and their allergenicity established (8–14). Blo t 11, a 2625 bp cDNA clone coding for an 875 amino acid protein homologous to paramyosin, was isolated from a Bt cDNA library (11). Recombinant and native Blo t 11 bind up to 59% of IgE from Bt allergic patients tested (15). The full-length (FL) Blo t 11 allergen contains multiple IgE and IgG epitopes scattered throughout the molecule and the major IgE epitopes were located in an immunodominant peptide, fD (16). Native and recombinant Blo t 11 are susceptible to degradation and the isolation and expression of the allergen is problematic (11, 15, 16). Generation of monoclonal antibodies (mAbs) is important in the identification, quantitation and purifi- cation of proteins, identifying antigenic sites, and in assessing the antigenic similarity between recombinant and native proteins. In HDM allergy, mAbs had been extensively used in the development of immunoassays such as sandwich enzyme-linked immunosorbent assay (ELISA) to monitor and determine environmental aller- gens, quantitation and standardization of allergen extracts, epitope mapping of allergens, comparison of IgE-binding determinants, immunolocalization of aller- gens, assessment of cross-reactivity, and in the isolation of native allergens. The conventional method of antibody production requires significant amount of purified proteins for immunization (17). In the case of low abundance proteins or recombinant proteins that lack post-translational modifications, mAb production may be problematic and challenging. DNA-based immunization is a promising new strategy for the production of mAbs bypassing the Background: Blo t 11 is a high molecular weight allergen from Blomia tropicalis with significant immunoglobulin (Ig)E binding frequency. Native and recom- binant 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 gen- erated 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. J. D. A. Ramos 1,2 , A. S. M. Teo 3 , B. W. Lee 1 , N. Cheong 3 , K. Y. Chua 1 1 Department of Paediatrics, National University of Singapore, Singapore; 2 Research Center for the Natural Sciences, University of Santo Tomas, Manila, Philippines; 3 Bioprocessing Technology Centre, Biomedical Science Institutes, Agency for Science Technology and Research (A*STAR), Singapore Key words: Blo t 11; Blomia tropicalis; DNA immunization; house dust mite; monoclonal antibody; paramyosin; sandwich enzyme-linked immunosorbent assay. Kaw Yan Chua Department of Paediatrics Faculty of Medicine National University of Singapore Lower Kent Ridge Singapore 119074 Accepted for publication 8 September 2003 Allergy 2004: 59: 539–547 Printed in UK. All rights reserved Copyright Ó Blackwell Munksgaard 2004 ALLERGY 539