DNA immunization for the production of monoclonal antibodies to Blo t 11, a paramyosin homolog from Blomia tropicalis

• Published in: Allergy (Copenhagen) Vol. 59; no. 5; pp. 539 - 547
• Main Authors: Ramos, J. D. A., Teo, A. S. M., Lee, B. W., Cheong, N., Chua, K. Y.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Munksgaard International Publishers 01.05.2004; Blackwell
• Subjects: Allergens - analysis; Allergens - chemistry; Allergens - immunology; Allergens - isolation & purification; Allergic diseases; Amino Acid Sequence; Animals; Antibodies, Monoclonal - immunology; Antibody Formation; Antigens, Plant; Biological and medical sciences; Blo t 11; Blomia tropicalis; DNA - immunology; DNA immunization; Dust; Enzyme-Linked Immunosorbent Assay - methods; Epitopes; Female; house dust mite; Humans; Immune Sera - immunology; Immunization; Immunoglobulin E - immunology; Immunopathology; Medical sciences; Mice; Mice, Inbred BALB C; Mites - chemistry; Mites - immunology; Molecular Sequence Data; monoclonal antibody; paramyosin; Recombinant Proteins - immunology; sandwich enzyme‐linked immunosorbent assay; Tropomyosin - analogs & derivatives; Prevention; Allergy; Immunopathology; Immunization; Immunology; Paramyosin; DNA; Monoclonal antibody; Blomia tropicalis
• ISSN: 0105-4538; 1398-9995
• DOI: 10.1046/j.1398-9995.2003.00409.x

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.