Journal
CLINICAL AND EXPERIMENTAL ALLERGY
Volume 34, Issue 4, Pages 604-610Publisher
WILEY
DOI: 10.1111/j.1365-2222.2004.1922.x
Keywords
Blo t 1; Blomia tropicalis; cysteine protease; DNA immunization; house dust mite; monoclonal antibody; sandwich ELISA
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Background Blo t 1 is a cysteine protease-like allergen from Blomia tropicalis. Recombinant Blo t 1 binds up to 90% of IgE from allergic patients and shows limited cross-reactivity to Der p 1. The generation of monoclonal antibodies (mAbs) against Blo t 1 is important for the detection, isolation and characterization of the native form of the allergen. Methods Mice were immunized intramuscularly with naked plasmid DNA encoding Blo t 1 gene with in vivo electroporation and boosted intraperitoneally with recombinant Blo t 1. mAbs against Blo t 1 were generated using a methylcellulose-based hybridoma cloning kit. The native Blo t 1 was isolated by mAb affinity purification and its allergenicity was determined by ELISA. A two-site ELISA for Blo t 1 was developed using the mAbs generated. Results A DNA-based immunization protocol induced high titre Blo t 1-specific antibodies in mice. Six stable hybridoma clones secreting mAbs recognizing the native and recombinant Blo t 1 were generated. The native Blo t 1 was affinity-purified from a B. tropicalis extract and its allergenicity was determined at 63% using a panel of Singaporean and Malaysian mite allergic patients' sera. A two-site ELISA was developed, which showed a detection limit of 10 ng/mL of Blot t 1. Conclusion Six Blo t 1 mAbs were successfully generated by DNA immunization. These mAbs are useful for nBlo t 1 immunoaffinity isolation and quantitative immunoassays for Blo t 1 in mite and environmental dust extracts.
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