Abstract:
BACKGROUND:Blomia tropicalis is an important mite species in the tropical and sub-tropical regions of the world. Blo t 5 is the major allergen with up to 70% sensitization rates in B. tropicalis allergic populations. METHODS:Mice were immunized intramuscularly with naked plasmid DNA encoding Blo t 5 gene with in vivo electroporation. Blo t 5 monoclonal antibodies were generated using methylcellulose-based hybridoma kit. Monoclonal antibody (mAb) 4A7 was characterized by two-dimensional electrophoresis immunoblotting. A specific quantitative two-site enzyme-linked immunosorbent assay (ELISA) was developed with mAb 4A7 and guinea pigs Blo t 5 polyclonal antibody as capture and detection antibodies, respectively. This system was tested with Blo t 5 in crude extracts and dust samples. RESULTS:A high-affinity mAb 4A7 recognizing several isoforms of Blo t 5 has been generated. Monoclonal antibody 4A7 is useful for immunoblotting and two-site ELISA. The two-site ELISA developed has a high sensitivity, with a detection limit of 10 pg/ml. The assay is species-specific and recognized the same epitopes on both native and recombinant Blo t 5. The assay developed is able to detect Blo t 5 in commercial diagnostic and therapeutic B. tropicalis extract. Blo t 5 quantification in dust samples showed that Blo t 5 is present in a high quantity in Singapore dust. CONCLUSIONS:A highly sensitive and specific two-site ELISA has been developed. The assay system developed is useful for the quantification of Blo t 5 in mite and environmental dust extracts.
journal_name
Allergyjournal_title
Allergyauthors
Yi FC,Lee BW,Cheong N,Chua KYdoi
10.1111/j.1398-9995.2004.00597.xkeywords:
subject
Has Abstractpub_date
2005-01-01 00:00:00pages
108-12issue
1eissn
0105-4538issn
1398-9995pii
ALL597journal_volume
60pub_type
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