Quantification of Blo t 5 in mite and dust extracts by two‐site ELISA
2004; Wiley; Volume: 60; Issue: 1 Linguagem: Inglês
10.1111/j.1398-9995.2004.00597.x
ISSN1398-9995
AutoresFong Cheng Yi, B. W. Lee, N. Cheong, K. Y. Chua,
Tópico(s)Contact Dermatitis and Allergies
ResumoBackground: 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.
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