Quantification of Blo t 5 in mite and dust extracts by two-site ELISA.
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.
Yi
F C
FC
Department of Paediatrics, National University of Singapore, Singapore.
Lee
B W
BW
Cheong
N
N
Chua
K Y
KY
D000485:Allergens / Q000032:analysis*; D000818:Animals; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D004391:Dust / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D020033:Protein Isoforms / Q000032:analysis; D039981:Pyroglyphidae / Q000737:chemistry / Q000276:immunology*; D011933:Reagent Kits, Diagnostic; D014020:Tissue Extracts / Q000276:immunology
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D004391:Dust; D020033:Protein Isoforms; D011933:Reagent Kits, Diagnostic; D014020:Tissue Extracts; C094701:allergen Blo t 5