Production of monoclonal antibodies for immunoaffinity purification and quantitation of Blo t 1 allergen in mite and dust extracts.
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.


CONCLUSIONS
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.
Ramos
J D A
JD
Department of Paediatrics, National University of Singapore, Singapore.
Cheong
N
N
Teo
A S M
AS
Kuo
I-C
IC
Lee
B W
BW
Chua
K Y
KY
D000485:Allergens / Q000032:analysis / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000917:Antibody Formation; D052179:Antigens, Plant; D002846:Chromatography, Affinity / Q000379:methods; D004391:Dust / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D008925:Mites / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D019444:Vaccines, DNA / Q000276:immunology
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; C473655:Blo t 1allergen, Blomia tropicalis; D004391:Dust; D011994:Recombinant Proteins; D019444:Vaccines, DNA