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

Summary 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.