Monoclonal antibodies to recombinant Der p 2, a major house dust mite allergen: specificity, epitope analysis and development of two-site capture ELISA (1)
The predominant species are only D. farinae and D. pteronyssinus. With the characterization of the allergens from two species of mites, especially the group 1 and 2 allergens are the most significant causative agents for the sensitization, invoking IgE and IgG antibody responses in 80% to 95% of patients allergic to HDMs, and each has been cloned and sequenced.
Four monoclonal antibodies were confirmed to be highly species-specific. They reacted only with the rDer p 2 and D. pteronyssinus extracts, and did not cross-react with neither of any other arthropod extracts, especially D. farinae extract. Two of the monoclonal antibodies (3B12 and 2B6) were IgG1 and the others (5D10 and 4A5) were IgM.
Recombinant polypeptides that were purified using a GST-affinity column. Three monoclonal antibodies, 3B12, 2B6, and 4A5, showed reactivity to both B polypeptide and the full-length rDer p 2. Interestingly, however, monoclonal antibody 5D10 reacted only to the full-length rDer p 2. Reactivities were confirmed by immunoblot analysis. Monoclonal antibodies were used for two-site capture ELISA which detects only Der p 2. The ELISA control curves were produced by the assay using rDer p 2. The limit of sensitivity of this assay was approximately 4 ng/ml with rDer p 2 and 8 µg/ml with the D. pteronyssinus crude extract.
The poor antibody response of BALB/c mice to group 2 allergens has been reported previously. The polypeptide comprising amino acid residues 44-93 of rDer p 2 was highly reactive with most monoclonal antibodies as well as human IgE.