Identification and characterization of allergens of Chironomus flaviplumus adults (Chironomidae, Diptera) in mice

Abstract

Non-biting midges (Chironomidae, Diptera) are one of the largest insect families, which are distributed worldwidely and are found in nearly all types of inland waters. They are known to be aggressive inhalant allergens which cause allergenic diseases. In this study, the crude antigens of Chironomus flaviplumus adults which are most widely distributed in Korea were extracted, and their allergens were analyzed with the sera from experimentally sensitized mice. The mice were immunized with 1 microg or 10 microg of the crude antigens, respectively, and the specific serum IgE levels were measured by both ELISA and passive cutaneous anaphylaxis (PCA) techniques. The highest levels of both total IgE and chironomid-specific IgE were found in the mouse sera obtained after 9 weeks of the first injection with 1 mu g crude antigen. The crude antigen was separated into 16-18 protein bands on gel by SDS-PAGE. The crude extract was assessed by SDS-PAGE/immunoblot analysis. One IgE-binding band (65 kDa) was detected by developing with colorimetric substrate, and 4 IgE-binding bands (65, 52, 35 IgE-binding and 25 kDa) by developing with CSPD chemiluminescent substrate. The SDS-PAGE gel of the crude extract of chironomid adults was equally cut into 30 pieces and each of them was eluted to isolate proteins by molecular weight, and the allergenicity of each eluate was assessed by applying P-K test on rats. Proteins of 65, 35 and 15 kDa showed the highest P-K titer (x 512) which was 16 times higher than that of the crude extract (x 32). The P-K titer of 52 kDa protein was also 4 times higher (x 128) than that of the crude extract, whereas the 25 kDa protein poorly responded, which seemed not antigenic. In conclusion, the present result in mice demonstrated that adults of Chironomus flaviplumus, a predominent species in Korea, cause allergenic diseases and the main allergens are 65, 52, 35 and 15 kDa proteins, of which 65 kDa protein seems to be a main allergen.