Ree H I, Lee S H, Kim Y K, Jeon S, Chang J K, Kim Y S
Department of Parasitology, College of Medicine, Yonsei University, Seoul, Korea.
Korean J Parasitol. 1996 Mar;34(1):35-47. doi: 10.3347/kjp.1996.34.1.35.
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.
非吸血蠓（双翅目：摇蚊科）是最大的昆虫类群之一，广泛分布于世界各地，几乎在所有类型的内陆水域中都能发现它们。已知它们是具有侵袭性的吸入性过敏原，可引发过敏性疾病。在本研究中，提取了韩国分布最广的黄色摇蚊成虫的粗抗原，并用实验致敏小鼠的血清对其过敏原进行了分析。分别用1微克或10微克粗抗原对小鼠进行免疫，通过酶联免疫吸附测定（ELISA）和被动皮肤过敏反应（PCA）技术检测特异性血清IgE水平。首次注射1微克粗抗原9周后获得的小鼠血清中，总IgE和摇蚊特异性IgE水平最高。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），粗抗原在凝胶上分离为16 - 18条蛋白带。通过SDS-PAGE/免疫印迹分析对粗提物进行评估。用比色底物显色检测到1条IgE结合带（65 kDa），用CSPD化学发光底物显色检测到4条IgE结合带（65、52、35 IgE结合和25 kDa）。将摇蚊成虫粗提物的SDS-PAGE凝胶等分成30块，每块洗脱以按分子量分离蛋白质，通过对大鼠进行普拉茨克（P-K）试验评估各洗脱物的致敏性。65、35和15 kDa的蛋白质显示出最高的P-K滴度（×512），比粗提物（×32）高16倍。52 kDa蛋白质的P-K滴度也比粗提物高4倍（×128），而25 kDa蛋白质反应较弱，似乎没有抗原性。总之，本研究在小鼠中的结果表明，韩国的优势种黄色摇蚊成虫可引发过敏性疾病，主要过敏原是65、52、35和15 kDa的蛋白质，其中65 kDa蛋白质似乎是主要过敏原。
