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Korean J Parasitol > Volume 34(1):1996 > Article

Original Article
Korean J Parasitol. 1996 Mar;34(1):35-47. English.
Published online Mar 20, 1996.  http://dx.doi.org/10.3347/kjp.1996.34.1.35
Copyright © 1996 by The Korean Society for Parasitology
Identification and characterization of allergens of Chironomus flaviplumus adults (Chironomidae, Diptera) in mice
Han-Il Ree,*1Sang-Hwa Lee,1Yu-Kyoung Kim,1Soungmin Jeon,1Jae-Kyung Chang,1 and Yoon-Suk Kim2
1Department of Parasitology, College of Medicine, Yonsei University, Seoul 120-752, Korea.
1Department of Clinical Pathology, College of Health science, Yonsei University, Wonju 220-701, Korea.
Received December 22, 1995; Accepted January 13, 1996.

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 µg or 10 µg 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 (×512) which was 16 times higher than that of the crude extract (×32). The P-K titer of 52 kDa protein was also 4 times higher (×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.

Figures


Fig. 1
Specific IgE values of the mouse sera immunized with 1 µg (A) or 10 µg (B) crude extracts of Chironomus flaviplumus. The bar represents the average.


Fig. 2
Ion exchange chromatography of Chironomus flaviplumus crude lysate on QAE sephadex A-50 column. A, Fractions eluted by non-salt elution buffer; B, Fractions eluted by a elution buffer with 0-0.5 N gradient salt.


Fig. 3
SDS-polyacrylamide gel electrophoresis of Chironomus flaviplumus crude extract and fractions from QAE sephadex A-50 column chromatography. The proteins were run on 10% SDS-PAGE. Lane 1, Molecular weigh standard; lane 2, Chironomus flaviplumus crude extract ; lane 3, a fraction eluted by non-salt elution buffer from QAE sephadex A-50 column chromatography; lane 4, a fraction eluted by elution buffer with 0-0.5 N gradient salt.


Fig. 4
Immunoblotting analysis of Chironomus flaviplumus crude extract and fraction from QAE sephadex A-50 column chromatography. The proteins were run on 10% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was visualized by chemiluminescent substrate in lane 1 and by colorimetric substrate in lane 2, 3 and 4. Lane 1 and 2, Chironomus flaviplumus crude extract; lane 3, a fraction eluted by non-salt elution buffer from QAE sephadex A-50 column chromatography; lane 4, a fraction eluted by elution buffer with 0-0.5 N gradient salt.


Fig. 5
SDS-PAGE analysis of proteins eluted from Chironomus flaviplumus crude extract on 10% SDS-polyacrylamide gel. The gel was cut into 30 pieces and each eluate was re-run on 5-20% gradient SDS-polyacrylamide gel. H, high molecular weight protein standard; L, low molecular weight protein standard; I, immunoblotting analysis of crude lysate; C: Chironomus flaviplumus crude extract.


Fig. 6
Reactivities of the elutions of Chironomus flaviplumus antigen by P-K type skin test. *Reactivity was represented as reciprocal P-K titers. **CE, crude extract.


Fig. 7
P-K skin tests. A, P-K titer (×32) of elution No. 4; B, Negative P-K titer elution No. 8.

Tables


Table 1
Total IgE values of the mouse sera immunized with 1 µg or 10 µg crude extracts of Chironomus flaviplumus


Table 2
Specific IgE values of the mouse sera immunized with 1 µg or 10 µg crude extracts of Chironomus flaviplumus


Table 3
Reciprocal PCA titer of the mouse sera immunized with 1 µg (A) or 10 µg (B) crude extracts of Chironomus flaviplumus


Table 4
Reciprocal P-K titers of Chironomus flaviplumus crude extract and the proteins fractionated by ion exchange chromatography

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