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Background: Chi t 1.01, a hemoglobin of the midge Chironomus thummi thummi, is a widespread environmental and occupational allergen. The aim of the present investigation was to identify and compare peptides involved in B-cell epitopes of Chi t 1.01 recognized by 15 human IgE sera, six murine monoclonal antibodies (mAbs), and a polyclonal rabbit antiserum., Methods: Synthetic peptides 19-21 amino acids long covering the whole Chi t 1.01-sequence were covalently coupled to activated paper disks as well as adsorbed to wells of immunoplates and used for enzyme-linked immunosorbent assay. For fine epitope mapping, we used overlapping synthetic octapeptides with one amino-acid offset., Results: Peptides containing the amino acids 13-17, 23-29, and 40-50 were recognized by three of the mAbs, while three other mAbs reacting with none of the peptides obviously recognized conformational epitopes. Binding sites for rabbit antibodies and for human IgE antibodies were scattered over the whole molecule. The peptide 80-100 seemed to comprise at least one important IgE epitope. Depending on the method of antigen binding to the solid phase, differing results were obtained., Conclusions: Several linear epitopes in Chi t 1.01 are recognized by human IgE antibodies, by mAbs, and by polyclonal rabbit antibodies. In addition, the results indicate the presence of conformational epitopes.

Lymphocyte cultures of persons sensitized to the hemoglobin allergen Chi t I show a highly significant response to the allergen measured in the lymphocyte stimulation assay by (3H)-thymidine uptake. In this study, we investigated by flow cytometry the expression of different cell surface markers on lymphocytes after in vitro stimulation for 7 d with or without the allergen Chi t I. We determined the expression of the low-affinity receptor for IgE (CD23) on lymphocytes of Chi t I-sensitized patients and Chi t I-exposed as well as nonexposed controls. CD23 expression was significantly higher in patients than in nonexposed controls. Exposed but healthy subjects showed intermediate values. We also determined the expression of activation markers CD25 (IL-2 receptor) and HLA-DR on the lymphocytes of patients and nonexposed controls. HLA-DR expression on non-T cells (CD3-) was significantly higher in patients than in controls. HLA-DR on T cells (CD3+), and CD25 as well as CD23 expression, could be significantly enhanced after antigen-specific stimulation in patients but not in controls, whereas alpha/beta-T-cell-receptor expression was significantly reduced in patients. Differences between patients and controls were not observed in response to tetanus toxoid (TT) and phytohemagglutinin (PHA). Our results demonstrate antigen-specific influences on the expression of cell surface molecules. These findings may be valuable diagnostic information.

Hemoglobins of the Diptera (insect) family Chironomidae have been identified as causative allergens in asthmatic patients. In this study, 229 Japanese, 17 Taiwanese, and 92 Swedish sera from atopic patients were tested for antibodies against Chi t I, the hemoglobin from the European midge species Chironomus thummi, and against crude extracts from the Japanese midges Tokunagayusurika akamusi (T. akamusi) and Cricotopus sylvestris (Cr. sylvestris). Nearly 40% of patients showed a positive reaction to Cr. sylvestris extract, which contains no hemoglobin. This result is probably due to the presence of other partially cross-reacting allergens than hemoglobin. Nearly all tested Japanese serum samples showed cross-reactivity between Chi t I and Cr. sylvestris, a finding which is evidence for common epitopes in both midge species. Furthermore, an overall good correlation between the amounts of IgE antibodies against Chi t I and Chi t I component III was found in sera from Swedish, Japanese, and German patients.

Polyclonal human IgE antibodies of patients sensitized to one species of the Diptera family Chironomidae as well as polyclonal rabbit anti-Chi t I hyperimmune serum recognize antigenic sites present in nearly all of the 33 species of this insect family. Evolutionary distantly related genera usually show weaker antibody binding. According to the pattern of reactivity of rabbit anti-Chi t I-component III, expression of the epitopes of this molecule varies considerably in the genera Chironomus and Glyptotendipes; it appears to be almost totally absent in all other species. Of five monoclonal antibodies raised against Chi t I-component III, two recognize an epitope which is expressed in nearly all closely related species. Three monoclonal antibodies recognize epitopes which are expressed in only a few species of the same genus. Our results demonstrate the presence of common as well as species-specific epitopes in chironomid hemoglobins which behave as potent inhalant allergens.

Chironomid midges are small (2-15 mm) non-biting flies, characteristically seen swarming by water at dusk. Allergens of the "green nimitti" midge, Cladotanytarsus lewisi (Freeman) (Diptera: Chironomidae), a cause of widespread hypersensitivity in the Sudan, were isolated and partially characterized by Sephacryl S200 chromatography. The allergenicity of the fractions was identified by "rocket" autoradiography, RAST inhibition, skin "prick" tests, and the immunoblot technique. The fractions were further analysed by isoelectric focusing and SDS-PAGE. Two major allergens with pI's ranging from 4.3 to 6.0 were identified and had molecular weights of approximately 17,000 and 32,000 daltons, sizes compatible with their being monomeric and dimeric haemoglobins. Since chironomids occur in nuisance numbers worldwide and their haemoglobins have been shown to produce severe hypersensitivity reactions in man, they should be seen as an important potential cause of environmental and occupational allergy.