Background: Silkworm pupa (SWP) food anaphylaxis has been described frequently in Asian countries. However, false-positive reactions by skin pricks and serum IgE (sIgE) tests to the extract complicate diagnosis, requiring identification of clinically relevant major allergens. Objectives:In this study, we characterized a novel SWP allergen, Bomb m 4, a 30-kDa lipoprotein, and evaluated its diagnostic sensitivity. Methods: Bomb m 4 was identified by a proteomic analysis. This recombinant (r)Bomb m 4 was overexpressed in Escherichia coli, and the IgE reactivity by ELISA was compared with other reported allergenic proteins: Bomb m 1 (arginine kinase), 27-kDa glycoprotein, Bomb m 3 (tropomyosin) using the serum samples from 17 SWP allergic patients and 11 asymptomatic sensitized subjects. Results: rBomb m 4-specific IgE was recognized by all 17 SWP allergic patients. The 27-kDa glycoprotein and Bomb m 1 sIgE were found in 35.3% and 0%, respectively, in the SWP allergic patients. ELISA sIgE reactivity increased significantly, when 4 M urea was added in serum samples. However, only 16% inhibition of sIgE reactivity to the whole SWP extract was exhibited by rBomb m 4, whereas more than 93% of self-inhibition of rBomb m 4 sIgE was obtained, possibly due to the low abundance of Bomb m 4 in the extract. Three linear epitopes (81-95, 191-205 and 224-238 residues) of rBomb m 4 were identified. These epitopes are shown to be released by pepsin digestion. Receiver operator characteristic (ROC) analysis showed the highest diagnostic value of Bomb m 4 followed by Bomb m 1, 27-kDa glycoprotein and Bomb m 3. Conclusion: Bomb m 4 is the major allergen of SWP allergic patients. It has cryptic epitopes which are exposed to IgE antibodies with digestive enzymes. This recombinant Bomb m 4 allergen permits exact diagnosis of SWP allergy.

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House dust mite is common cause of atopic dermatitis (AD) both in human and dogs. Detection of serum IgE to allergen is commonly used to diagnose allergic diseases. However, false-positive reactions due to cross-reactivity and non-specific reactivity may lead to misdiagnosis. We compared human and canine IgE reactivities to mite component allergens. Canine IgE-reactive components of D. farinae and T. putrescentiae were identified by tandem mass spectrometry. Recombinant proteins were produced and IgE reactivities to component allergens were assessed by ELISA and inhibition assays using sera from AD patients and dogs. Canine IgE-reactive proteins (Der f 1, Der f 11, Tyr p 4, Tyr p 8, Tyr p 11, Tyr p 28) were identified by proteome analysis. The majority of patients were sensitized to Der f 1 (93.3%) and Der f 2 (86.7%). Dogs showed high sensitization to Der f 2 (94.1%) and Der f 18 (84.6%). Both patients and dogs showed low IgE binding frequency to Tyr p 8, 43.3% and 4% respectively. The ELISA inhibition study indicated that canine IgE reactivity to T. putrescentiae is mostly due to non-specific reaction and cross-reaction with D. farinae. Different IgE sensitization patterns were shown between allergic human and dogs with AD, especially to Der f 18 in Korea for the first time. Furthermore, non-specific canine IgE reactivity to storage mite indicates the possible misdiagnoses. Standardizations focused on the major canine allergen content of extracts should be developed. This will allow precision diagnosis and individuated treatments for each patient and atopic dog.

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