Allergenic characterization of Bomb m 4, a 30‐kDa Bombyx mori lipoprotein 6 from silkworm pupa

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. Silkworm pupa is an important cause of food anaphylaxis in Asian countries. The newly identified allergen, a 30‐kDa lipoprotein (Bmlp6) has cryptic epitopes. Three linear epitopes, which is hidden in native sate, were exposed after urea or pepsin treatment. Its recombinant protein showed highest diagnostic value compared to previously described silkworm allergens.