ANALYSIS OF ALLERGENS FROM BLACK TIGER SHRIMP (Penaeus monodon) BY COMBINING TWO-DIMENSIONAL GEL ELECTROPHORESIS WITH RS-ATL8 REPORTER CELL LINE

Abstract

Food allergy is an immediate hypersensitivity reaction. Specific IgE is generated against allergens, and binds to the specific IgE receptor on the surface of basophils and mast cells. Allergen binding induces IgE cross-linking that triggers these cells to release chemical mediators such as histamine, prostaglandins and leukotrienes. Symptoms of allergic reactions vary from mild irritation to anaphylaxis and life-threatening. Black tiger shrimp (Penaeus monodon) is an important aquaculture species in Asia and is the common cause of food allergy in Thailand. One of the important problems in management of shellfish allergy is the lack of accurate diagnostic assay because the biological and immunological properties of allergens in the black tiger shrimps have not been well characterized. This study aims to investigate the reactive pattern of serum IgE from shrimp allergic patients to raw and cooked protein extract from black tiger shrimps and to identify shrimp allergens that can trigger IgE crosslinking by combining two-dimensional gel electrophoresis (2-DE) and RS-ATL8 reporter cell line. ELISA using sera from 24 shrimp allergic subjects, indicated that there were significant differences in reactivity to the raw and cooked shrimp extracts (P= 0.0093). Allergic serum IgE reacted stronger to raw shrimp extract than cooked shrimp extract. Consistent with these results, in SDS-PAGE, raw shrimp extract contained more protein bands than in the cooked extract. Western blot demonstrated that there was the major IgE reactivity area at 32-39 kDa in both raw and cooked shrimp extract. Eighteen of 24 patients (75%) and all patients (100%) had specific IgE to proteins in the range of 32-39 kDa in cooked and raw shrimp, respectively. The minimum concentration of crude shrimp extract to induce IgE cross-linking as measured by RS-ATL8 cell line were 10 fg/ml and 100 fg/ml in raw and cooked shrimp extract, respectively. The ten spots excised from 2-DE did not induce IgE cross-linking in RS-ATL8 cell line. The eluted protein from one-dimensional gel electrophoresis at the 115 and 38 kDa bands from raw shrimp extract induced an IgE cross-linking and the proteins were analysed by mass spectrometry. Some novel proteins were identified with the possibility of novel allergen. These results may be useful for shrimp allergy diagnostic test in the future.