국내 소아 호두 알레르기: 임상적으로 중요한 성분 항원 분석 및 가열 조리 호두 내 혈청 특이 IgE 결합 단백 규명
Walnut allergy in Korean children: Analysis of clinically relevant allergenic component and identification of IgE-binding proteins in cooked walnut extract
- 주제(키워드) antigenicity , cooking , walnut , Jug r 1 , children
- 발행기관 아주대학교
- 지도교수 이수영
- 발행년도 2020
- 학위수여년월 2020. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029759
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Background: Walnuts (WN) are one of the main causes of tree nut allergies. However, the potential value of component resolved diagnosis (CRD) for WN allergy is controversial and the influence of different cooking methods on WN proteins have not been evaluated to date. Purpose: This study aimed to evaluate the clinical and immunological features of clinical WN allergy and the usefulness of CRD in young children, together with changes in WN antigenicity caused by common cooking methods. Methods: In study part A, twenty-nine participants with a history of ingesting WN who were assessed for serum-specific IgE to WN (WN-sIgE) and CRD (ImmunoCAP ISAC 112) at the Department of Pediatrics in Ajou University Hospital were enrolled and their demographic profiles, clinical symptoms, and laboratory findings were evaluated. Further, in study part B, the protein fractions of dry-fried and boiled WN extracts were compared with those of raw WNs using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2-dimentional gel electrophoresis (2DE) and a proteomic analysis using electrospray ionization (LC-MS). An immunoblotting analysis was conducted to examine IgE reactivity toward raw WNs using serum samples from 6 children with a clinical WN allergy. To determine the processed WN proteins with IgE-binding capacity, a 2D-immunoblotting analysis was performed using the pooled sera of 20 WN-sensitized children. Results: Twenty-eight out of 29 patients had specific immunoglobulin E antibodies to Jug r 1 (Jug r1-sIgE). Receiver-operating characteristic curves demonstrated that the Jug r 1-sIgE level was better for discriminating between children who were clinically allergic and tolerant to WN than the WN-sIgE level. Protein bands from raw WNs were identified at 9, 16, 28, 52, 58 and 64 kDa via SDS-PAGE. The 9-kDa and 16-kDa protein bands were enhanced by boiling, whereas the 52-kDa and 64-kDa bands were considerably diminished. On LC-MS analysis, of the 66 IgE-binding proteins present in raw WNs, 57 were found in dry-fried WNs, but only 4 in boiled WNs. A 2D-immunoblotting result confirmed the presence of different binding patterns among children who consumed cooked WNs. Conclusions: Jug r 1 is the major component allergen in young children with clinical WN allergy. Though the protein profile of cooked WNs is substantially different from that of raw WNs, 4 proteins, including Jug r 1, remained stable after dry-frying or boiling. Further studies are needed to evaluate the clinical relevance of these findings.
more목차
I. Introduction 1
II. Materials and Methods 4
Part A. Analysis of clinically relevant allergenic component 4
1. Study population 4
2. Microarray 4
3. Stastical analysis 5
Part B. Identification of Immunoglobulin E-binding proteins in cooked walnut extract 5
1. Study population 6
2. Walnut preparation and processing 6
3. One-dimensional polyacrylamide gel electrophoresis (PAGE) and immunoblotting analysis 7
4. Two-dimensional electrophoresis (2DE) and 2D-immunoblotting analysis 7
5. LC-MS/MS for peptide analysis 8
6. Database search 8
III. Results 12
Part A. Demographic, clinical and immunological characteristics of patients allergic to walnuts and the findings of clinically relevant allergenic component 12
1. Demographic and clinical characteristics of the patients 12
2. Immunological characteristics of patients and the diagnostic value of component resolved diagnosis 13
Part B. Identification of IgE-binding proteins in cooked walnut extract using sera of the patients sensitized to walnuts 14
1. Demographic and clinical characteristics of the patients 14
2. Comparison of the protein fractions of raw, dry-fried, and boiled WNs via SDS-PAGE and 2DE 14
3. IgE reactivity of each WN extract based on immunoblotting analysis 15
4. Protein profiles of differently processed WN extracts 15
IV. Discussion 31
V. Conclusion 35
References 36
