초록 ▼

◦ 세부과제명: 구제역 세가지 혈청형 항체 신속진단법 개발연구
- A형 및 Asia1형 FMDV P1 유전자와 3C 유전자를 이용하여 재조합단백질 항원제작
- A형 및 Asia1형(1A31) FMDV에 특이적으로 결합하는 단클론항체 제작
- A형 및 Asia1형 유전자재조합 항원과 단클론항체를 이용한 ELISA 확립
- 두가지 혈청형 ELISA 유효성 평가(국내사육 음성혈청, 백신접종혈청, 몽골감염혈청 ◦ 세부과제명: 구제역 백신접종 감별항체 동시진단법 개발연구
- 비구조단백질 진단항원(2AB, 3A, 3

◦ 세부과제명: 구제역 세가지 혈청형 항체 신속진단법 개발연구
- A형 및 Asia1형 FMDV P1 유전자와 3C 유전자를 이용하여 재조합단백질 항원제작
- A형 및 Asia1형(1A31) FMDV에 특이적으로 결합하는 단클론항체 제작
- A형 및 Asia1형 유전자재조합 항원과 단클론항체를 이용한 ELISA 확립
- 두가지 혈청형 ELISA 유효성 평가(국내사육 음성혈청, 백신접종혈청, 몽골감염혈청 ◦ 세부과제명: 구제역 백신접종 감별항체 동시진단법 개발연구
- 비구조단백질 진단항원(2AB, 3A, 3B, 3D) 및 진단항체(토끼항혈청) 제작
- 비구조단백질 동시액상분석조건 선정(bead 선정, 항원농도, 혈청희석배수)
- 구조단백질 및 비구조단백질 항체 동시액상분석조건 선정

Abstract ▼

A recombinant protein-based enzyme-linked immunosorbent assay (rP13C ELISA) was developed for the detection of antibodies to against foot-and-mouth disease virus (FMDV) serotype Asia 1. The recombinant protein (rP13C) derived from P1 precursor and 3C protease genes was expressed in insect cell. It e

A recombinant protein-based enzyme-linked immunosorbent assay (rP13C ELISA) was developed for the detection of antibodies to against foot-and-mouth disease virus (FMDV) serotype Asia 1. The recombinant protein (rP13C) derived from P1 precursor and 3C protease genes was expressed in insect cell. It elicited low titer of FMDV neutralizing antibodies in pigs. Its utility as a diagnostic antigen was explored in a blocking ELISA format with monoclonal antibodies. The rP13C ELISA yielded higher endpoint titers than liquid phase blocking (LPB) ELISA and virus neutralization test performed on sera from goats challenged with FMDV post vaccination. The rP13C ELISA scored FMD international reference weak positive serum correctly. The relative sensitivity between the rP13C ELISA and LPB ELISA was equivalent for vaccinated sera. With this comparable sensitivity, the rP13C ELISA exhibited a specificity of 99.7% for domestic naive swine, bovine and caprine sera. Taken together, the rP13C ELISA using recombinant proteins has a potential to replace LPB ELISA using inactivated FMDV antigen as a simple and robust serological tool for screening antibodies to FMDV serotype Asia 1.
A recombinant protein-based enzyme-linked immunosorbent assay (RP ELISA) was developed for the detection of antibodies to against foot-and-mouth disease virus (FMDV) serotype A. The detection limit of RP ELISA was found out to e less than titer 16 by virus neutralization test, implying its utility as an alternative to virus neutralization test. The RP ELISA scored FMD international reference weak positive serum correctly. The relative sensitivity between the rP13C ELISA was superiot to LPB ELISA for vaccinated sera collected in Mongolia. With this high comparable sensitivity, the RP ELISA exhibited a specificity of 98.8% for domestic naive swine, bovine and caprine sera. Taken all, RP ELISA using recombinant proteins has a potential to replace LPB ELISA using inactivated FMDV antigen as a simple and robust serological tool for screening antibodies to FMDV serotype A.
As an optimal bead, COOH bead was preferred to Penta(His-tag) bead in a preliminary test. The result of multiplex bead assay using 2AB, 3A, 3B, 3AB and 3D diangostic antigens together was the same as that of monoplex bead assay using individual antigen, implying that there was no interferences to detect antibodies to each nonstrucutral protein antigen. While bead assay in an indirect format to detect antibodies to nonstructural proteins was inferior to 3AB ELISA, 3AB bead assay in a competition format was equivalent to 3AB ELISA. In an effort to establish multiplex assay to detect antibodies to 3AB antibodies and O, A, Asia 1 strucutral proteins, optimal concentration of each serotype of antigen, biotin-conjugated monoclonal antibody and serum dilution factor were determined.
This bead assay to detect antibodies to nonstructural protein and strucutral proteins simultaneously would be useful as a simple and rapid serological diagnostic tool.

목차 Contents

• 연구과제 최종결과보고서 Final Report (FR) ... 1
• 연구과제 최종결과보고서 ... 2
• Ⅰ. 연구배경 및 목표 ... 2
• 1. 연구배경 ... 2
• 2. 연구최종목표 ... 3
• 3. 연차별 연구개발 목표 및 내용 ... 3
• Ⅱ. 연구방법 및 수행전략 ... 4
• 1. 재 료 ... 4
• 2. 연구전략 및 방법 ... 4
• Ⅲ. 연구결과 ... 5
• 가. 제1세부과제명 ... 5
• 나. 제2세부과제명 ... 11
• Ⅳ. 연구결과요약 ... 14
• 1. 유전자재조합단백질 항원과 중화단클론항체를 이용한 Asia1형 항체진단법 개발 ... 14
• 2. 유전자재조합단백질 항원과 중화단클론항체를 이용한 A형 항체진단법 개발 ... 15
• 3. 동시액상분석기법을 이용한 구제역 구조 및 비구조단백질 항체진단법 개발 ... 15
• Ⅴ. 고 찰 ... 15
• Ⅵ. 연구결과 활용실적 및 계획 ... 17
• Ⅶ. 참고문헌 ... 17
• Ⅹ. 영문초록 ... 18