KR20230056987A - Kit for diagnosis of Severe Fever with Thrombocytopenia Syndrome virus infection - Google Patents
Kit for diagnosis of Severe Fever with Thrombocytopenia Syndrome virus infection Download PDFInfo
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- KR20230056987A KR20230056987A KR1020210140885A KR20210140885A KR20230056987A KR 20230056987 A KR20230056987 A KR 20230056987A KR 1020210140885 A KR1020210140885 A KR 1020210140885A KR 20210140885 A KR20210140885 A KR 20210140885A KR 20230056987 A KR20230056987 A KR 20230056987A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/175—Bunyaviridae, e.g. California encephalitis virus, Rift valley fever virus, Hantaan virus
Abstract
Description
본 발명은 중증 열성 혈소판 감소 증후군(Severe Fever with Thrombocytopenia Syndrome; SFTS) 바이러스 감염 진단용 키트에 관한 것으로서, 더욱 상세하게는 SFTS 바이러스에 특이적으로 반응하는 2종의 단클론항체(Monoclonal antibody; mAb)의 선발, 이를 이용한 진단용 키트 및 SFTS 바이러스 감염 여부를 진단하기 위한 정보 제공 방법에 관한 것이다.The present invention relates to a kit for diagnosing Severe Fever with Thrombocytopenia Syndrome (SFTS) virus infection, and more particularly, to the selection of two types of monoclonal antibodies (mAb) that specifically react to the SFTS virus , a diagnostic kit using the same and a method for providing information for diagnosing SFTS virus infection.
중증 열성 혈소판 감소 증후군(Severe Fever with Thrombocytopenia Syndrome; SFTS)은 진드기가 매개하는 바이러스성 인수공통 전염병으로 국내에서 2013년 처음 환자가 발생한 이후 지속 발생하고 있다(‘19년 223명 발생, 40명 사망; 치사율 17.9%).Severe Fever with Thrombocytopenia Syndrome (SFTS) is a tick-mediated viral zoonotic epidemic that has been occurring continuously since the first patient occurred in Korea in 2013 (223 cases in 2019, 40 deaths; mortality rate 17.9%).
환자의 대부분(90% 이상)은 50대 이상으로, 연령별로 감염율 및 치사율이 상이한 특징을 보인다. 반면, 동물의 경우에는 무증상 감염이 대부분으로 임상증상 발현이나 이로 인한 폐사에 이르는 예는 극히 드물게 나타난다.Most of the patients (more than 90%) are in their 50s or older, and the infection rate and mortality rate are different according to age. On the other hand, in the case of animals, most cases of asymptomatic infection lead to clinical symptoms or death due to them, which are extremely rare.
최근 일본에서 바이러스에 감염된 고양이 및 개에서 사람과 유사한 임상증상 발현 및 폐사가 발생한 예가 다수 보고되기도 하였으며, 이들을 진료한 수의 의료진(수의사, 수의 간호사 등)에 전파된 예가 보고되기도 하였다.Recently, many cases of human-like clinical symptoms and deaths in cats and dogs infected with the virus have been reported, and cases of transmission to veterinary medical staff (veterinarians, veterinary nurses, etc.) who treated them have been reported.
국내에서도 ‘18년, ’20년 임상증상이 발현된 반려견으로부터 바이러스가 분리되는 등, 동물로부터의 바이러스 전파 가능성에 대한 우려가 증가하고 있어 신속진단을 통한 빠른 대처가 필요한 상황이다.In Korea, there is an increasing concern about the possibility of virus transmission from animals, such as the isolation of the virus from companion dogs with clinical symptoms in 2018 and 2020.
이에 본 발명자들은 중증 열성 혈소판 감소 증후군(Severe Fever with Thrombocytopenia Syndrome; SFTS) 바이러스 항원을 이용하여 단클론항체(Monoclonal antibody; mAb)를 선발하였으며, 이를 이용하여 SFTS 바이러스 감염 여부를 특이적으로 진단할 수 있음을 확인하였다.Accordingly, the present inventors selected a monoclonal antibody (mAb) using the Severe Fever with Thrombocytopenia Syndrome (SFTS) virus antigen, and using this, SFTS virus infection can be specifically diagnosed. confirmed.
이에, 본 발명의 목적은 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 10의 아미노산 서열로 이루어진 중쇄가변영역; 또는 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 18의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 단클론항체를 제공하는 것이다.Accordingly, an object of the present invention is a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10; Or to provide a monoclonal antibody comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18.
본 발명의 다른 목적은 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 10의 아미노산 서열로 이루어진 중쇄가변영역; 또는 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 18의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 단클론항체를 포함하는 SFTS 바이러스 검출용 조성물을 제공하는 것이다.Another object of the present invention is a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10; Or to provide a composition for detecting SFTS virus comprising a monoclonal antibody comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18.
본 발명의 또 다른 목적은 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 10의 아미노산 서열로 이루어진 중쇄가변영역; 또는 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 18의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 단클론항체를 포함하는 SFTS 바이러스 검출용 조성물을 포함하는 SFTS 바이러스 감염 진단용 키트를 제공하는 것이다.Another object of the present invention is a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10; Or a kit for diagnosing SFTS virus infection comprising a monoclonal antibody comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18.
본 발명의 또 다른 목적은 다음을 포함하는 SFTS 바이러스 감염 진단을 위한 정보 제공 방법을 제공하는 것이다:Another object of the present invention is to provide an information providing method for diagnosing SFTS virus infection, including:
서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 10의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 고정항체(capture mAb)로 코팅된 고정체에 시료를 접촉시키는 반응 단계; 및A reaction step of contacting a sample to a stationary body coated with a capture mAb comprising a light chain variable region composed of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region composed of the amino acid sequence of SEQ ID NO: 10; and
상기 고정체에 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 18의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 검출항체(detector Ab)를 반응시키는 검출 단계.A detection step of reacting a detection antibody (detector Ab) comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18 to the fixed body.
본 발명의 또 다른 목적은 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 10의 아미노산 서열로 이루어진 중쇄가변영역; 또는 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 18의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 단클론항체의 SFTS 바이러스 감염 진단 용도에 관한 것이다.Another object of the present invention is a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10; Or, it relates to the use of a monoclonal antibody comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18 for diagnosing SFTS virus infection.
본 발명은 중증 열성 혈소판 감소 증후군(Severe Fever with Thrombocytopenia Syndrome; SFTS) 바이러스 감염 진단용 키트에 관한 것으로, 본 발명에 따른 진단용 키트는 SFTS 바이러스 감염 여부를 특이적으로 진단할 수 있다.The present invention relates to a kit for diagnosing Severe Fever with Thrombocytopenia Syndrome (SFTS) virus infection, and the diagnostic kit according to the present invention can specifically diagnose SFTS virus infection.
본 발명자들은 SFTS 바이러스 항원을 이용하여 단클론항체(Monoclonal antibody; mAb)를 선발하였고, 이를 이용한 진단용 키트를 사용함으로써 신속하고 정확한 진단이 가능함을 확인하였다.The present inventors selected a monoclonal antibody (mAb) using the SFTS virus antigen, and confirmed that rapid and accurate diagnosis is possible by using a diagnostic kit using the mAb.
이하 본 발명을 더욱 자세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 양태는 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 10의 아미노산 서열로 이루어진 중쇄가변영역; 또는 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 18의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 단클론항체이다.One aspect of the present invention comprises a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10; or a monoclonal antibody comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18.
본 발명에 있어서 상기 단클론항체는 서열번호 4의 아미노산 서열로 이루어진 CDR-L1, 서열번호 6의 아미노산 서열로 이루어진 CDR-L2, 및 서열번호 8의 아미노산 서열로 이루어진 CDR-L3을 포함하는 경쇄가변영역; 및 서열번호 12의 아미노산 서열로 이루어진 CDR-H1, 서열번호 14의 아미노산 서열로 이루어진 CDR-H2, 및 서열번호 16의 아미노산 서열로 이루어진 CDR-H3을 포함하는 중쇄가변영역을 포함하는 것일 수 있다.In the present invention, the monoclonal antibody comprises a light chain variable region comprising CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 6, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 8. ; and a heavy chain variable region comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 12, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 14, and CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 16.
본 발명에 있어서 상기 단클론항체는 서열번호 4의 아미노산 서열로 이루어진 CDR-L1, 서열번호 6의 아미노산 서열로 이루어진 CDR-L2, 및 서열번호 8의 아미노산 서열로 이루어진 CDR-L3을 포함하는 경쇄가변영역; 및 서열번호 20의 아미노산 서열로 이루어진 CDR-H1, 서열번호 22의 아미노산 서열로 이루어진 CDR-H2, 및 서열번호 24의 아미노산 서열로 이루어진 CDR-H3을 포함하는 중쇄가변영역을 포함하는 것일 수 있다.In the present invention, the monoclonal antibody comprises a light chain variable region comprising CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 6, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 8. ; and a heavy chain variable region comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 20, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 22, and CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 24.
본 발명에 있어서 상기 단클론항체는 수탁번호 KCTC18912P로 기탁된 하이브리도마 세포주, 및 수탁번호 KCTC18913P로 기탁된 하이브리도마 세포주로 이루어진 군으로부터 선택되는 1종 이상에 의하여 생산되는 것일 수 있다.In the present invention, the monoclonal antibody may be produced by at least one selected from the group consisting of a hybridoma cell line deposited with accession number KCTC18912P and a hybridoma cell line deposited with accession number KCTC18913P.
본 발명의 다른 양태는 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 10의 아미노산 서열로 이루어진 중쇄가변영역; 또는 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 18의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 단클론항체를 포함하는 SFTS 바이러스 검출용 조성물이다.Another aspect of the present invention comprises a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10; or a composition for detecting SFTS virus comprising a monoclonal antibody comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18.
본 발명에 있어서 상기 단클론항체는 서열번호 4의 아미노산 서열로 이루어진 CDR-L1, 서열번호 6의 아미노산 서열로 이루어진 CDR-L2, 및 서열번호 8의 아미노산 서열로 이루어진 CDR-L3을 포함하는 경쇄가변영역; 및 서열번호 12의 아미노산 서열로 이루어진 CDR-H1, 서열번호 14의 아미노산 서열로 이루어진 CDR-H2, 및 서열번호 16의 아미노산 서열로 이루어진 CDR-H3을 포함하는 중쇄가변영역을 포함하는 것일 수 있다.In the present invention, the monoclonal antibody comprises a light chain variable region comprising CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 6, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 8. ; and a heavy chain variable region comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 12, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 14, and CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 16.
본 발명에 있어서 상기 단클론항체는 서열번호 4의 아미노산 서열로 이루어진 CDR-L1, 서열번호 6의 아미노산 서열로 이루어진 CDR-L2, 및 서열번호 8의 아미노산 서열로 이루어진 CDR-L3을 포함하는 경쇄가변영역; 및 서열번호 20의 아미노산 서열로 이루어진 CDR-H1, 서열번호 22의 아미노산 서열로 이루어진 CDR-H2, 및 서열번호 24의 아미노산 서열로 이루어진 CDR-H3을 포함하는 중쇄가변영역을 포함하는 것일 수 있다.In the present invention, the monoclonal antibody comprises a light chain variable region comprising CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 6, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 8. ; and a heavy chain variable region comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 20, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 22, and CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 24.
본 발명에 있어서 상기 단클론항체는 수탁번호 KCTC18912P로 기탁된 하이브리도마 세포주, 및 수탁번호 KCTC18913P로 기탁된 하이브리도마 세포주로 이루어진 군으로부터 선택되는 1종 이상에 의하여 생산되는 것일 수 있다.In the present invention, the monoclonal antibody may be produced by at least one selected from the group consisting of a hybridoma cell line deposited with accession number KCTC18912P and a hybridoma cell line deposited with accession number KCTC18913P.
본 발명의 또 다른 양태는 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 10의 아미노산 서열로 이루어진 중쇄가변영역; 또는 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 18의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 단클론항체를 포함하는 SFTS 바이러스 검출용 조성물을 포함하는 SFTS 바이러스 감염 진단용 키트이다.Another aspect of the present invention comprises a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10; Or a kit for diagnosing SFTS virus infection comprising a monoclonal antibody comprising a light chain variable region composed of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region composed of the amino acid sequence of SEQ ID NO: 18.
본 발명에 있어서 상기 키트는 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 10의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 고정항체(capture mAb); 및 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 18의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 검출항체(detector Ab)를 포함하는 것일 수 있다.In the present invention, the kit includes a fixed antibody (capture mAb) comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10; and a detector Ab comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18.
상기 키트를 제조함에 있어서 고정항체와 검출항체를 서로 달리하여, 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 18의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 고정항체; 및 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 10의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 검출항체를 이용할 수 있으나, 반응성이 상대적으로 저조하게 나타난다.In preparing the kit, a fixed antibody comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18 by differentiating the fixed antibody and the detection antibody from each other; And a detection antibody comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10 may be used, but the reactivity is relatively low.
본 발명에 있어서 상기 검출항체는 콜로이달 골드(colloidal gold), HRP(horseradish peroxidase), 알칼리성 인산분해효소(alkaline phosphatase), 형광물질(fluorescein) 및 색소(dye)로 이루어진 군으로부터 선택되는 1종 이상의 표지체와의 콘쥬게이트 형태인 것일 수 있다.In the present invention, the detection antibody is at least one selected from the group consisting of colloidal gold, horseradish peroxidase (HRP), alkaline phosphatase, fluorescein, and dye It may be in the form of a conjugate with a marker.
본 발명의 일 구현예에서, 상기 키트는 샘플 패드, 콘쥬게이트 패드, 멤브레인, 및 흡수 패드의 순서로 나열되어 인접한 패드끼리 중첩되도록 부착된 스트립을 포함한다.In one embodiment of the present invention, the kit includes strips arranged in the order of a sample pad, a conjugate pad, a membrane, and an absorbent pad and attached so that adjacent pads overlap each other.
상기 스트립의 제조 순서에 있어서, 백킹 카드(backing card) 또는 이와 같은 지지체에 고정항체를 고정시킨 검사선이 구비된 멤브레인을 가장 먼저 부착하고, 멤브레인과 한 쪽 말단이 중첩되도록 골드 콘쥬게이트 패드를 부착하고, 멤브레인과 중첩되지 않은 골드 콘쥬게이트 패드의 나머지 말단이 중첩되도록 샘플 패드를 부착할 수 있다. 이어서 골드 콘쥬게이트 패드와 중첩되지 않은 멤브레인의 나머지 말단이 중첩되도록 흡수 패드를 부착할 수 있으나, 흡수 패드의 부착 순서는 골드 콘쥬게이트 패드 또는 샘플 패드의 부착 순서보다 선행되어도 무관하다.In the manufacturing sequence of the strip, the membrane equipped with the test line immobilized with the immobilized antibody is first attached to a backing card or such a support, and then a gold conjugate pad is attached so that one end overlaps the membrane. Then, the sample pad may be attached so that the other end of the gold conjugate pad, which does not overlap with the membrane, overlaps. Subsequently, the absorption pad may be attached so that the other end of the membrane that does not overlap with the gold conjugate pad overlaps, but the order of attachment of the absorption pad may precede the order of attachment of the gold conjugate pad or sample pad.
상기 멤브레인에는 검사선과 이격되어 위치하는 대조선이 추가적으로 구비될 수 있고, 멤브레인 상에 전개되는 물질은 샘플 패드에 투입되어 흡수 패드 방향으로 전개됨으로써 멤브레인 상의 검사선을 지나 대조선에 도달할 수 있다.A control line spaced apart from the test line may be additionally provided on the membrane, and a material spread on the membrane may pass through the test line on the membrane and reach the control line by being introduced into the sample pad and spread in the direction of the absorption pad.
상기 골드 콘쥬게이트 패드는 표지체와 콘쥬게이트 된 형태의 검출항체를 포함하는 것일 수 있고, 멤브레인 상에 전개되는 물질과 함께 멤브레인 상의 검사선 또는 대조선에 도달한다.The gold conjugate pad may include a detection antibody conjugated with a marker, and reaches a test line or control line on the membrane together with a material spread on the membrane.
본 발명의 또 다른 양태는 다음을 포함하는 SFTS 바이러스 감염 진단을 위한 정보 제공 방법이다:Another aspect of the present invention is an informational method for diagnosing SFTS virus infection comprising:
서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 10의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 고정항체로 코팅된 고정체에 시료를 접촉시키는 반응 단계; 및A reaction step of contacting a sample with a stationary body coated with a fixed antibody comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10; and
상기 고정체에 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 18의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 검출항체를 반응시키는 검출 단계.A detection step of reacting a detection antibody comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18 to the fixed body.
상기 반응 단계 및 검출 단계는 순차적으로 이루어지는 것일 수 있으나, 고정항체, 시료 내 표적물질, 및 검출항체의 반응이 동시에 일어나는 것일 수도 있다. 다만, 시료 내 표적물질에 앞서 검출항체의 고정체에 대한 반응이 선행되지는 않는다.The reaction step and the detection step may be performed sequentially, but the reaction of the immobilized antibody, the target substance in the sample, and the detection antibody may occur simultaneously. However, the reaction to the stationary body of the detection antibody does not precede the target substance in the sample.
본 발명에 있어서 상기 검출항체는 콜로이달 골드, HRP, 알칼리성 인산분해효소, 형광물질 및 색소로 이루어진 군으로부터 선택되는 1종 이상의 표지체와의 콘쥬게이트 형태인 것일 수 있다.In the present invention, the detection antibody may be in the form of a conjugate with one or more markers selected from the group consisting of colloidal gold, HRP, alkaline phosphatase, fluorescent substances and dyes.
본 발명은 중증 열성 혈소판 감소 증후군(Severe Fever with Thrombocytopenia Syndrome; SFTS) 바이러스 감염 진단용 키트에 관한 것으로서, 상기 키트는 SFTS 바이러스 항원을 이용하여 선발된 단클론항체(Monoclonal antibody; mAb)를 이용하여 특이적이고 신속하게 SFTS 바이러스를 검출하는 효과를 발휘하므로, 이를 효과적으로 SFTS 바이러스 감염 여부의 진단에 이용할 수 있다.The present invention relates to a kit for diagnosing Severe Fever with Thrombocytopenia Syndrome (SFTS) virus infection, wherein the kit uses a monoclonal antibody (mAb) selected using an SFTS virus antigen to detect specific and rapid Since it exhibits the effect of detecting the SFTS virus, it can be effectively used for diagnosing whether or not the SFTS virus is infected.
도 1은 본 발명의 일 실시예에 따라 제조된 진단용 키트를 이용하여 바이러스 검출 효과를 확인한 결과이다.1 is a result of confirming the virus detection effect using a diagnostic kit manufactured according to an embodiment of the present invention.
이하, 본 발명을 하기의 실시예에 의하여 더욱 상세히 설명한다. 그러나 이들 실시예는 본 발명을 예시하기 위한 것일 뿐이며, 본 발명의 범위가 이들 실시예에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
본 명세서 전체에 걸쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 "%"는 별도의 언급이 없는 경우, 고체/고체는 (중량/중량)%, 고체/액체는 (중량/부피)%, 그리고 액체/액체는 (부피/부피)%이다.Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight/weight)% for solids/solids, (weight/volume)% for solids/liquids, and liquid/liquid is (volume/volume) %.
실시예 1: SFTS 바이러스에 대한 단클론항체의 제작Example 1: Construction of monoclonal antibodies against SFTS virus
1-1. 면역원의 준비1-1. preparation of immunogens
중증 열성 혈소판 감소 증후군(Severe Fever with Thrombocytopenia Syndrome; SFTS) 바이러스(개 유래 국내분리주, CBCB01/2018)를 Vero E6 세포에 접종 후 37℃에서 5-7일간 배양을 통해 증폭된 SFTS 바이러스를 수거하였다.After inoculating Vero E6 cells with Severe Fever with Thrombocytopenia Syndrome (SFTS) virus (dog-derived domestic isolate, CBCB01/2018), the amplified SFTS virus was collected through incubation at 37°C for 5-7 days.
그 다음 바이너리 에칠렌이민(Binary Ethylenimine; BEI)을 0.001 M(=1 mM) 농도로 처리한 후 37℃에서 26-30시간 동안 진탕배양하여 불활화 후, 티오황산나트륨(sodium thiosulfate) 2 mM을 첨가하여 중화시켰다.Then, after treatment with binary ethylenimine (BEI) at a concentration of 0.001 M (= 1 mM), shaking culture at 37 ° C. for 26-30 hours to inactivate it, and then sodium thiosulfate (2 mM sodium thiosulfate) was added to Neutralized.
불활화 배양액을 4,000 rpm에서 30분간 원심분리한 후 상층액을 다시 18,000 RPM에서 4시간 동안 초원심분리하였다. 펠릿(Pellet)은 PBS(pH 7.4)로 잘 풀어준 후 설탕 밀도 기울기 원심 분리법(sucrose density gradient centrifugation)으로 초원심 분리(27,000 rpm, 2.5시간)하여 순도 높은 SFTS 바이러스 분획을 수득하였다.The inactivated culture medium was centrifuged at 4,000 rpm for 30 minutes, and the supernatant was further ultracentrifuged at 18,000 RPM for 4 hours. The pellet was well dissolved in PBS (pH 7.4) and then subjected to ultracentrifugation (27,000 rpm, 2.5 hours) by sucrose density gradient centrifugation to obtain a highly pure SFTS virus fraction.
1-2. 면역화1-2. immunization
상기 1-1에서 수득한 불활화된 SFTS 바이러스 항원을 이용하여 실험동물(balb/c mouse)에 면역(immunization)을 실시하였다. 구체적으로, 항원으로서 불활화 STFS 바이러스 항원을 0.25 mg/ml 농도로 200 ul 준비하고 FA(Freund's Adjuvant) 200 ul와 혼합하여 마우스 복강에 2주 간격으로 주입하여 3차 면역까지 실시하였다.Immunization was performed on experimental animals (balb/c mice) using the inactivated SFTS virus antigen obtained in 1-1 above. Specifically, 200 ul of an inactivated STFS virus antigen was prepared as an antigen at a concentration of 0.25 mg/ml, mixed with 200 ul of FA (Freund's Adjuvant), and injected into the mouse peritoneal cavity at 2-week intervals until the third immunization.
퓨전 면역 검사를 위해 3차 면역 1주 후, 마우스 혈청을 이용하여 간접(indirect) ELISA 검사를 실시하였다. 최종 부스팅을 위해 불활화된 SFTS 바이러스 항원 100 ul를 추가적으로 복강에 주입하여 면역(4차)하고 4일 후 세포융합에 사용하였다.For the fusion immunoassay, an indirect ELISA test was performed using mouse serum 1 week after the third immunization. For final boosting, 100 ul of inactivated SFTS virus antigen was additionally injected intraperitoneally to immunize (4th) and used for cell fusion after 4 days.
1-3. 세포융합 및 하이브리도마 세포의 제조1-3. Fusion of cells and preparation of hybridoma cells
면역화된 마우스 몸통 좌측에 위치한 비장(spleen)을 적출하여 DMEM(Dulbecco’s modified Eagle’s medium) 배지에 부유시켰다. 적출한 비장을 메쉬로 갈아서 세포를 분리하고, 배양배지 DMEM과 혼합하여 비장세포 현탁액을 제조하였다. 그 다음, 상기 현탁액을 1,200 rpm에서 5분 동안 원심 분리하여 상층액을 제거하고, 비장세포와 골수세포주(SP2/0 Ag14 (ATCC, USA))를 1:5의 세포 수 비율로 혼합한 후, 1,200 rpm에서 5분 동안 원심 분리하여 세포를 침전시켜 상층액은 제거하였다.The spleen located on the left side of the body of the immunized mouse was removed and suspended in DMEM (Dulbecco's modified Eagle's medium) medium. The cells were separated by grinding the extracted spleen with a mesh, and mixed with the culture medium DMEM to prepare a spleen cell suspension. Then, the suspension was centrifuged at 1,200 rpm for 5 minutes to remove the supernatant, and the splenocytes and the bone marrow cell line (SP2/0 Ag14 (ATCC, USA)) were mixed at a cell count ratio of 1:5, Cells were precipitated by centrifugation at 1,200 rpm for 5 minutes, and the supernatant was removed.
원심 분리된 세포를 천천히 분산시킨 후 폴리에틸렌 글리콜 1500(PEG1500, Roche) 1.0 ml을 처리하고, 37℃에서 1분 동안 유지시킨 후 DMEM 1 ml을 첨가하였다. 그 다음, DMEM 10 ml을 1분 동안 서서히 첨가하고, 37℃의 물에서 5분 동안 반응시킨 후 50.0 ml로 맞추고, 1,200 rpm에서 5분 동안 원심 분리하였다.After slowly dispersing the centrifuged cells, 1.0 ml of polyethylene glycol 1500 (PEG1500, Roche) was treated, maintained at 37° C. for 1 minute, and then 1 ml of DMEM was added. Then, 10 ml of DMEM was slowly added for 1 minute, reacted in 37° C. water for 5 minutes, adjusted to 50.0 ml, and centrifuged at 1,200 rpm for 5 minutes.
세포침전물을 분리배지(HAT 배지)에 1 X 105 내지 2 X 105 cells/ml 농도로 재현탁시키고, 96웰 플레이트에 0.1 ml씩 분주한 후 37℃ 이산화탄소 배양기에서 배양하여 하이브리도마 세포를 제조하였다.The cell precipitate was resuspended in a separation medium (HAT medium) at a concentration of 1 X 10 5 to 2 X 10 5 cells/ml, dispensed in 0.1 ml each in a 96-well plate, and then cultured in a 37°C carbon dioxide incubator to obtain hybridoma cells. manufactured.
1-4. 하이브리도마 세포의 선별1-4. Selection of hybridoma cells
상기 제조한 하이브리도마 세포군 중에서 불활화된 SFTS 바이러스 항원에 특이적으로 반응하는 하이브리도마 세포를 선별하기 위하여 ELISA 분석 방법을 사용하였다.An ELISA analysis method was used to select hybridoma cells that specifically reacted to the inactivated SFTS virus antigen among the prepared hybridoma cell population.
구체적으로, 목적으로 하는 불활화된 SFTS 바이러스 항원을 96웰 마이크로플레이트의 한 웰당 각각 100 ul(1 ug/ml)씩 가하여 플레이트 표면에 부착시키고, 반응하지 않은 항원은 세척하여 제거하였다. 그 다음, 0.1% 블로킹(casein-PBS blocking) 용액을 웰 당 100 ul씩 분주한 후 37℃에서 2시간 동안 반응 후 인산 완충용액-트윈20(0.05% tween-20(v/v)이 포함되어 있는 인산완충용액, pH 7.4, 이하 PBS-T)로 3회 세척하였다.Specifically, 100 ul (1 ug/ml) of the inactivated SFTS virus antigen of interest was added to each well of a 96-well microplate and attached to the surface of the plate, and unreacted antigens were removed by washing. Then, after dispensing 100 ul of 0.1% blocking (casein-PBS blocking) solution per well and reacting at 37 ° C for 2 hours, phosphate buffer-Tween 20 (0.05% tween-20 (v / v) is included) washed three times with a phosphate buffered solution, pH 7.4, hereinafter PBS-T).
그 다음, 하이브리도마 세포의 배양액을 각 웰에 100 ul씩을 가하여 1시간 동안 실온에서 반응시킨 후, PBS-T 용액으로 3회 세척하여 반응하지 않은 배양액을 제거하였다. 여기에 염소 항-마우스 IgG-호스레디쉬 퍼옥시다제(Goat anti-mouse IgG-HRP)를 가하여 1시간 동안 실온에서 반응시킨 다음, PBS-T로 3회 세척하였다. 세척한 후 퍼옥시다제의 기질용액(TMB)을 가하여 반응시키고, 황산 0.5 M를 플레이트 각 웰에 50 ul씩 분주한 뒤 Sunrise ELISA reader(Tecan)를 이용하여 450 nm에서 흡광도를 측정하여 목적으로 하는 불활화된 SFTS 바이러스 항원에 반응성이 높은 하이브리도마 세포주들을 선별하였다.Then, 100 ul of the hybridoma cell culture medium was added to each well, reacted at room temperature for 1 hour, and washed three times with a PBS-T solution to remove unreacted culture medium. Goat anti-mouse IgG-HRP was added thereto, reacted at room temperature for 1 hour, and then washed three times with PBS-T. After washing, peroxidase substrate solution (TMB) was added for reaction, 0.5 M sulfuric acid was dispensed into each well of the plate in an amount of 50 ul, and absorbance was measured at 450 nm using a Sunrise ELISA reader (Tecan) to obtain the desired Hybridoma cell lines highly reactive to the inactivated SFTS virus antigen were selected.
선별된 하이브리도마 세포주를 제한 희석하여 클로닝을 진행하여 단클론화 하였고, 이후 위와 같은 방법으로 목적 항체를 분비하는 하이브리도마 세포주를 선별하였다. 위와 같은 방법으로 불활화된 SFTS 바이러스에 특이적으로 반응하는 2종의 단클론항체(Monoclonal antibody; mAb) 2D10 및 3A10이 하기 표 1과 같이 선발되었다.The selected hybridoma cell line was monocloned by cloning by limited dilution, and then a hybridoma cell line secreting the target antibody was selected in the same manner as above. Two types of monoclonal antibodies (mAb) 2D10 and 3A10 that specifically reacted to the SFTS virus inactivated in the above manner were selected as shown in Table 1 below.
표 2에서 확인할 수 있듯이, 불활화된 SFTS 바이러스 항원에 특이적으로 반응하고, 면역 글로불린 아형 IgG2a인 단클론항체 2D10 및 3A10을 생산할 수 있는 하이브리도마 세포주를 선발하여 한국세포주은행에 각각 수탁번호 KCTC18912P, KCTC18913P로 기탁하였다.As can be seen in Table 2, hybridoma cell lines capable of reacting specifically to the inactivated SFTS virus antigen and producing monoclonal antibodies 2D10 and 3A10, which are immunoglobulin subtype IgG2a, were selected, and accession numbers KCTC18912P and Deposited as KCTC18913P.
실시예 2: 래피드 진단 키트의 제작Example 2: Fabrication of Rapid Diagnostic Kit
실시예 1에서 선발된 단클론항체 2종을 사용하여 래피드 진단 키트를 제조하였다. 골드 콘쥬게이트는 항체 및 골드(gold) 입자의 결합체를 뜻하며 콘쥬게이트 제조 후 흡착 방법을 이용하였다.A rapid diagnostic kit was prepared using the two monoclonal antibodies selected in Example 1. The gold conjugate refers to a conjugate of an antibody and gold particles, and an adsorption method was used after preparing the conjugate.
2-1. 고정항체의 분주 및 고정2-1. Dispensing and immobilization of immobilized antibody
항-말라리아 항체(Anti-malaria Mab 12D85(메디안디노스틱; Cat.No. 12003)) 1 mg/ml과 SFTS mAb 2D10 1.5 mg/ml이 되도록 각각 1 x PBS(Phosphate buffered saline)에 넣고, 양 쪽 모두 0.5%(v/v) 수크로오스(sucrose)와 0.1%(v/v) NaN3를 첨가하여 항체 용액을 준비하였다. 이어서 백킹 카드(backing card)에 멤브레인을 붙이고 대조선(control line)에는 항-말라리아 항체 용액을, 검사선(test line)에는 2D10 항체 용액을 분주하였다. 그 후 제습 캐비닛에서 24시간 이상 건조하여 항체를 멤브레인에 고정시켰다.Anti-malaria Mab 12D85 (Mediandinostik; Cat.No. 12003) 1 mg/ml and SFTS mAb 2D10 1.5 mg/ml were added to 1 x PBS (Phosphate buffered saline), respectively, and both sides Antibody solutions were prepared by adding 0.5% (v/v) sucrose and 0.1% (v/v) NaN 3 . Subsequently, the membrane was attached to a backing card, and an anti-malaria antibody solution was dispensed on a control line and a 2D10 antibody solution was dispensed on a test line. Thereafter, the antibody was fixed to the membrane by drying for more than 24 hours in a dehumidifying cabinet.
2-2. 검출항체의 준비2-2. Preparation of detection antibody
구체적으로, 상기 실시예 1-4에 따라 정제 완료된 SFTS 3A10 항체를 준비하였다. 콜로이달 골드(colloidal gold)를 분광광도계(Spectrophotometer)를 이용하여 525 nm 파장에서 OD 값 11이 되도록 만들어 골드 용액(gold solution)을 준비하였다.Specifically, the purified SFTS 3A10 antibody was prepared according to Examples 1-4. A gold solution was prepared by making colloidal gold to have an OD value of 11 at a wavelength of 525 nm using a spectrophotometer.
검사선에서의 검출을 위한 SFTS 3A10 항체 골드 콘쥬게이트를 제조하기 위하여 0.2 M K2CO3를 5 ul/ml 넣은 골드 용액에 준비한 SFTS 3A10 항체를 20 ug/ml이 되도록 섞고 상온에서 3분 30초간 반응시켰다. 동일한 방법으로 대조선에서의 검출을 위하여 말라리아 항원으로 콘쥬게이트된 말라리아 항원 골드 콘쥬게이트 또한 제조하였다.To prepare the SFTS 3A10 antibody gold conjugate for detection on the test line, mix the prepared SFTS 3A10 antibody in a gold solution containing 5 ul/ml of 0.2 MK 2 CO 3 to 20 ug/ml and react at room temperature for 3 minutes and 30 seconds made it In the same manner, a malaria antigen gold conjugate conjugated to the malaria antigen was also prepared for detection in a control line.
제조된 골드 콘쥬게이트에서 SFTS 3A10 항체와 반응하지 못한 부분을 블로킹(Blocking)하기 위해 소 혈청 알부민(Bovine Serum Albumin; BSA)을 0.5%(v/v)의 농도가 되도록 첨가 후 볼텍싱(voltexing)하여 1분간 반응시켰다.In the prepared gold conjugate, bovine serum albumin (BSA) was added to a concentration of 0.5% (v / v) to block the part that did not react with the SFTS 3A10 antibody, followed by vortexing and reacted for 1 minute.
이후 5%(v/v) 수크로오스를 더 첨가하고, 골드 콘쥬게이트를 분광광도계 OD 값 기준 7이 되도록 골드 희석 버퍼(2 mM Tris-base buffer)에 넣어 폴리에틸렌 패드(polyethylene pad)에 흡착시킨 후 제습 공간에서 건조하였다.After that, 5% (v/v) sucrose was further added, and the gold conjugate was put in a gold dilution buffer (2 mM Tris-base buffer) so that the OD value of the spectrophotometer was 7, adsorbed on a polyethylene pad, and then dehumidified. dried in space.
2-3. 스트립의 제조 및 조립2-3. Manufacture and assembly of strips
스트립(strip)은 샘플 패드, 골드 콘쥬게이트 패드, 멤브레인, 흡수 패드를 중첩되게 하여 일정한 방향으로 흘러가면서 고정(capture)항체와 반응하도록 하는 형태를 갖는다.The strip has a shape in which the sample pad, the gold conjugate pad, the membrane, and the absorption pad are overlapped so that the strip flows in a certain direction and reacts with the capture antibody.
멤브레인의 검사선 위치에 고정항체를 고정시킨 후 한 쪽 말단에 골드 콘쥬게이트 패드 및 샘플 패드, 다른 쪽 말단에 흡수 패드를 각각 정해진 위치에 중첩되도록 부착하고 로터리 슬리터(rotary slitter)를 이용하여 4 mm 간격으로 절단(cutting)하였다.After immobilizing the immobilized antibody at the position of the test line on the membrane, attach the gold conjugate pad and sample pad to one end and the absorption pad to the other end so that they overlap each other at predetermined positions, and use a rotary slitter to 4 It was cut at intervals of mm.
완성된 스트립을 하우징에 넣어 조립하고 알루미늄 파우치에 실리카겔과 함께 밀봉하여 포장하였다. 검출 용액으로는 50 mM 보랙스 버퍼(borax buffer, pH 9.2)에 1%(v/v) Tween-20, 0.01%(v/v) NaN3를 넣어 제조하였다.The completed strip was put into a housing, assembled, and sealed with silica gel in an aluminum pouch to be packaged. The detection solution was prepared by adding 1% (v/v) Tween-20 and 0.01% (v/v) NaN 3 to 50 mM borax buffer (pH 9.2).
실시예 3: 진단키트의 사용Example 3: Use of diagnostic kit
3-1. 진단키트의 검출능 확인3-1. Checking the detection ability of the diagnostic kit
실시예 2에서 제조된 래피드 진단 키트를 이용하여 SFTS 바이러스와의 반응성을 아래와 같은 과정에 따라 확인할 수 있다.Using the rapid diagnostic kit prepared in Example 2, reactivity with the SFTS virus can be confirmed according to the following procedure.
a. 시험(test)하고자 하는 샘플 20 μl(전혈) 또는 10 μl(혈장 또는 혈청)을 진단 키트의 샘플 투입구에 투입시킨다.a. 20 μl (whole blood) or 10 μl (plasma or serum) of the sample to be tested is put into the sample inlet of the diagnostic kit.
b. 검출 용액을 샘플 투입구에 수직으로 3방울(100 μl) 적하한다.b. 3 drops (100 μl) of the detection solution are dropped vertically into the sample inlet.
c. 상온에서 10분 동안 반응시킨 후 대조선과 검사선 위치에 붉은 색 선이 나타나면 양성, 대조선에만 붉은 선이 나타나면 음성으로 육안 판정한다. 대조선에 붉은 색 선이 나타나지 않는 경우 재실험한다.c. After reacting at room temperature for 10 minutes, if red lines appear at the control and test lines, it is positive, and if red lines appear only on the control line, it is visually determined as negative. If the red line does not appear on the control line, repeat the test.
상기 진단 키트 사용 방법에 따라 하기 표 3과 같은 A, B, D, 및 F 각 유형(type)의 SFTS 바이러스 배양액 및 SFTS 음성 희석액인 음성 개 혈청 및 전혈을 준비하고 래피드 진단키트의 용법용량에 따라 시험하였다.According to the method of using the diagnostic kit, SFTS virus culture medium of each type of A, B, D, and F as shown in Table 3 below and SFTS negative dilution, negative dog serum and whole blood were prepared, and according to the dosage of the rapid diagnostic kit tested.
(TCID50/0.1mL)virus titer
(TCID 50 /0.1mL)
표 3 및 도 1에서 확인할 수 있듯이, 모든 SFTS 바이러스형(type)에서 래피드 진단키트가 양성으로 관찰되었다.As can be seen in Table 3 and FIG. 1, the rapid diagnostic kit was observed positively in all SFTS virus types.
3-2. 교차반응성 평가3-2. Cross-reactivity evaluation
SFTS 바이러스 외에 매개체성 질병을 일으키는 다른 바이러스들과의 교차반응을 확인하기 위해 래피드 진단 키트를 이용하여 시험하였다.In addition to the SFTS virus, it was tested using a rapid diagnostic kit to confirm cross-reactivity with other viruses that cause vector-borne diseases.
표 4에서 확인할 수 있듯이, 시험 결과 다른 매개체성 질병을 일으키는 다른 바이러스들과의 교차반응은 없는 것으로 측정되었다.As can be seen in Table 4, as a result of the test, it was determined that there was no cross-reaction with other viruses causing other vector-borne diseases.
<110> MEDIAN Diagnostics Inc. REPUBLIC OF KOREA(Animal and Plant Quarantine Agency) <120> Kit for diagnosis of Severe Fever with Thrombocytopenia Syndrome virus infection <130> PN210350 <160> 24 <170> KoPatentIn 3.0 <210> 1 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> 2D10, 3A10 light chain Variable region DNA <400> 1 gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60 atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120 caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180 ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240 cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300 tcggaggggg gaccaagctg gaaataaaac 330 <210> 2 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> 2D10, 3A10 light chain Variable region amino acid <400> 2 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg 85 90 95 Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys *** Asn 100 105 110 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 2D10, 3A10 light chain CDR-L1 DNA <400> 3 aaaagtgtca gtacatctgg ctatagttat 30 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 2D10, 3A10 light chain CDR-L1 amino acid <400> 4 Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr 1 5 10 <210> 5 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> 2D10, 3A10 light chain CDR-L2 DNA <400> 5 cttgtatcc 9 <210> 6 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> 2D10, 3A10 light chain CDR-L2 amino acid <400> 6 Leu Val Ser 1 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 2D10, 3A10 light chain CDR-L3 DNA <400> 7 cagcacatta gggagcttac acg 23 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 2D10, 3A10 light chain CDR-L3 amino acid <400> 8 Gln His Ile Arg Glu Leu Thr 1 5 <210> 9 <211> 355 <212> DNA <213> Artificial Sequence <220> <223> 2D10 heavy chain Variable region DNA <400> 9 caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggggcttc agtgaggctg 60 tcctgcaagg cttctggcta caccttcact aactactgga tgaactgggt gatacagagg 120 cctggacaag gccttgagtg gatcggaaat atttttcctt ctgacagtta tactgactac 180 aatctaaagt tcaagaacaa ggccacattg actgtagaca aatcctccag cacagcctac 240 atgcaactca gcagcccgac atctgaagac tctgcggtct attactgtac cttcggttac 300 gacgggtggt acttcgatgt ctggggcgca gggaccacgg tcaccgtctc ctcag 355 <210> 10 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> 2D10 heavy chain Variable region amino acid <400> 10 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met Asn Trp Val Ile Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Phe Pro Ser Asp Ser Tyr Thr Asp Tyr Asn Leu Lys Phe 50 55 60 Lys Asn Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Phe Gly Tyr Asp Gly Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 2D10 heavy chain CDR-H1 DNA <400> 11 ggctacacct tcactaacta ctgg 24 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 2D10 heavy chain CDR-H1 amino acid <400> 12 Gly Tyr Thr Phe Thr Asn Tyr Trp 1 5 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 2D10 heavy chain CDR-H2 DNA <400> 13 atttttcctt ctgacagtta tact 24 <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 2D10 heavy chain CDR-H2 amino acid <400> 14 Ile Phe Pro Ser Asp Ser Tyr Thr 1 5 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> 2D10 heavy chain CDR-H3 DNA <400> 15 accttcggtt acgacgggtg gtacttcgat gtc 33 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 2D10 heavy chain CDR-H3 amino acid <400> 16 Thr Phe Gly Tyr Asp Gly Trp Tyr Phe Asp Val 1 5 10 <210> 17 <211> 358 <212> DNA <213> Artificial Sequence <220> <223> 3A10 heavy chain Variable region DNA <400> 17 gaagtgaagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cgctttcagt agctatgaca tgtcttgggt tcgccagact 120 ccggagaaga ggctggagtg ggtcgcagtc attagtagtg gtggtaatta cacctactgt 180 ccagacagtt tgaagggccg attcaccatc tccagagaca atgccaggaa caccctgtac 240 ctgcaaatga gcagtctgag gtctgaggac acggccttgt attactgtgg aagacaagat 300 ggtaactacg gagggtttcc ttactggggc caagggactc tggtcactgt ctctgcag 358 <210> 18 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> 3A10 heavy chain Variable region amino acid <400> 18 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Ser Gly Gly Asn Tyr Thr Tyr Cys Pro Asp Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Gly Arg Gln Asp Gly Asn Tyr Gly Gly Phe Pro Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 3A10 heavy chain CDR-H1 DNA <400> 19 ggattcgctt tcagtagcta tgac 24 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 3A10 heavy chain CDR-H1 amino acid <400> 20 Gly Phe Ala Phe Ser Ser Tyr Asp 1 5 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 3A10 heavy chain CDR-H2 DNA <400> 21 attagtagtg gtggtaatta cacc 24 <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 3A10 heavy chain CDR-H2 amino acid <400> 22 Ile Ser Ser Gly Gly Asn Tyr Thr 1 5 <210> 23 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 3A10 heavy chain CDR-H3 DNA <400> 23 ggaagacaag atggtaacta cggagggttt ccttac 36 <210> 24 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> 3A10 heavy chain CDR-H3 amino acid <400> 24 Gly Arg Gln Asp Gly Asn Tyr Gly Gly Phe Pro Tyr 1 5 10 <110> MEDIAN Diagnostics Inc. REPUBLIC OF KOREA (Animal and Plant Quarantine Agency) <120> Kit for diagnosis of Severe Fever with Thrombocytopenia Syndrome virus infection <130> PN210350 <160> 24 <170> KoPatentIn 3.0 <210> 1 <211> 330 <212> DNA <213> artificial sequence <220> <223> 2D10, 3A10 light chain variable region DNA <400> 1 gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60 atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120 caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180 ggggtccctg ccaggttcag tggcagtggg tctggggacag acttcaccct caacatccat 240 cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300 tcggaggggg gaccaagctg gaaataaaac 330 <210> 2 <211> 110 <212> PRT <213> artificial sequence <220> <223> 2D10, 3A10 light chain variable region amino acid <400> 2 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg 85 90 95 Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys *** Asn 100 105 110 <210> 3 <211> 30 <212> DNA <213> artificial sequence <220> <223> 2D10, 3A10 light chain CDR-L1 DNA <400> 3 aaaagtgtca gtacatctgg ctatagttat 30 <210> 4 <211> 10 <212> PRT <213> artificial sequence <220> <223> 2D10, 3A10 light chain CDR-L1 amino acid <400> 4 Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr 1 5 10 <210> 5 <211> 9 <212> DNA <213> artificial sequence <220> <223> 2D10, 3A10 light chain CDR-L2 DNA <400> 5 cttgtatcc 9 <210> 6 <211> 3 <212> PRT <213> artificial sequence <220> <223> 2D10, 3A10 light chain CDR-L2 amino acid <400> 6 Leu Val Ser One <210> 7 <211> 23 <212> DNA <213> artificial sequence <220> <223> 2D10, 3A10 light chain CDR-L3 DNA <400> 7 cagcacatta gggagcttac acg 23 <210> 8 <211> 7 <212> PRT <213> artificial sequence <220> <223> 2D10, 3A10 light chain CDR-L3 amino acid <400> 8 Gln His Ile Arg Glu Leu Thr 1 5 <210> 9 <211> 355 <212> DNA <213> artificial sequence <220> <223> 2D10 heavy chain variable region DNA <400> 9 caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggggcttc agtgaggctg 60 tcctgcaagg cttctggcta caccttcact aactactgga tgaactgggt gatacagagg 120 cctggacaag gccttgagtg gatcggaaat atttttcctt ctgacagtta tactgactac 180 aatctaaagt tcaagaacaa ggccacattg actgtagaca aatcctccag cacagcctac 240 atgcaactca gcagcccgac atctgaagac tctgcggtct attactgtac cttcggttac 300 gacgggtggt acttcgatgt ctggggcgca gggaccacgg tcaccgtctc ctcag 355 <210> 10 <211> 118 <212> PRT <213> artificial sequence <220> <223> 2D10 heavy chain variable region amino acid <400> 10 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met Asn Trp Val Ile Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Phe Pro Ser Asp Ser Tyr Thr Asp Tyr Asn Leu Lys Phe 50 55 60 Lys Asn Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Phe Gly Tyr Asp Gly Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 11 <211> 24 <212> DNA <213> artificial sequence <220> <223> 2D10 heavy chain CDR-H1 DNA <400> 11 ggctaccct tcactaacta ctgg 24 <210> 12 <211> 8 <212> PRT <213> artificial sequence <220> <223> 2D10 heavy chain CDR-H1 amino acid <400> 12 Gly Tyr Thr Phe Thr Asn Tyr Trp 1 5 <210> 13 <211> 24 <212> DNA <213> artificial sequence <220> <223> 2D10 heavy chain CDR-H2 DNA <400> 13 atttttcctt ctgacagtta tact 24 <210> 14 <211> 8 <212> PRT <213> artificial sequence <220> <223> 2D10 heavy chain CDR-H2 amino acid <400> 14 Ile Phe Pro Ser Asp Ser Tyr Thr 1 5 <210> 15 <211> 33 <212> DNA <213> artificial sequence <220> <223> 2D10 heavy chain CDR-H3 DNA <400> 15 accttcggtt acgacgggtg gtacttcgat gtc 33 <210> 16 <211> 11 <212> PRT <213> artificial sequence <220> <223> 2D10 heavy chain CDR-H3 amino acid <400> 16 Thr Phe Gly Tyr Asp Gly Trp Tyr Phe Asp Val 1 5 10 <210> 17 <211> 358 <212> DNA <213> artificial sequence <220> <223> 3A10 heavy chain variable region DNA <400> 17 gaagtgaagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cgctttcagt agctatgaca tgtcttgggt tcgccagact 120 ccggagaaga ggctggagtg ggtcgcagtc attagtagtg gtggtaatta cacctactgt 180 ccagacagtt tgaagggccg attcaccatc tccagagaca atgccaggaa caccctgtac 240 ctgcaaatga gcagtctgag gtctgaggac acggccttgt attactgtgg aagacaagat 300 ggtaactacg gagggtttcc ttactggggc caagggactc tggtcactgt ctctgcag 358 <210> 18 <211> 119 <212> PRT <213> artificial sequence <220> <223> 3A10 heavy chain variable region amino acid <400> 18 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Ser Gly Gly Asn Tyr Thr Tyr Cys Pro Asp Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Gly Arg Gln Asp Gly Asn Tyr Gly Gly Phe Pro Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 19 <211> 24 <212> DNA <213> artificial sequence <220> <223> 3A10 heavy chain CDR-H1 DNA <400> 19 ggattcgctt tcagtagcta tgac 24 <210> 20 <211> 8 <212> PRT <213> artificial sequence <220> <223> 3A10 heavy chain CDR-H1 amino acid <400> 20 Gly Phe Ala Phe Ser Ser Tyr Asp 1 5 <210> 21 <211> 24 <212> DNA <213> artificial sequence <220> <223> 3A10 heavy chain CDR-H2 DNA <400> 21 attagtagtg gtggtaatta cacc 24 <210> 22 <211> 8 <212> PRT <213> artificial sequence <220> <223> 3A10 heavy chain CDR-H2 amino acid <400> 22 Ile Ser Ser Gly Gly Asn Tyr Thr 1 5 <210> 23 <211> 36 <212> DNA <213> artificial sequence <220> <223> 3A10 heavy chain CDR-H3 DNA <400> 23 ggaagacaag atggtaacta cggagggttt ccttac 36 <210> 24 <211> 12 <212> PRT <213> artificial sequence <220> <223> 3A10 heavy chain CDR-H3 amino acid <400> 24 Gly Arg Gln Asp Gly Asn Tyr Gly Gly Phe Pro Tyr 1 5 10
Claims (10)
서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 18의 아미노산 서열로 이루어진 중쇄가변영역
을 포함하는 단클론항체(Monoclonal antibody; mAb).a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10; or
The light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18
A monoclonal antibody (mAb) including a.
서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 18의 아미노산 서열로 이루어진 중쇄가변영역
을 포함하는 단클론항체(Monoclonal antibody; mAb)를 포함하는 중증 열성 혈소판 감소 증후군(Severe Fever with Thrombocytopenia Syndrome; SFTS) 바이러스 검출용 조성물.a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10; or
The light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18
A composition for detecting Severe Fever with Thrombocytopenia Syndrome (SFTS) virus comprising a monoclonal antibody (mAb) comprising a.
서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 18의 아미노산 서열로 이루어진 중쇄가변영역
을 포함하는 단클론항체(Monoclonal antibody; mAb)를 포함하는 중증 열성 혈소판 감소 증후군(Severe Fever with Thrombocytopenia Syndrome; SFTS) 바이러스 검출용 조성물을 포함하는 SFTS 바이러스 감염 진단용 키트.a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10; or
The light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18
A kit for diagnosing SFTS virus infection comprising a composition for detecting Severe Fever with Thrombocytopenia Syndrome (SFTS) virus including a monoclonal antibody (mAb) comprising a.
서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 10의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 고정항체(capture mAb); 및
서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 18의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 검출항체(detector Ab)
를 포함하는 것인, SFTS 바이러스 감염 진단용 키트.The method of claim 6, wherein the kit
A fixed antibody (capture mAb) comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10; and
Detector Ab comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18
A kit for diagnosing SFTS virus infection comprising a.
서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 10의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 고정항체(capture mAb)로 코팅된 고정체에 시료를 접촉시키는 반응 단계; 및
상기 고정체에 서열번호 2의 아미노산 서열로 이루어진 경쇄가변영역 및 서열번호 18의 아미노산 서열로 이루어진 중쇄가변영역을 포함하는 검출항체(detector Ab)를 반응시키는 검출 단계.How to provide information for the diagnosis of Severe Fever with Thrombocytopenia Syndrome (SFTS) viral infection, including:
A reaction step of contacting a sample to a stationary body coated with a capture mAb comprising a light chain variable region composed of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region composed of the amino acid sequence of SEQ ID NO: 10; and
A detection step of reacting a detection antibody (detector Ab) comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18 to the fixed body.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20160054161A (en) * | 2014-11-05 | 2016-05-16 | 대한민국(농림축산식품부 농림축산검역본부장) | Production and application of a monoclonal antibody and the development of a competitive enzyme-linked immunosorbent assay(c-ELISA) for detection of severe fever with thrombocytopenia syndrome virus(SFTSV) |
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