KR102368133B1 - Rapid Detection Method of COVID-19 Immunoglobulin G - Google Patents

Rapid Detection Method of COVID-19 Immunoglobulin G Download PDF

Info

Publication number
KR102368133B1
KR102368133B1 KR1020200053003A KR20200053003A KR102368133B1 KR 102368133 B1 KR102368133 B1 KR 102368133B1 KR 1020200053003 A KR1020200053003 A KR 1020200053003A KR 20200053003 A KR20200053003 A KR 20200053003A KR 102368133 B1 KR102368133 B1 KR 102368133B1
Authority
KR
South Korea
Prior art keywords
covid
virus
immunoglobulin
ser
leu
Prior art date
Application number
KR1020200053003A
Other languages
Korean (ko)
Other versions
KR20210134452A (en
Inventor
윤성준
윤경원
설재구
이진우
강상현
Original Assignee
주식회사 인트론바이오테크놀로지
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 주식회사 인트론바이오테크놀로지 filed Critical 주식회사 인트론바이오테크놀로지
Priority to KR1020200053003A priority Critical patent/KR102368133B1/en
Publication of KR20210134452A publication Critical patent/KR20210134452A/en
Application granted granted Critical
Publication of KR102368133B1 publication Critical patent/KR102368133B1/en

Links

Images

Classifications

    • 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
    • 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
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • 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
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

본 발명은 COVID-19 바이러스에 대한 항체 보유 여부 조사나 COVID-19 바이러스에 대한 노출 동향을 모니터링하는 목적으로 활용할 수 있는, 재조합 COVID-19 바이러스 항원과 항-인간 면역글로불린 G 항체를 이용한 면역크로마토그래피법에 기반한 항원 처리된 2개의 검사선을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G를 검출하는 신속검출기술 및 신속검출 진단키트에 관한 것이다. The present invention is immunochromatography using recombinant COVID-19 virus antigen and anti-human immunoglobulin G antibody, which can be used for the purpose of investigating whether or not possessing antibodies to the COVID-19 virus or monitoring exposure trends to the COVID-19 virus It relates to a rapid detection technology and rapid detection diagnostic kit that detects immunoglobulin G for COVID-19 virus, which is characterized by two antigen-treated test lines based on the law.

Description

COVID-19 바이러스에 대한 면역글로불린 G 신속검출기술{Rapid Detection Method of COVID-19 Immunoglobulin G}Rapid Detection Method of COVID-19 Immunoglobulin G

본 발명은 COVID-19 바이러스에 대한 면역글로불린 G를 검출하는 신속검출기술에 관한 것으로, 더욱 상세하게는 재조합 COVID-19 바이러스 항원과 항-인간 면역글로불린 G 항체를 이용한 면역크로마토그래피법에 기반한 항원 처리된 2개의 검사선을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 기술 및 이에 활용될 수 있는 신속검출 진단키트에 관한 것이다.The present invention relates to a rapid detection technology for detecting immunoglobulin G against COVID-19 virus, and more particularly, antigen processing based on immunochromatography using recombinant COVID-19 virus antigen and anti-human immunoglobulin G antibody. It relates to a rapid detection technology for immunoglobulin G for COVID-19 virus, characterized by two test lines, and a rapid detection diagnostic kit that can be used therefor.

최근 중증급성호흡기증후군 코로나바이러스 2 (Severe acute respiratory syndrome coronavirus 2; SARS-CoV-2; 이하 ‘COVID-19 바이러스’라 함)에 의한 감염질환 (코로나바이러스감염증-19; 이하 ‘COVID-19’라 함)이 전세계적으로 큰 문제가 되고 있다. Recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; hereinafter referred to as 'COVID-19 virus') caused by an infectious disease (coronavirus infection-19; hereinafter referred to as 'COVID-19') ) is becoming a global problem.

COVID-19는 2019년 11월 중국 후베이성 우한시에서 시작된 이래 전세계적으로 확산되면서 보건사회적 뿐만 아니라 경제적으로도 큰 문제를 초래하고 있다. COVID-19는 Coronaviridae에 속하는 RNA 바이러스인 SARS-CoV-2, 즉 COVID-19 바이러스 감염에 의한 호흡기 감염증을 의미한다. 전파경로로는 비말 (침방울) 및 접촉을 통해 전파된다고 알려져 있으며, 상세하게는 기침이나 재채기를 할 때 생긴 비말을 통해 전파되거나 COVID-19 바이러스에 오염된 물건을 만진 뒤 눈, 코, 입을 만져서 전파된다고 알려져 있다.Since COVID-19 started in Wuhan City, Hubei Province, China in November 2019, it has spread worldwide, causing great problems not only in health and society but also economically. COVID-19 refers to respiratory infection caused by SARS-CoV-2, that is, COVID-19 virus infection, which is an RNA virus belonging to Coronaviridae. It is known that the transmission route is through droplets (saliva) and contact. Specifically, it is spread through droplets generated when coughing or sneezing, or by touching an object contaminated with the COVID-19 virus and then touching your eyes, nose, and mouth. is known to be

COVID-19 바이러스의 잠복기는 1~14일 (평균 4~7일)이며, 검체에서 COVID-19 바이러스가 분리되거나 검체에서 COVID-19 바이러스의 특이 유전자가 검출될 때 감염이 되었다고 진단한다. COVID-19에 의해 나타나는 일반적인 증상으로는 발열, 권태감, 기침, 호흡곤란 및 폐렴 등 경증에서 중증까지 다양한 호흡기 감염증이 있으며, 그 외에 가래, 인후통, 두통, 객혈과 오심, 설사 등도 나타난다.The incubation period of the COVID-19 virus is 1 to 14 days (average 4 to 7 days), and infection is diagnosed when the COVID-19 virus is isolated from the sample or a specific gene for the COVID-19 virus is detected in the sample. Common symptoms caused by COVID-19 include various respiratory infections ranging from mild to severe, such as fever, malaise, cough, shortness of breath, and pneumonia.

현재까지 알려진 치료법으로는 수액 보충, 해열제 등의 대증 치료가 보조수단으로 사용되고 있으나, COVID-19 바이러스에 대응 가능한 백신이나 항바이러스제는 개발되지 않은 상황이다. 세계보건기구 (World Health Organization; WHO)에 의하면 COVID-19로 인한 전세계 치명률은 약 3.4%이며, 고령, 면역기능이 저하된 환자, 기저질환을 가진 환자들이 주로 중증에 걸리거나 사망에 이르게 된다. Symptomatic treatments such as fluid supplementation and fever-reducing drugs are used as auxiliary means as known treatments, but vaccines or antiviral agents that can respond to the COVID-19 virus have not been developed. According to the World Health Organization (WHO), the global fatality rate due to COVID-19 is about 3.4%, and the elderly, immunocompromised patients, and patients with underlying diseases mainly become seriously ill or die.

아직도 COVID-19가 확산 일로에 있지만 우리는 다시 일상을 준비할 필요가 있다. 이러한 측면에서 COVID-19에 노출된 이력이 있어 COVID-19 바이러스에 대한 항체를 가지고 있는 사람들을 신속하고 정확하며 용이하게 판별 및 구분할 수 있는 기술이 필요하다. 이는 COVID-19 바이러스에 대한 방어력을 가진 사람들을 선별하기 위해서도 필요하며, 방역 측면에서 COVID-19 바이러스에 노출된 사람의 분포 정도를 모니터링하여 적절한 정책을 수립하기 위해서도 필요하다.Although COVID-19 is still spreading, we need to prepare for our daily life again. In this respect, there is a need for a technology that can quickly, accurately, and easily identify and distinguish people who have antibodies to the COVID-19 virus with a history of exposure to COVID-19. This is necessary in order to screen those who have defenses against the COVID-19 virus, and in terms of quarantine, it is necessary to establish an appropriate policy by monitoring the distribution of people exposed to the COVID-19 virus.

한편, 방어면역 측면을 개략적으로 파악하거나 방역 정책 수립을 위해서는 COVID-19 바이러스에 대한 면역글로불린 (Immunoglobulin)을 신속하고 정확하게 검출할 수 있는 기술이 필요하다. 면역글로불린 중에서도 면역글로불린 M (Immunoglobulin M; IgM)은 감염 초기에만 검출될 수 있는 면역글로불린이므로 그 활용성이 제한적이다. 반면, 면역글로불린 G (Immunoglobulin G; IgG)는 항체 중 큰 비율을 차지하며, 감염 초기부터 생성되기 시작하여 감염 후기까지 지속적으로 증가하므로 면역글로불린 G를 신속하고 정확하게 검출할 수 있는 진단기술 및 진단키트의 개발은 매우 중요하다.On the other hand, in order to outline the aspects of defense immunity or establish quarantine policies, a technology capable of rapidly and accurately detecting immunoglobulin against the COVID-19 virus is required. Among immunoglobulins, immunoglobulin M (Immunoglobulin M; IgM) is an immunoglobulin that can be detected only in the early stages of infection, so its utility is limited. On the other hand, immunoglobulin G (Immunoglobulin G; IgG) accounts for a large proportion of antibodies, and is produced from the early stage of infection and continues to increase until late infection. development is very important.

한편, 면역글로불린 G는 폴리클로날 항체 (Polyclonal antibody)이므로 위양성 (False-positive)과 위음성 (False-negative)의 문제 발생 가능성이 높아 검출 정확도가 높은 진단기술 및 진단키트의 개발이 필요하다. 이에 더하여 바이러스 변종에 대한 대비도 가능하다면 더욱 바람직하다 할 것이다.On the other hand, since immunoglobulin G is a polyclonal antibody, there is a high possibility of false-positive and false-negative problems, so it is necessary to develop a diagnostic technology and diagnostic kit with high detection accuracy. In addition to this, it would be more preferable if possible to prepare for virus variants.

이에, COVID-19 바이러스에 대한 면역글로불린 G를 신속검출하는 기술로서, 본 발명자들은 재조합 COVID-19 바이러스 항원과 항-인간 면역글로불린 G 항체를 이용한 면역크로마토그래피법 (Immunochromatography)에 기반한 항원 처리된 2개의 검사선 (Test line)을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G를 검출하는 신속검출기술을 제공하고자 한다.Accordingly, as a technology for rapid detection of immunoglobulin G against COVID-19 virus, the present inventors have prepared antigen-treated 2 based on immunochromatography using recombinant COVID-19 virus antigen and anti-human immunoglobulin G antibody. It is intended to provide a rapid detection technology that detects immunoglobulin G against the COVID-19 virus, which is characterized by a test line in dogs.

따라서, 본 발명의 목적은 COVID-19 바이러스에 대한 면역글로불린 G를 검출하는 신속검출기술을 제공하는 것이다.Accordingly, an object of the present invention is to provide a rapid detection technology for detecting immunoglobulin G against the COVID-19 virus.

본 발명의 다른 목적은 상기 COVID-19 바이러스에 대한 면역글로불린 G를 검출하는 신속검출기술을 구현하는 데에 활용될 수 있는, 항원 처리된 2개의 검사선을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출용 진단키트를 제공하는 것이다.Another object of the present invention is immunity to COVID-19 virus characterized by two antigen-treated test lines that can be utilized to implement a rapid detection technology for detecting immunoglobulin G for the COVID-19 virus To provide a diagnostic kit for rapid detection of globulin G.

상기 목적들을 달성하고자, 본 발명의 발명자들은 공지의 정보를 활용하여 COVID-19 바이러스에 대한 면역글로불린 G를 검출하는 목적으로 활용될 수 있는 COVID-19 바이러스 특이 항원 (Antigen)들을 확보하고, COVID-19 바이러스에 대한 면역글로불린 G를 검출하는 목적으로 활용될 수 있는 항-인간 면역글로불린 G 항체를 확보하고, 상기 확보한 COVID-19 바이러스 특이 항원들과 항-인간 면역글로불린 G 항체를 이용하여 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 목적의 항원 처리된 2개의 검사선을 특징으로 하는 검출용 진단키트를 개발하였고, 상기 검출용 진단키트가 COVID-19 바이러스에 대한 면역글로불린 G를 신속하게 검출하는 데에 효과적으로 활용될 수 있음을 확인함으로써 본 발명을 완성하였다.In order to achieve the above objects, the inventors of the present invention use known information to secure COVID-19 virus-specific antigens (Antigen) that can be used for the purpose of detecting immunoglobulin G for COVID-19 virus, and 19 Obtaining an anti-human immunoglobulin G antibody that can be used for the purpose of detecting immunoglobulin G against the virus, and using the obtained COVID-19 virus-specific antigens and anti-human immunoglobulin G antibody 19 We have developed a detection diagnostic kit featuring two antigen-treated test lines for the purpose of rapid detection of immunoglobulin G against virus, and the detection kit is capable of rapidly detecting immunoglobulin G for COVID-19 virus. The present invention was completed by confirming that it can be effectively utilized for

따라서, 본 발명의 일 양태에 따르면, 본 발명은 COVID-19 바이러스에 대한 면역글로불린 G를 검출하는 데에 활용될 수 있는 COVID-19 바이러스 특이 항원으로 서열번호 1 내지 서열번호 4의 아미노산 서열을 갖는 COVID-19 바이러스 특이 항원 단백질을 제공한다. 상기 COVID-19 바이러스 특이 항원 단백질들은 통상의 재조합 단백질 제조기술을 이용하여 자체적으로 제조하여 이용할 수도 있고 또한 상업적으로 구입하여 활용할 수도 있다. Therefore, according to one aspect of the present invention, the present invention is a COVID-19 virus-specific antigen that can be utilized to detect immunoglobulin G for COVID-19 virus having the amino acid sequence of SEQ ID NOs: 1 to 4 Provides a COVID-19 virus-specific antigen protein. The COVID-19 virus-specific antigen proteins may be prepared and used by themselves using conventional recombinant protein production techniques, or may be commercially purchased and used.

본 발명의 다른 일 양태에 따르면, 본 발명은 서열번호 1내지 서열번호 4로 표시되는 아미노산 서열로 특징지어지는 COVID-19 바이러스 특이 항원을 활용하여 제작되어진, 항원 처리된 2개의 검사선을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트를 제공한다. According to another aspect of the present invention, the present invention is characterized by two antigen-treated test lines prepared using a COVID-19 virus-specific antigen characterized by the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 4 Provides a diagnostic kit for rapid detection of immunoglobulin G against the COVID-19 virus.

본 발명의 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트에 포함되는 항-인간 면역글로불린 G 항체는 자체적으로 제조하여 이용할 수도 있고 또한 상업적으로 구입하여 활용할 수도 있다.The anti-human immunoglobulin G antibody included in the diagnostic kit for rapid detection of immunoglobulin G against COVID-19 virus of the present invention may be manufactured and used by itself or may be commercially purchased and utilized.

본 발명의 항원 처리된 2개의 검사선을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트는 면역크로마토그래피법에 기반하여 작동하는 검출용 진단키트이다.The immunoglobulin G rapid detection diagnostic kit for COVID-19 virus characterized by two antigen-treated test lines of the present invention is a diagnostic kit for detection that operates based on immunochromatography.

본 발명의 항원 처리된 2개의 검사선을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트는 액체 시료가 가해지는 샘플 패드 (Sample pad), 면역글로불린 G 감지용 접합체 패드 (Conjugate pad), COVID-19 바이러스에 대한 면역글로불린 G 캡쳐용 항원이 고정된 멤브레인 (Membrane) 및 액체 시료를 흡수할 수 있는 흡수 패드 (Adsorbent pad; absorption pad)가 순차적으로 연결된 구조를 포함한다.The immunoglobulin G rapid detection diagnostic kit for COVID-19 virus, characterized by two antigen-treated test lines of the present invention, is a sample pad to which a liquid sample is applied, and a conjugate pad for immunoglobulin G detection. ), a structure in which an antigen for capturing immunoglobulin G for COVID-19 virus is immobilized and an absorbent pad capable of absorbing a liquid sample are sequentially connected.

본 발명의 항원 처리된 2개의 검사선을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트에서 상기 “샘플 패드”는 분석을 행할 생물학적 시료를 수용하여 확산 흐름이 가능한 패드를 의미하며, 분석을 행할 생물학적 시료를 수용하여 함유하기에 충분한 다공성을 갖는 물질로 구성된다. 이러한 다공성 물질로는 섬유성 종이, 셀룰로오스 물질로 된 미세 가공 멤브레인, 셀룰로오스, 셀룰로오스 아세테이트와 같은 셀룰로오스 유도체, 니트로셀룰로오스, 유리섬유, 천연발생의 면 (Cotton), 나일론과 같은 직물 또는 다공성 겔 등이 있으나, 이에 한정되지 않는다.In the immunoglobulin G rapid detection diagnostic kit for COVID-19 virus characterized by two antigen-treated test lines of the present invention, the "sample pad" means a pad capable of diffusion flow by receiving a biological sample to be analyzed, , consisting of a material having sufficient porosity to receive and contain the biological sample to be analyzed. Such porous materials include fibrous paper, microfabricated membranes made of cellulosic materials, cellulose, cellulose derivatives such as cellulose acetate, nitrocellulose, glass fibers, naturally occurring cotton, fabrics such as nylon, or porous gels. , but is not limited thereto.

본 발명의 항원 처리된 2개의 검사선을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트에서 상기 “접합체 패드”는 샘플 패드로부터 확산되어 이동되는 시료를 수용하며, 동시에 나노입자와 항체가 연결된 “나노입자-항체 콘쥬게이트”가 포함되어 있는 패드이다. 접합체 패드는 샘플 패드와 마찬가지로 확산 흐름이 가능한 물질로 구성된다. 이러한 물질로는 섬유성 종이, 셀룰로오스 물질로 된 미세 가공 멤브레인, 셀룰로오스, 셀룰로오스 아세테이트와 같은 셀룰로오스 유도체, 니트로셀룰로오스, 유리섬유, 천연발생의 면, 나일론과 같은 직물 또는 다공성 겔 등이 있으나, 이에 한정되지 않는다.In the immunoglobulin G rapid detection diagnostic kit for COVID-19 virus, characterized by two antigen-treated test lines of the present invention, the "conjugate pad" receives the sample that is diffused from the sample pad, and at the same time receives the nanoparticles and It is a pad containing a “nanoparticle-antibody conjugate” to which an antibody is linked. The conjugate pad, like the sample pad, is made of a material capable of diffusion flow. Such materials include, but are not limited to, fibrous paper, microfabricated membranes made of cellulosic materials, cellulose, cellulose derivatives such as cellulose acetate, nitrocellulose, glass fibers, naturally occurring cotton, fabrics such as nylon, or porous gels. does not

본 발명의 항원 처리된 2개의 검사선을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트에서 상기 “멤브레인”은 시료 물질이 통과할 수 있는 어떠한 다양한 물질로도 제조될 수 있다. 예를 들어, 천연, 합성, 또는 합성에 의해 변형된 천연 발생 물질, 예를 들어 폴리사카라이드 (예: 셀룰로오스 물질, 종이, 셀룰로오스 아세테이트 및 니트로셀룰로오스와 같은 셀룰로오스 유도체); 폴리에테르 술폰; 폴리에틸렌; 나일론; 폴리비닐리덴 플루오라이드 (PVDF); 폴리에스테르; 폴리프로필렌; 실리카; 비닐 클로라이드, 비닐 클로라이드-프로필렌 공중합체 및 비닐 클로라이드-비닐 아세테이트 공중합체와 같은 중합체와 함께 다공성 중합체 매트릭스에 균일하게 분산된 무기 물질, 예를 들어 불활성화된 알루미나, 규조토, MgSO4, 또는 다른 무기 미분 물질; 자연 발생 (예: 면) 및 합성 (예: 나일론 또는 레이온) 천; 다공성 겔, 예를 들어 실리카겔, 아가로스, 덱스트란 및 젤라틴; 중합체 필름, 예를 들어 폴리아크릴아미드; 등의 물질로부터 형성될 수 있다. 바람직하게는 상기 멤브레인은 중합체 물질, 예를 들어 니트로셀룰로오스, 폴리에테르술폰, 폴리에틸렌, 나일론, 폴리비닐리덴 플루오라이드, 폴리에스테르 및 폴리프로필렌을 포함한다. In the diagnostic kit for rapid detection of immunoglobulin G for COVID-19 virus, which is characterized by two antigen-treated test lines of the present invention, the “membrane” may be made of any various materials through which the sample material can pass. For example, naturally occurring, synthetic, or synthetically modified materials, such as polysaccharides (eg, cellulosic materials, paper, cellulose derivatives such as cellulose acetate and nitrocellulose); polyether sulfone; polyethylene; nylon; polyvinylidene fluoride (PVDF); Polyester; polypropylene; silica; An inorganic material uniformly dispersed in a porous polymer matrix with a polymer such as vinyl chloride, vinyl chloride-propylene copolymer and vinyl chloride-vinyl acetate copolymer, for example deactivated alumina, diatomaceous earth, MgSO 4 , or other inorganic fines matter; naturally occurring (eg cotton) and synthetic (eg nylon or rayon) fabrics; porous gels such as silica gel, agarose, dextran and gelatin; polymer films such as polyacrylamide; It may be formed from materials such as Preferably the membrane comprises a polymeric material, for example nitrocellulose, polyethersulfone, polyethylene, nylon, polyvinylidene fluoride, polyester and polypropylene.

본 발명의 항원 처리된 2개의 검사선을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트에서 상기 “흡수패드”는 상기 멤브레인의 말단에 또는 그 가까이에 인접해서 위치할 수 있다. 흡수 패드는 일반적으로 전체 멤브레인을 통해 이동하는 유체 시료를 받아들인다. 흡수 패드는 멤브레인을 통한 모세관 작용 및 유체의 확산 유동을 촉진하는 데 도움을 줄 수 있다.In the immunoglobulin G rapid detection diagnostic kit for COVID-19 virus characterized by two antigen-treated test lines of the present invention, the "absorbent pad" may be located adjacent to or near the end of the membrane. The absorbent pad generally receives a fluid sample that travels through the entire membrane. Absorbent pads can help promote capillary action and diffuse flow of fluids through the membrane.

본 발명의 항원 처리된 2개의 검사선을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트는 상기한 샘플 패드, 접합체 패드, 멤브레인 및 흡수 패드가 동일한 지지체상에 순차적으로 연결되어 구성된다.The immunoglobulin G rapid detection diagnostic kit for COVID-19 virus, characterized by two antigen-treated test lines of the present invention, is configured by sequentially connecting the above-described sample pad, conjugate pad, membrane and absorbent pad on the same support. do.

상기 지지체는 상기한 샘플 패드, 접합체 패드, 멤브레인 및 흡수 패드를 지지 및 운반할 수 있다면 어떠한 물질로도 형성될 수 있으나, 일반적으로 상기 멤브레인을 통해 확산하는 시료의 유체가 지지체를 통해 누출되지 않도록 액체 불투과성인 것이 바람직하다. 예컨대, 유리; 중합체 물질, 예를 들어 폴리스티렌, 폴리프로필렌, 폴리에스테르, 폴리부타디엔, 폴리비닐클로라이드, 폴리아미드, 폴리카르보네이트, 에폭시드, 메타크릴레이트 및 폴리멜라민 등을 포함하지만, 이에 한정되는 것은 아니다. The support may be formed of any material as long as it can support and transport the sample pad, the conjugate pad, the membrane, and the absorbent pad. It is preferably impermeable. For example, glass; polymeric materials such as polystyrene, polypropylene, polyester, polybutadiene, polyvinylchloride, polyamide, polycarbonate, epoxide, methacrylate, polymelamine, and the like.

본 발명의 항원 처리된 2개의 검사선을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트에서 상기 멤브레인 상에는 상기 접합체 패드로부터 상기 흡수 패드 방향으로 항원이 처리된 2개의 검사선 및 시료가 전개되었다는 것을 확인하기 위한 대조선이 순차적으로 형성되어 있다.In the diagnostic kit for rapid detection of immunoglobulin G for COVID-19 virus of the present invention, characterized by the two antigen-treated test lines of the present invention, two antigen-treated test lines and a sample from the conjugate pad to the absorption pad on the membrane Control lines are sequentially formed to confirm that the has been developed.

본 발명의 항원 처리된 2개의 검사선을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트는 COVID-19 바이러스에 대한 면역글로불린 G 유무 판정, COVID-19 바이러스에 노출된 이력이 있는 사람들에 대한 모니터링 목적으로 활용될 수 있다. Immunoglobulin G rapid detection diagnostic kit for COVID-19 virus, characterized by two antigen-treated test lines of the present invention, determines the presence or absence of immunoglobulin G for COVID-19 virus, It can be used for monitoring purposes.

본 명세서에서 COVID-19 바이러스는 2019년 11월 중국 후베이성 우한시에서 시작된 바이러스 감염의 원인 바이러스를 지칭하나, 이에 국한되지 않고 이로부터 변형된 변종 바이러스까지를 포함한다.In the present specification, the COVID-19 virus refers to a virus that causes a viral infection that started in Wuhan, Hubei Province, China in November 2019, but is not limited thereto, and includes a modified virus therefrom.

본 발명에 따른 항원 처리된 2개의 검사선을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트를 활용하면 COVID-19 바이러스에 대한 면역글로불린 G 보유 여부를 신속하게 그리고 높은 정확도로 판정할 수 있다. 본 발명에 따른 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트는 항원 처리된 2개의 검사선을 가지고 있어 위양성 (False-positive) 발생을 낮출 수 있고, 특히 2개의 검사선에 코팅하는 항원의 종류를 달리할 경우에는 다양한 에피토프 (Epitope)를 제공해 줄 수 있어 폴리클로날 (Polyclonal) 면역글로불린 G 검출에 있어 검출 성공 확률을 높여줄 수 있으면서 추가적으로 위양성 발생 가능성을 획기적으로 낮추어 줄 수 있다. 즉, 폴리클로날 항체 검출에 있어 특히 장점을 제공해 줄 수 있다. 또한, 서로 다른 종류의 항원으로 처리된 2개의 검사선은 COVID-19 바이러스 변종에 대한 항체 검사에서도 위음성 (False-negative) 발생을 낮출 수 있다. 이에 더하여, 본 발명의 진단키트에서는 검사선에는 항원을 처리하고 골드 콘쥬게이트 (Conjugate)에는 항체를 처리하는 방식으로 구현된다. 통상적으로 검사선 처리에 사용되는 성분이 골드 콘쥬게이트에 결합하는 성분보다 진단키트 제작에 있어 재료적으로 많은 양이 사용된다. 본 발명은 항체와 비교하여 제조에 있어 상대적으로 적은 비용이 소요되는 항원을 검사선 처리용 성분으로 채택함으로써 진단키트의 제조비용을 낮출 수 있다는 이점을 가진다. 이는 산업적 활용에 있어 매우 중요한 요소라 할 수 있다. 또, 골드 콘쥬게이트에 결합하는 성분을 항원으로 하지 않고 항체로 함으로써 본 발명의 진단키트에서는 시그널 (Signal)의 강도 (Intensity)를 보다 높일 수 있다. 골드 콘쥬게이트에 결합하는 성분을 항원으로 하게 되면 항원 처리된 골드 콘쥬게이트가 면역글로불린 G 외에도 면역글로불린 M 등에도 결합하게 되어 결과적으로 골드 콘쥬게이트 (발색체)의 낭비를 초래하게 되어 시그널의 약화가 초래될 수 있다.By utilizing the rapid detection kit of immunoglobulin G for COVID-19 virus, which features two antigen-treated test lines according to the present invention, it is possible to quickly and with high accuracy determine whether or not to have immunoglobulin G for COVID-19 virus. can do. Immunoglobulin G rapid detection diagnostic kit for COVID-19 virus according to the present invention has two antigen-treated test lines, so it is possible to lower the occurrence of false-positives. In the case of different types, various epitopes can be provided, which can increase the probability of successful detection in polyclonal immunoglobulin G detection, and further reduce the possibility of false positives. That is, it may provide a particular advantage in the detection of polyclonal antibodies. In addition, two test lines treated with different types of antigens can lower the incidence of false-negatives in antibody tests against the COVID-19 virus strain. In addition, in the diagnostic kit of the present invention, the test line is embodied in a way that the antigen is treated and the gold conjugate is treated with the antibody. In general, a larger amount of material is used in manufacturing the diagnostic kit than the component used for processing the test line, rather than the component binding to the gold conjugate. The present invention has the advantage that the manufacturing cost of a diagnostic kit can be lowered by adopting an antigen, which is relatively inexpensive to manufacture compared to an antibody, as a component for processing a test line. This can be said to be a very important factor for industrial application. In addition, in the diagnostic kit of the present invention, the intensity of the signal can be further increased by using the component binding to the gold conjugate as an antibody rather than as an antigen. When a component that binds to the gold conjugate is used as an antigen, the antigen-treated gold conjugate binds to immunoglobulin M in addition to immunoglobulin G, resulting in wastage of the gold conjugate (chromophore), resulting in signal attenuation. can be brought about

도 1은 본 발명의 항원 처리된 2개의 검사선을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 목적의 검출용 진단키트의 구조도를 나타낸 것이다.
도 2는 본 발명의 동일 종류의 항원 처리된 2개의 검사선을 갖는, COVID-19 바이러스에 대한 면역글로불린 G 신속검출 목적의 검출용 진단키트의 결과 예시이다. 1 shows a structural diagram of a diagnostic kit for the purpose of rapid detection of immunoglobulin G for COVID-19 virus, characterized by two antigen-treated test lines of the present invention.
2 is an example of the results of the diagnostic kit for rapid detection of immunoglobulin G for COVID-19 virus, which has two test lines treated with the same type of antigen of the present invention.

이하, 실시예에 의거하여 본 발명을 보다 구체적으로 설명하지만, 이들 실시예는 본 발명의 예시일 뿐이며 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail based on Examples, but these Examples are merely illustrative of the present invention and the scope of the present invention is not limited to these Examples.

실시예Example 1: One: COVIDCOVID -19 바이러스 특이 항원 제조 및 항체 확보-19 Virus-specific antigen production and antibody acquisition

COVID-19 바이러스 특이 항원은 서열번호 1 [COVID-19 spike protein S1 subunit (이하 ‘S1 단백질 (S1 protein)’이라 함)], 서열번호 2 [COVID-19 spike protein S1 subunit의 receptor binding domain (이하 ‘S1’ 단백질 (S1’ protein)’이라 함)], 서열번호 3 [COVID-19 spike protein S2 subunit (이하 ‘S2 단백질 (S2 protein)’이라 함)], 또는 서열번호 4 [COVID-19 nucleocapsid protein (이하 ‘N 단백질 (N protein)’이라 함)]의 아미노산 서열로 표시되는 단백질을 통상의 재조합 단백질 생산법으로 제조하여 확보하든지 상업적으로 구입하여 사용하면 된다. 재조합 단백질로의 제조는 통상적으로 흔히 이용되는 대장균, 동물세포, 곤충세포 등을 사용하여 제작할 수 있는데, 본 실시예에서는 예시로 대장균을 사용한 서열번호 4의 N 단백질 제조를 제시한다. 물론 이에 국한되지 않음은 당업자에게 당연하다. 본 실시예의 N 단백질 제조에서는 용이한 분리정제를 위하여 C-말단에 His-tag이 붙은 형태로 제조했으며 서열번호 8의 유전자 서열을 이용하여 발현 플라스미드를 제작하여 사용하였다. S1 단백질의 발현에서는 서열번호 5의 유전자 서열을, S1’ 단백질의 발현에서는 서열번호 6의 유전자 서열을, S2 단백질의 발현에서는 서열번호 7의 유전자 서열을 이용하면 된다.COVID-19 virus-specific antigen is SEQ ID NO: 1 [COVID-19 spike protein S1 subunit (hereinafter referred to as 'S1 protein')], SEQ ID NO: 2 [COVID-19 spike protein S1 subunit receptor binding domain (hereinafter referred to as 'S1 protein)'] 'S1' protein')], SEQ ID NO: 3 [COVID-19 spike protein S2 subunit (hereinafter referred to as 'S2 protein')], or SEQ ID NO: 4 [COVID-19 nucleocapsid Protein (hereinafter referred to as 'N protein (N protein)')] can be prepared and secured by a conventional recombinant protein production method or commercially purchased and used. Preparation of the recombinant protein can be prepared using commonly used E. coli, animal cells, insect cells, etc. In this example, the preparation of the N protein of SEQ ID NO: 4 using E. coli is presented as an example. Of course, it is obvious to those skilled in the art that the present invention is not limited thereto. In the preparation of the N protein of this example, it was prepared in the form of a His-tag attached to the C-terminus for easy separation and purification, and an expression plasmid was prepared and used using the gene sequence of SEQ ID NO: 8. For the expression of the S1 protein, the gene sequence of SEQ ID NO: 5, the gene sequence of SEQ ID NO: 6 for the expression of S1' protein, and the gene sequence of SEQ ID NO: 7 for the expression of the S2 protein may be used.

COVID-19 바이러스의 N 단백질을 발현시키기 위해, 서열번호 8의 유전자 서열을 이용하여 발현 플라스미드를 제작하였다. 이때 C-말단에 히스티딘 표지 (Histidine tag)가 도입되게 유전자를 합성하여 이용하였다. 합성된 유전자는 pIBA-3KN vector로 클로닝 하였고, 이렇게 제작한 발현 플라스미드를 이용하여 통상의 방법대로 대장균 Top10 (DH10B competent cell, Thermo Fisher Scientific)을 형질전환시켜 생산균주 TOP10-pIBA3KN-NP를 제작하였다. 이러한 과정은 당업자에게 자명하므로 상세한 설명은 생략한다. 이렇게 제작한 균주를 생산균주로 하여 N 단백질 제조를 다음과 같이 실시하였다. In order to express the N protein of the COVID-19 virus, an expression plasmid was prepared using the gene sequence of SEQ ID NO: 8. At this time, a gene was synthesized and used so that a histidine tag was introduced at the C-terminus. The synthesized gene was cloned into pIBA-3KN vector, and E. coli Top10 (DH10B competent cell, Thermo Fisher Scientific) was transformed using the expression plasmid prepared in this way to prepare the production strain TOP10-pIBA3KN-NP. Since this process is obvious to those skilled in the art, a detailed description thereof will be omitted. Using the thus-prepared strain as a production strain, the N protein was prepared as follows.

50 μg/ml 카나마이신이 포함된 LB배지 (트립톤 10 g/L, 효모 추출물 5 g/L, 염화나트륨 10 g/L) 20 ml에 TOP10-pIBA3KN-NP를 20 μl 첨가하여 접종한 다음 37℃에서 한밤 동안 진탕 배양하였다. 다음날, 50 μg/ml 카나마이신이 포함된 LB배지 0.2 L에 한밤 배양한 배양액을 1/100 부피비로 첨가하였다. 220 rpm의 교반속도로, 37℃ 온도 조건에서 배양을 실시하였다. 세포 농도가 600 nm에서의 흡광도 기준으로 0.5가 되었을 때, 최종 농도가 0.2 μg/mL이 되도록 언하이드로테트라사이클린 (Anhydrotetracycline)을 첨가하여 COVID-19 바이러스의 N 단백질의 발현을 유도한 후에 3시간 배양을 추가 실시하였다.Add 20 μl of TOP10-pIBA3KN-NP to 20 ml of LB medium (trypton 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L) containing 50 μg/ml kanamycin and inoculate it at 37°C Incubated with shaking overnight. The next day, the overnight culture solution was added to 0.2 L of LB medium containing 50 μg/ml kanamycin in a 1/100 volume ratio. At a stirring speed of 220 rpm, culture was performed at 37°C. When the cell concentration reached 0.5 based on the absorbance at 600 nm, anhydrotetracycline was added so that the final concentration was 0.2 μg/mL to induce the expression of the N protein of the COVID-19 virus, followed by incubation for 3 hours was additionally carried out.

배양 종료 후, 세포 배양액을 취하여 7,000 rpm에서 15분간 4℃에서 원심분리하여 세포 침전물을 회수하였고, 회수한 세포 침전물은 20 ml의 buffer A (20 mM Tris-Cl, 0.5 M NaCl, 5 mM imidazole, pH 7.9)에 부유시켰다. 이렇게 준비된 세포 부유액을 초음파 분쇄법을 이용하여 세포를 파쇄하였고, 상기 초음파 분쇄법은 3초간 초음파를 가하여 세포를 깨고, 3초간 멈추는 조건을 총 15분간 반복하여 실시하며, 이는 얼음조 (Ice bath)에서 실시하였다.After completion of the culture, the cell culture solution was taken and centrifuged at 7,000 rpm for 15 minutes at 4° C. to collect cell precipitates, and the recovered cell precipitates were prepared in 20 ml of buffer A (20 mM Tris-Cl, 0.5 M NaCl, 5 mM imidazole, pH 7.9). The cell suspension thus prepared was disrupted by sonication, and the sonication method was repeated for a total of 15 minutes in which the ultrasonic wave was applied for 3 seconds to break the cells, and the condition was stopped for 3 seconds, which was performed in an ice bath. was carried out in

세포 파쇄 후에 세포 파쇄액을 7,000 rpm에서 15분간 4℃에서 원심분리하여 얻어진 상등액을 통상의 히스티딘-친화 크로마토그래피 (Histidine affinity chromatography) 정제 공정을 통하여 정제하였다.After cell disruption, the supernatant obtained by centrifuging the cell disruption solution at 7,000 rpm for 15 minutes at 4° C. was purified through a conventional histidine-affinity chromatography purification process.

정제 공정을 간단히 설명하면 다음과 같다. 히스티딘-친화 수지로 5 ml의 HisTrapTM HP (GE Healthcare사)를 사용하였다. 크로마토그래피는 칼럼을 Buffer A (20 mM Tris-Cl, 0.5 M NaCl, 5 mM imidazole, pH 7.9)로 미리 평형화시킨 후에 실시하였고, 시료를 칼럼에 적하한 다음에는 5 ml/분의 유량 (Flow rate)으로 상기 buffer A를 10 CV (Column Volume) 흘려주어 세척을 실시하였다. 세척 후에는 5 ml/분의 유량으로 buffer B (20 mM Tris-Cl, 0.5 M NaCl, 5 mM imidazole, pH 7.9)의 농도가 4%가 되게 하여 10 CV를 흘려준 후, 농도 기울기 (Gradient)가 100%가 되게 하는 조건으로 크로마토그래피를 수행하였다. 이 과정에서 목적하는 COVID-19 바이러스의 N 단백질의 용출이 달성되었다. 용출액은 투석 완충액 (10 mM PBS, pH 7.2)에 대하여 4℃에서 24시간 동안 투석을 실시하여 항원 단백질 용액을 준비하였다.A brief description of the purification process is as follows. 5 ml of HisTrap ™ HP (GE Healthcare) was used as the histidine-friendly resin. Chromatography was performed after the column was pre-equilibrated with Buffer A (20 mM Tris-Cl, 0.5 M NaCl, 5 mM imidazole, pH 7.9), and after dropping the sample onto the column, a flow rate of 5 ml/min (Flow rate) ), the buffer A was washed by flowing 10 CV (Column Volume). After washing, the concentration of buffer B (20 mM Tris-Cl, 0.5 M NaCl, 5 mM imidazole, pH 7.9) was 4% at a flow rate of 5 ml/min, and 10 CV was flowed, and then the concentration gradient (Gradient) Chromatography was performed under conditions such that the In this process, the desired elution of the N protein of the COVID-19 virus was achieved. The eluate was dialyzed against dialysis buffer (10 mM PBS, pH 7.2) at 4° C. for 24 hours to prepare an antigen protein solution.

면역크로마토그래피법에 기반한 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트를 제작하기 위해서는 항-인간 면역글로불린 G 항체와 이 항-인간 면역글로불린 G 항체에 대한 항체의 조합이 필요하다. 이러한 조합의 선택은 당업자에게는 일반적이므로 이에 대한 상세한 설명은 생략한다. 본 발명의 실시예에서는 염소 (Goat) 항-인간 면역글로불린 G 항체와 토끼 (Rabbit) 항-염소 항체 (Anti-goat antibody)를 사용하였다. 그러나 이에 국한되지 않음은 자명하다. A combination of an anti-human immunoglobulin G antibody and an antibody against the anti-human immunoglobulin G antibody is required to produce a diagnostic kit for rapid detection of immunoglobulin G against COVID-19 virus based on immunochromatography. Selection of such a combination is common to those skilled in the art, so a detailed description thereof will be omitted. In the examples of the present invention, goat anti-human immunoglobulin G antibody and rabbit anti-goat antibody were used. However, it is obvious that the present invention is not limited thereto.

실시예Example 2: 40 nm2: 40 nm 콜로이드 골드용액의 제조 Preparation of colloidal gold solution

Sigma사의 gold(Ⅲ) chloride trihydrate (제품번호: 520918)를 사용하여 콜로이드 골드 (Colloidal gold) 용액을 제조하였다. Gold(Ⅲ) chloride trihydrate를 증류수에 1% (w/v)가 되게 녹인 후, 3구 플라스크에 증류수 1 L를 넣고 30분간 끓여 주었다. 1% gold 20 ml을 넣은 후 5분간 끓여준 후, 1% sodium citrate 20 ml을 넣고 30분간 끓여 주었다. 이렇게 제조된 콜로이드 골드 용액을 식힌 후 분광광도계 (spectrophotometer)를 이용하여 콜로이드 골드의 크기가 40 nm임을 확인하였다.A colloidal gold solution was prepared using Sigma's gold(III) chloride trihydrate (Product Number: 520918). Gold(III) chloride trihydrate was dissolved in distilled water to 1% (w/v), and then 1 L of distilled water was added to a three-necked flask and boiled for 30 minutes. After adding 20 ml of 1% gold and boiling for 5 minutes, 20 ml of 1% sodium citrate was added and boiled for 30 minutes. After cooling the colloidal gold solution thus prepared, it was confirmed that the size of the colloidal gold was 40 nm using a spectrophotometer.

실시예Example 3: 항-인간 면역글로불린 G 항체 3: anti-human immunoglobulin G antibody 콘쥬게이트conjugate 제작 produce

2 mM sodium tetraborate (pH 9.0-9.2)에 항-인간 면역글로불린 G 항체를 투석하여 버퍼를 교환했다. 투석한 항체를 정량한 후 1 mg/ml로 희석한 후 K2CO3를 이용하여 콜로이드 골드 용액의 pH를 4, 5, 6, 7, 8, 9로 제조하였다. pH별로 제조된 콜로이드 골드 용액을 1.5 ml tube에 1 ml씩 분주하였다. 각각의 콜로이드 골드 용액에 투석한 항체를 1, 5, 10, 20 μl을 넣어 상온에서 30분간 반응시켰다. Blocking을 위해 BSA를 최종농도로 1%가 되게 첨가한 후 상온에서 30분간 반응시켰다. 반응 후, 12,000 rpm에서 20분간 원심분리 한 다음에 상층액을 덜어내고 저장 완충액 (storage buffer; 1× PBS, 0.1% Tween 20, 0.1% BSA, 0.1% NaN3)으로 펠렛을 완전히 부유시켜 냉장보관하면서 접합체 패드 전처리에 사용하였다.The buffer was exchanged by dialysis of anti-human immunoglobulin G antibody against 2 mM sodium tetraborate (pH 9.0-9.2). After quantifying the dialyzed antibody, it was diluted to 1 mg/ml, and then the pH of the colloidal gold solution was prepared at 4, 5, 6, 7, 8, 9 using K 2 CO 3 . 1 ml of the colloidal gold solution prepared for each pH was dispensed into a 1.5 ml tube. 1, 5, 10, 20 μl of the dialyzed antibody was added to each colloidal gold solution and reacted at room temperature for 30 minutes. For blocking, BSA was added to a final concentration of 1% and reacted at room temperature for 30 minutes. After the reaction, after centrifugation at 12,000 rpm for 20 minutes, the supernatant is removed, and the pellet is completely suspended in storage buffer (storage buffer; 1× PBS, 0.1% Tween 20, 0.1% BSA, 0.1% NaN 3 ) and stored refrigerated. It was used for pre-treatment of the bonding body pad.

실시예Example 4: 4: 검출키트의detection kit 각 부분별for each part 전처리 및 조립 Pretreatment and assembly

샘플 패드 (밀리포어사)(10)는 PBS에 0.5％ BSA와 0.5％ Tween 20을 혼합하여 전처리하였다. 접합체 패드 (밀리포어사)(20)는 항-인간 면역글로불린 G 항체 콘쥬게이트가 4 OD가 되도록 조정하였고, 5％ 트레할로스 (Trehalose)를 첨가하였다. DCI-400 (ZETA, KOREA)을 이용하여 니트로셀룰로오스 멤브레인 (Nitrocellulose membrane) (30)에 검사선(40)과 대조선 (Control line) (50)을 전처리하였다. 즉, 검사선(40)은 실시예 1에서 제작된 항원 단백질 용액 1 ㎕가 니트로셀룰로오스 멤브레인(30) 1 cm에 분사되도록 농도를 조정하였으며, 분사속도는 초당 5 cm의 속도로 하였다. 대조선(50)도 검사선과 동일한 조건으로 토끼 (Rabbit) 항-염소 항체 (Anti-goat antibody)를 분사하였다. 검사선과 대조선이 분사 완료된 니트로셀룰로오스 멤브레인은 37℃에서 2시간 건조시켜 항원 단백질 용액과 항-염소 항체 용액이 충분히 니트로셀룰로오스 멤브레인에 부착되도록 처리하였다. 전처리가 완료된 샘플 패드 (Sample pad) (10), 접합체 패드 (Conjugate pad) (20), 니트로셀룰로오스 멤브레인(30), 흡수 패드 (Adsorbent pad) (60)들을 도 1에 나타난 것처럼 백킹 카드 (Backing card) (70) 위에서 2 mm씩 서로 중첩되도록 순차적으로 부착시킨 후 폭 4 mm로 절단하여 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트(100)를 제작하였다.The sample pad (Millipore) 10 was pretreated by mixing 0.5% BSA and 0.5% Tween 20 in PBS. Conjugate pads (Millipore) (20) were adjusted to 4 OD of anti-human immunoglobulin G antibody conjugate and 5% Trehalose was added. A test line 40 and a control line 50 were pretreated on a nitrocellulose membrane 30 using DCI-400 (ZETA, KOREA). That is, the concentration of the test line 40 was adjusted so that 1 μl of the antigen protein solution prepared in Example 1 was sprayed on 1 cm of the nitrocellulose membrane 30, and the spraying speed was 5 cm per second. The control line 50 was also sprayed with a rabbit anti-goat antibody under the same conditions as the test line. The nitrocellulose membrane sprayed with the test line and the control line was dried at 37° C. for 2 hours and treated so that the antigen protein solution and the anti-goat antibody solution were sufficiently attached to the nitrocellulose membrane. As shown in FIG. 1, the sample pad 10, the conjugate pad 20, the nitrocellulose membrane 30, and the absorbent pad 60, after which the pre-treatment is completed, a backing card (Backing card) ) (70) was sequentially attached to overlap each other by 2 mm, and then cut to 4 mm in width to prepare an immunoglobulin G rapid detection diagnostic kit 100 for COVID-19 virus.

실시예Example 5: 5: COVIDCOVID -19 바이러스에 대한 면역글로불린 G 신속검출 - Rapid detection of immunoglobulin G against viruses 진단키트의of the diagnostic kit 성능 평가 performance evaluation

환자 혈청 20건을 대상으로 표준측정법인 효소결합면역흡착검사 (Enzyme-linked immunosorbent assay, ELISA)법과 본 발명의 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트를 동시에 적용하여 COVID-19 바이러스에 대한 면역글로불린 G 검출양상을 비교하고 그 결과를 아래의 표 1에 나타내었다. 대조 진단키트로는 상용 COVID-19 바이러스에 대한 면역글로불린 G 검출 진단키트 2종을 사용하였다.For 20 patient sera, enzyme-linked immunosorbent assay (ELISA) method, which is a standard measurement method, and the immunoglobulin G rapid detection diagnostic kit for COVID-19 virus of the present invention were simultaneously applied to prevent COVID-19 virus. The immunoglobulin G detection patterns were compared and the results are shown in Table 1 below. As a control diagnostic kit, two commercially available immunoglobulin G detection diagnostic kits for COVID-19 were used.

그 결과를 표 1에 제시하였다. The results are presented in Table 1.

검출법detection method 구분division ELISAELISA 본 발명의 진단키트
(검사선 1: S1’ 단백질; 검사선 2: N 단백질)Diagnostic kit of the present invention
(Test line 1: S1'protein; Test line 2: N protein) 본 발명의 진단키트
(검사선 1: N 단백질; 검사선 2: S1’ 단백질)Diagnostic kit of the present invention
(Test line 1: N protein; Test line 2: S1' protein) 본 발명의 진단키트
(검사선 1: N 단백질; 검사선 2: N 단백질)Diagnostic kit of the present invention
(Test line 1: N protein; Test line 2: N protein) 상용제품 Acommercial product A 상용제품 Bcommercial product B 양성시료positive sample 1010 1010 1010 1010 88 77 음성시료voice sample 1010 1010 1010 1010 1212 1313

표 1에 제시된 결과가 본 발명에 따라 제조된 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트의 장점을 모두 보여주기에는 시험대상 시료수가 작았지만 최소한 표준측정법인 효소결합면역흡착검사에 상응하는 성능 수준을 제공함을 알 수 있었다. Although the number of samples to be tested was small enough to show all the advantages of the immunoglobulin G rapid detection diagnostic kit for COVID-19 virus prepared according to the present invention, the results presented in Table 1 are at least equivalent to the standard assay method, enzyme-linked immunosorbent test. It was found to provide a level of performance.

이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

10: 샘플 패드 (Sample pad)
20: 접합체 패드 (Conjugate pad)
30: 니트로셀룰로오스 멤브레인 (Nitrocellulose membrane)
40: 검사선 (Test line)
50: 대조선 (Control line)
60: 흡수 패드 (Adsorbent pad)
70: 백킹 카드 (Backing card)
100: COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트10: Sample pad
20: conjugate pad (Conjugate pad)
30: nitrocellulose membrane (Nitrocellulose membrane)
40: test line
50: Control line
60: absorbent pad (Adsorbent pad)
70: Backing card
100: Immunoglobulin G rapid detection diagnostic kit for COVID-19 virus

SEQUENCE LISTING <110> iNtRON Biotechnology, Co. Ltd. <120> Rapid Detection Method of COVID-19 Immunoglobulin G <130> KP090693 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 674 <212> PRT <213> Artificial Sequence <220> <223> COVID-19 spike protein S1 subunit <400> 1 Ser Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala 1 5 10 15 Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe 20 25 30 Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe 35 40 45 Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly 50 55 60 Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr 65 70 75 80 Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly 85 90 95 Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala 100 105 110 Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro 115 120 125 Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser 130 135 140 Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val 145 150 155 160 Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys 165 170 175 Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile 180 185 190 Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly 195 200 205 Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile 210 215 220 Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro 225 230 235 240 Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val 245 250 255 Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly 260 265 270 Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr 275 280 285 Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr 290 295 300 Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn 305 310 315 320 Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe 325 330 335 Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala 340 345 350 Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys 355 360 365 Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val 370 375 380 Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala 385 390 395 400 Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp 405 410 415 Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser 420 425 430 Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser 435 440 445 Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala 450 455 460 Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro 465 470 475 480 Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro 485 490 495 Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr 500 505 510 Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val 515 520 525 Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser 530 535 540 Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp 545 550 555 560 Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile 565 570 575 Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn 580 585 590 Thr Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu 595 600 605 Val Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val 610 615 620 Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile 625 630 635 640 Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly 645 650 655 Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg 660 665 670 Ala Arg <210> 2 <211> 194 <212> PRT <213> Artificial Sequence <220> <223> receptor binding domain for COVID-19 spike protein S1 subunit <400> 2 Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg 1 5 10 15 Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val 20 25 30 Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys 35 40 45 Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn 50 55 60 Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile 65 70 75 80 Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro 85 90 95 Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp 100 105 110 Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys 115 120 125 Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln 130 135 140 Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe 145 150 155 160 Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln 165 170 175 Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala 180 185 190 Thr Val <210> 3 <211> 588 <212> PRT <213> Artificial Sequence <220> <223> COVID-19 spike protein S2 subunit <400> 3 Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala 1 5 10 15 Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn 20 25 30 Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys 35 40 45 Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys 50 55 60 Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg 65 70 75 80 Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val 85 90 95 Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe 100 105 110 Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser 115 120 125 Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala 130 135 140 Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala 145 150 155 160 Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu 165 170 175 Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu 180 185 190 Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala 195 200 205 Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile 210 215 220 Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn 225 230 235 240 Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr 245 250 255 Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln 260 265 270 Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile 275 280 285 Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala 290 295 300 Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln 305 310 315 320 Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser 325 330 335 Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser 340 345 350 Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 355 360 365 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro 370 375 380 Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly 385 390 395 400 Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His 405 410 415 Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr 420 425 430 Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val 435 440 445 Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys 450 455 460 Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp 465 470 475 480 Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys 485 490 495 Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu 500 505 510 Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro 515 520 525 Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met 530 535 540 Val Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys 545 550 555 560 Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser 565 570 575 Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr 580 585 <210> 4 <211> 419 <212> PRT <213> Artificial Sequence <220> <223> COVID-19 nucleocapsid protein <400> 4 Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr 1 5 10 15 Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg 20 25 30 Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn 35 40 45 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu 50 55 60 Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro 65 70 75 80 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 85 90 95 Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr 100 105 110 Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln 145 150 155 160 Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser 165 170 175 Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn 180 185 190 Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala 195 200 205 Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu 210 215 220 Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln 225 230 235 240 Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys 245 250 255 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln 260 265 270 Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp 275 280 285 Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile 290 295 300 Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile 305 310 315 320 Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala 325 330 335 Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu 340 345 350 Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro 355 360 365 Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln 370 375 380 Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu 385 390 395 400 Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser 405 410 415 Thr Gln Ala <210> 5 <211> 2028 <212> DNA <213> Artificial Sequence <220> <223> COVID-19 spike protein S1 subunit <400> 5 atgtctagtc agtgtgttaa tcttacaacc agaactcaat taccccctgc atacactaat 60 tctttcacac gtggtgttta ttaccctgac aaagttttca gatcctcagt tttacattca 120 actcaggact tgttcttacc tttcttttcc aatgttactt ggttccatgc tatacatgtc 180 tctgggacca atggtactaa gaggtttgat aaccctgtcc taccatttaa tgatggtgtt 240 tattttgctt ccactgagaa gtctaacata ataagaggct ggatttttgg tactacttta 300 gattcgaaga cccagtccct acttattgtt aataacgcta ctaatgttgt tattaaagtc 360 tgtgaatttc aattttgtaa tgatccattt ttgggtgttt attaccacaa aaacaacaaa 420 agttggatgg aaagtgagtt cagagtttat tctagtgcga ataattgcac ttttgaatat 480 gtctctcagc cttttcttat ggaccttgaa ggaaaacagg gtaatttcaa aaatcttagg 540 gaatttgtgt ttaagaatat tgatggttat tttaaaatat attctaagca cacgcctatt 600 aatttagtgc gtgatctccc tcagggtttt tcggctttag aaccattggt agatttgcca 660 ataggtatta acatcactag gtttcaaact ttacttgctt tacatagaag ttatttgact 720 cctggtgatt cttcttcagg ttggacagct ggtgctgcag cttattatgt gggttatctt 780 caacctagga cttttctatt aaaatataat gaaaatggaa ccattacaga tgctgtagac 840 tgtgcacttg accctctctc agaaacaaag tgtacgttga aatccttcac tgtagaaaaa 900 ggaatctatc aaacttctaa ctttagagtc caaccaacag aatctattgt tagatttcct 960 aatattacaa acttgtgccc ttttggtgaa gtttttaacg ccaccagatt tgcatctgtt 1020 tatgcttgga acaggaagag aatcagcaac tgtgttgctg attattctgt cctatataat 1080 tccgcatcat tttccacttt taagtgttat ggagtgtctc ctactaaatt aaatgatctc 1140 tgctttacta atgtctatgc agattcattt gtaattagag gtgatgaagt cagacaaatc 1200 gctccagggc aaactggaaa gattgctgat tataattata aattaccaga tgattttaca 1260 ggctgcgtta tagcttggaa ttctaacaat cttgattcta aggttggtgg taattataat 1320 tacctgtata gattgtttag gaagtctaat ctcaaacctt ttgagagaga tatttcaact 1380 gaaatctatc aggccggtag cacaccttgt aatggtgttg aaggttttaa ttgttacttt 1440 cctttacaat catatggttt ccaacccact aatggtgttg gttaccaacc atacagagta 1500 gtagtacttt cttttgaact tctacatgca ccagcaactg tttgtggacc taaaaagtct 1560 actaatttgg ttaaaaacaa atgtgtcaat ttcaacttca atggtttaac aggcacaggt 1620 gttcttactg agtctaacaa aaagtttctg cctttccaac aatttggcag agacattgct 1680 gacactactg atgctgtccg tgatccacag acacttgaga ttcttgacat tacaccatgt 1740 tcttttggtg gtgtcagtgt tataacacca ggaacaaata cttctaacca ggttgctgtt 1800 ctttatcagg atgttaactg cacagaagtc cctgttgcta ttcatgcaga tcaacttact 1860 cctacttggc gtgtttattc tacaggttct aatgtttttc aaacacgtgc aggctgttta 1920 ataggggctg aacatgtcaa caactcatat gagtgtgaca tacccattgg tgcaggtata 1980 tgcgctagtt atcagactca gactaattct cctcggcggg cacgttaa 2028 <210> 6 <211> 588 <212> DNA <213> Artificial Sequence <220> <223> receptor binding domain for COVID-19 spike protein S1 subunit <400> 6 atgaatatta caaacttgtg cccttttggt gaagttttta acgccaccag atttgcatct 60 gtttatgctt ggaacaggaa gagaatcagc aactgtgttg ctgattattc tgtcctatat 120 aattccgcat cattttccac ttttaagtgt tatggagtgt ctcctactaa attaaatgat 180 ctctgcttta ctaatgtcta tgcagattca tttgtaatta gaggtgatga agtcagacaa 240 atcgctccag ggcaaactgg aaagattgct gattataatt ataaattacc agatgatttt 300 acaggctgcg ttatagcttg gaattctaac aatcttgatt ctaaggttgg tggtaattat 360 aattacctgt atagattgtt taggaagtct aatctcaaac cttttgagag agatatttca 420 actgaaatct atcaggccgg tagcacacct tgtaatggtg ttgaaggttt taattgttac 480 tttcctttac aatcatatgg tttccaaccc actaatggtg ttggttacca accatacaga 540 gtagtagtac tttcttttga acttctacat gcaccagcaa ctgtttaa 588 <210> 7 <211> 1770 <212> DNA <213> Artificial Sequence <220> <223> COVID-19 spike protein S2 subunit <400> 7 atgagtgtag ctagtcaatc catcattgcc tacactatgt cacttggtgc agaaaattca 60 gttgcttact ctaataactc tattgccata cccacaaatt ttactattag tgttaccaca 120 gaaattctac cagtgtctat gaccaagaca tcagtagatt gtacaatgta catttgtggt 180 gattcaactg aatgcagcaa tcttttgttg caatatggca gtttttgtac acaattaaac 240 cgtgctttaa ctggaatagc tgttgaacaa gacaaaaaca cccaagaagt ttttgcacaa 300 gtcaaacaaa tttacaaaac accaccaatt aaagattttg gtggttttaa tttttcacaa 360 atattaccag atccatcaaa accaagcaag aggtcattta ttgaagatct acttttcaac 420 aaagtgacac ttgcagatgc tggcttcatc aaacaatatg gtgattgcct tggtgatatt 480 gctgctagag acctcatttg tgcacaaaag tttaacggcc ttactgtttt gccacctttg 540 ctcacagatg aaatgattgc tcaatacact tctgcactgt tagcgggtac aatcacttct 600 ggttggacct ttggtgcagg tgctgcatta caaataccat ttgctatgca aatggcttat 660 aggtttaatg gtattggagt tacacagaat gttctctatg agaaccaaaa attgattgcc 720 aaccaattta atagtgctat tggcaaaatt caagactcac tttcttccac agcaagtgca 780 cttggaaaac ttcaagatgt ggtcaaccaa aatgcacaag ctttaaacac gcttgttaaa 840 caacttagct ccaattttgg tgcaatttca agtgttttaa atgatatcct ttcacgtctt 900 gacaaagttg aggctgaagt gcaaattgat aggttgatca caggcagact tcaaagtttg 960 cagacatatg tgactcaaca attaattaga gctgcagaaa tcagagcttc tgctaatctt 1020 gctgctacta aaatgtcaga gtgtgtactt ggacaatcaa aaagagttga tttttgtgga 1080 aagggctatc atcttatgtc cttccctcag tcagcacctc atggtgtagt cttcttgcat 1140 gtgacttatg tccctgcaca agaaaagaac ttcacaactg ctcctgccat ttgtcatgat 1200 ggaaaagcac actttcctcg tgaaggtgtc tttgtttcaa atggcacaca ctggtttgta 1260 acacaaagga atttttatga accacaaatc attactacag acaacacatt tgtgtctggt 1320 aactgtgatg ttgtaatagg aattgtcaac aacacagttt atgatccttt gcaacctgaa 1380 ttagactcat tcaaggagga gttagataaa tattttaaga atcatacatc accagatgtt 1440 gatttaggtg acatctctgg cattaatgct tcagttgtaa acattcaaaa agaaattgac 1500 cgcctcaatg aggttgccaa gaatttaaat gaatctctca tcgatctcca agaacttgga 1560 aagtatgagc agtatataaa atggccatgg tacatttggc taggttttat agctggcttg 1620 attgccatag taatggtgac aattatgctt tgctgtatga ccagttgctg tagttgtctc 1680 aagggctgtt gttcttgtgg atcctgctgc aaatttgatg aagacgactc tgagccagtg 1740 ctcaaaggag tcaaattaca ttacacataa 1770 <210> 8 <211> 1260 <212> DNA <213> Artificial Sequence <220> <223> COVID-19 nucleocapsid protein <400> 8 atgtctgata atggacccca aaatcagcga aatgcacccc gcattacgtt tggtggaccc 60 tcagattcaa ctggcagtaa ccagaatgga gaacgcagtg gggcgcgatc aaaacaacgt 120 cggccccaag gtttacccaa taatactgcg tcttggttca ccgctctcac tcaacatggc 180 aaggaagacc ttaaattccc tcgaggacaa ggcgttccaa ttaacaccaa tagcagtcca 240 gatgaccaaa ttggctacta ccgaagagct accagacgaa ttcgtggtgg tgacggtaaa 300 atgaaagatc tcagtccaag atggtatttc tactacctag gaactgggcc agaagctgga 360 cttccctatg gtgctaacaa agacggcatc atatgggttg caactgaggg agccttgaat 420 acaccaaaag atcacattgg cacccgcaat cctgctaaca atgctgcaat cgtgctacaa 480 cttcctcaag gaacaacatt gccaaaaggc ttctacgcag aagggagcag aggcggcagt 540 caagcctctt ctcgttcctc atcacgtagt cgcaacagtt caagaaattc aactccaggc 600 agcagtaggg gaacttctcc tgctagaatg gctggcaatg gcggtgatgc tgctcttgct 660 ttgctgctgc ttgacagatt gaaccagctt gagagcaaaa tgtctggtaa aggccaacaa 720 caacaaggcc aaactgtcac taagaaatct gctgctgagg cttctaagaa gcctcggcaa 780 aaacgtactg ccactaaagc atacaatgta acacaagctt tcggcagacg tggtccagaa 840 caaacccaag gaaattttgg ggaccaggaa ctaatcagac aaggaactga ttacaaacat 900 tggccgcaaa ttgcacaatt tgcccccagc gcttcagcgt tcttcggaat gtcgcgcatt 960 ggcatggaag tcacaccttc gggaacgtgg ttgacctaca caggtgccat caaattggat 1020 gacaaagatc caaatttcaa agatcaagtc attttgctga ataagcatat tgacgcatac 1080 aaaacattcc caccaacaga gcctaaaaag gacaaaaaga agaaggctga tgaaactcaa 1140 gccttaccgc agagacagaa gaaacagcaa actgtgactc ttcttcctgc tgcagatttg 1200 gatgatttct ccaaacaatt gcaacaatcc atgagcagtg ctgactcaac tcaggcctaa 1260 SEQUENCE LISTING <110> iNtRON Biotechnology, Co. Ltd. <120> Rapid Detection Method of COVID-19 Immunoglobulin G <130> KP090693 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 674 <212> PRT <213> Artificial Sequence <220> <223> COVID -19 spike protein S1 subunit <400> 1 Ser Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala 1 5 10 15 Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe 20 25 30 Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe 35 40 45 Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly 50 55 60 Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr 65 70 75 80 Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly 85 90 95 Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala 100 105 110 Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro 115 120 125 Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser 130 135 140 Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val 145 150 155 160 Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys 165 170 175 Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile 180 185 190 Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly 195 200 205 Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile 210 215 220 Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro 225 230 235 240 Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val 245 250 255 Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly 260 265 270 Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr 275 280 285 Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr 290 295 300 Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn 305 310 315 320 Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe 325 330 335 Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala 340 345 350 Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys 355 360 365 Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val 370 375 380 Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala 385 390 395 400 Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp 405 410 415 Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser 420 425 430 Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser 435 440 445 Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala 450 455 460 Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro 465 470 475 480 Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro 485 490 495 Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr 500 505 510 Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val 515 520 525 Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser 530 535 540 Asn Lys Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp 545 550 555 560 Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile 565 570 575 Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn 580 585 590 Thr Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu 595 600 605 Val Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val 610 615 620 Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile 625 630 635 640 Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly 645 650 655 Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg 660 665 670 Ala Arg <210> 2 <211> 194 <212> PRT <213> Artificial Sequence <220> <223> receptor binding domain for COVID-19 spike protein S1 subunit <400> 2 Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg 1 5 10 15 Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val 20 2 5 30 Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys 35 40 45 Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn 50 55 60 Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile 65 70 75 80 Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro 85 90 95 Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp 100 105 110 Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys 115 120 125 Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln 130 135 140 Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe 145 150 155 160 Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln 165 170 175 Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala 180 185 190 Thr Val <210> 3 <211> 588 <212> PRT <213> Artificial Sequence <220> <223> COVID-19 spike protein S2 subunit <400> 3 Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala 1 5 10 15 Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn 20 25 30 Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys 35 40 45 Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys 50 55 60 Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg 65 70 75 80 Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val 85 90 95 Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe 100 105 110 Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser 115 120 125 Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala 130 135 140 Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala 145 150 155 160 Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu 165 170 175 Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu 180 185 190 Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala 195 200 205 Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile 210 215 220 Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn 225 230 235 240 Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr 245 250 255 Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln 260 265 270 Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile 275 280 285 Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala 290 295 300 Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln 305 310 315 320 Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser 325 330 335 Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser 340 345 350 Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 355 360 365 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro 370 375 380 Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly 385 390 395 400 Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His 405 410 415 Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr 420 425 430 Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val 435 440 445 Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys 450 455 460 Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp 465 470 475 480 Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys 485 490 495 Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu 500 505 510 Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro 515 520 525 Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met 530 535 540 Val Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys 545 550 555 560 Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser 565 570 575 Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr 580 585 <210> 4 <211> 419 <212> PRT <213> Artificial Sequence <220> <223> COVID-19 nucleocapsid protein <400> 4 Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr 1 5 10 15 Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg 20 25 30 Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn 35 40 45 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu 50 55 60 Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro 65 70 75 80 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 85 90 95 Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr 100 105 110 Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln 145 150 155 160 Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser 165 170 175 Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn 180 185 190 Ser Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala 195 200 205 Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu 210 215 220 Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln 225 230 235 240 Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys 245 250 255 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln 260 265 270 Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp 275 280 285 Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile 290 295 300 Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile 305 310 315 320 Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala 325 330 335 Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu 340 345 350 Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro 355 360 365 Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln 370 375 380 Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu 385 390 395 400 Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser 405 410 415 Thr Gln Ala <210> 5 <211> 2028 <212> DNA <213> Artificial Sequence < 220> <223> COVID-19 spike protein S1 subunit <400> 5 atgtctagtc agtgtgttaa tcttacaacc agaactcaat taccccctgc atacactaat 60 tctttcacac gtggtgttta ttaccctgac aaagt tttca gatcctcagt tttacattca 120 actcaggact tgttcttacc tttcttttcc aatgttactt ggttccatgc tatacatgtc 180 tctgggacca atggtactaa gaggtttgat aaccctgtcc taccatttaa tgatggtgtt 240 tattttgctt ccactgagaa gtctaacata ataagaggct ggatttttgg tactacttta 300 gattcgaaga cccagtccct acttattgtt aataacgcta ctaatgttgt tattaaagtc 360 tgtgaatttc aattttgtaa tgatccattt ttgggtgttt attaccacaa aaacaacaaa 420 agttggatgg aaagtgagtt cagagtttat tctagtgcga ataattgcac ttttgaatat 480 gtctctcagc cttttcttat ggaccttgaa ggaaaacagg gtaatttcaa aaatcttagg 540 gaatttgtgt ttaagaatat tgatggttat tttaaaatat attctaagca cacgcctatt 600 aatttagtgc gtgatctccc tcagggtttt tcggctttag aaccattggt agatttgcca 660 ataggtatta acatcactag gtttcaaact ttacttgctt tacatagaag ttatttgact 720 cctggtgatt cttcttcagg ttggacagct ggtgctgcag cttattatgt gggttatctt 780 caacctagga cttttctatt aaaatataat gaaaatggaa ccattacaga tgctgtagac 840 tgtgcacttg accctctctc agaaacaaag tgtacgttga aatccttcac tgtagaaaaa 900 ggaatctatc aaacttctaa ctttagagtc caaccaacag aatctattgt tag atttcct 960 aatattacaa acttgtgccc ttttggtgaa gtttttaacg ccaccagatt tgcatctgtt 1020 tatgcttgga acaggaagag aatcagcaac tgtgttgctg attattctgt cctatataat 1080 tccgcatcat tttccacttt taagtgttat ggagtgtctc ctactaaatt aaatgatctc 1140 tgctttacta atgtctatgc agattcattt gtaattagag gtgatgaagt cagacaaatc 1200 gctccagggc aaactggaaa gattgctgat tataattata aattaccaga tgattttaca 1260 ggctgcgtta tagcttggaa ttctaacaat cttgattcta aggttggtgg taattataat 1320 tacctgtata gattgtttag gaagtctaat ctcaaacctt ttgagagaga tatttcaact 1380 gaaatctatc aggccggtag cacaccttgt aatggtgttg aaggttttaa ttgttacttt 1440 cctttacaat catatggttt ccaacccact aatggtgttg gttaccaacc atacagagta 1500 gtagtacttt cttttgaact tctacatgca ccagcaactg tttgtggacc taaaaagtct 1560 actaatttgg ttaaaaacaa atgtgtcaat ttcaacttca atggtttaac aggcacaggt 1620 gttcttactg agtctaacaa aaagtttctg cctttccaac aatttggcag agacattgct 1680 gacactactg atgctgtccg tgatccacag acacttgaga ttcttgacat tacaccatgt 1740 tcttttggtg gtgtcagtgt tataacacca ggaacaaata cttctaacca ggttgctgtt 1800 ctttatcagg atgttaactg cacagaagtc cctgttgcta ttcatgcaga tcaacttact 1860 cctacttggc gtgtttattc tacaggttct aatgtttttc aaacacgtgc aggctgttta 1920 ataggggctg aacatgtcaa caactcatat gagtgtgaca tacccattgg tgcaggtata 1980 tgcgctagtt atcagactca gactaattct cctcggcggg cacgttaa 2028 <210> 6 <211> 588 <212> DNA <213> Artificial Sequence <220> <223 > receptor binding domain for COVID-19 spike protein S1 subunit <400> 6 atgaatatta caaacttgtg cccttttggt gaagttttta acgccaccag atttgcatct 60 gtttatgctt ggaacaggaa gagaatcagc aactgtgttg ctgattattc tgtcctatat 120 aattccgcat cattttccac ttttaagtgt tatggagtgt ctcctactaa attaaatgat 180 ctctgcttta ctaatgtcta tgcagattca tttgtaatta gaggtgatga agtcagacaa 240 atcgctccag ggcaaactgg aaagattgct gattataatt ataaattacc agatgatttt 300 acaggctgcg ttatagcttg gaattctaac aatcttgatt ctaaggttgg tggtaattat 360 aattacctgt atagattgtt taggaagtct aatctcaaac cttttgagag agatatttca 420 actgaaatct atcagggtccgg tagcacttt tgttttaat 480 taggtt tgttttaat accc actaatggtg ttggttacca accatacaga 540 gtagtagtac tttcttttga acttctacat gcaccagcaa ctgtttaa 588 <210> 7 <211> 1770 <212> DNA <213> Artificial Sequence <220> <223> COVID-19 spike protein S2 subunit <400> 7 atgagtgtag ctagtcaatc catcattgcc tacactatgt cacttggtgc agaaaattca 60 gttgcttact ctaataactc tattgccata cccacaaatt ttactattag tgttaccaca 120 gaaattctac cagtgtctat gaccaagaca tcagtagatt gtacaatgta catttgtggt 180 gattcaactg aatgcagcaa tcttttgttg caatatggca gtttttgtac acaattaaac 240 cgtgctttaa ctggaatagc tgttgaacaa gacaaaaaca cccaagaagt ttttgcacaa 300 gtcaaacaaa tttacaaaac accaccaatt aaagattttg gtggttttaa tttttcacaa 360 atattaccag atccatcaaa accaagcaag aggtcattta ttgaagatct acttttcaac 420 aaagtgacac ttgcagatgc tggcttcatc aaacaatatg gtgattgcct tggtgatatt 480 gctgctagag acctcatttg tgcacaaaag tttaacggcc ttactgtttt gccacctttg 540 ctcacagatg aaatgattgc tcaatacact tctgcactgt tagcgggtac aatcacttct 600 ggttggacct ttggtgcagg tgctgcatta caaataccat ttgctatgca aatggcttat 660 aggtttaatg gtattggagt tacacagaat gttctctatg agaaccaaaa attgattgcc 720 aaccaattta atagtgctat tggcaaaatt caagactcac tttcttccac agcaagtgca 780 cttggaaaac ttcaagatgt ggtcaaccaa aatgcacaag ctttaaacac gcttgttaaa 840 caacttagct cc aattttgg tgcaatttca agtgttttaa atgatatcct ttcacgtctt 900 gacaaagttg aggctgaagt gcaaattgat aggttgatca caggcagact tcaaagtttg 960 cagacatatg tgactcaaca attaattaga gctgcagaaa tcagagcttc tgctaatctt 1020 gctgctacta aaatgtcaga gtgtgtactt ggacaatcaa aaagagttga tttttgtgga 1080 aagggctatc atcttatgtc cttccctcag tcagcacctc atggtgtagt cttcttgcat 1140 gtgacttatg tccctgcaca agaaaagaac ttcacaactg ctcctgccat ttgtcatgat 1200 ggaaaagcac actttcctcg tgaaggtgtc tttgtttcaa atggcacaca ctggtttgta 1260 acacaaagga atttttatga accacaaatc attactacag acaacacatt tgtgtctggt 1320 aactgtgatg ttgtaatagg aattgtcaac aacacagttt atgatccttt gcaacctgaa 1380 ttagactcat tcaaggagga gttagataaa tattttaaga atcatacatc accagatgtt 1440 gatttaggtg acatctctgg cattaatgct tcagttgtaa acattcaaaa agaaattgac 1500 cgcctcaatg aggttgccaa gaatttaaat gaatctctca tcgatctcca agaacttgga 1560 aagtatgagc agtatataaa atggccatgg tacatttggc taggttttat agctggcttg 1620 attgccatag taatggtgac aattatgctt tgctgtatga ccagttgctg tagttgtctc 1680 aagggctgtt gttcttgtgg atcctgctgc aaatttgatg aagacgactc tgagccagtg 1740 ctcaaaggag tcaaattaca ttacacataa 1770 <210> 8 <211> 1260 <212> DNA <213> Artificial Sequence <220> <223> COVID-19 nucleocapsid protein <400> 8 accgta accagta atggcaccc acctgta accagta atggt ccagaatgga gaacgcagtg gggcgcgatc aaaacaacgt 120 cggccccaag gtttacccaa taatactgcg tcttggttca ccgctctcac tcaacatggc 180 aaggaagacc ttaaattccc tcgaggacaa ggcgttccaa ttaacaccaa tagcagtcca 240 gatgaccaaa ttggctacta ccgaagagct accagacgaa ttcgtggtgg tgacggtaaa 300 atgaaagatc tcagtccaag atggtatttc tactacctag gaactgggcc agaagctgga 360 cttccctatg gtgctaacaa agacggcatc atatgggttg caactgaggg agccttgaat 420 acaccaaaag atcacattgg cacccgcaat cctgctaaca atgctgcaat cgtgctacaa 480 cttcctcaag gaacaacatt gccaaaaggc ttctacgcag aagggagcag aggcggcagt 540 caagcctctt ctcgttcctc atcacgtagt cgcaacagtt caagaaattc aactccaggc 600 agcagtaggg gaacttctcc tgctgaatg gctggcaatg gcctgctgatgatt g acctgtgctagatgc 660 ttgctgatgattg t caaaa tgtctggtaa aggccaacaa 720 caacaaggcc aaactgtcac taagaaatct gctgctgagg cttctaagaa gcctcggcaa 780 aaacgtactg ccactaaagc atacaatgta acacaagctt tcggcagacg tggtccagaa 840 caaacccaag gaaattttgg ggaccaggaa ctaatcagac aaggaactga ttacaaacat 900 tggccgcaaa ttgcacaatt tgcccccagc gcttcagcgt tcttcggaat gtcgcgcatt 960 ggcatggaag tcacaccttc gggaacgtgg ttgacctaca caggtgccat caaattggat 1020 gacaaagatc caaatttcaa agatcaagtc attttgctga ataagcatat tgacgcatac 1080 aaaacattcc caccaacaga gcctaaaaag gacaaaaaga agaaggctga tgaaactcaa 1140 gccttaccgc agagacagaa gaaacagcaa actgtgactc ttcttcctgc tgcagatttg 1200gatgatttct ccaaacaatt gcaacaatcc atgagcagtg ctgactcaac tcaggcctaa 1260

Claims (11)

백킹 카드 위에 면역글로불린 G 검출목적의 COVID-19 바이러스 특이 항원이 처리된 2개의 검사선과 항-인간 면역글로불린 G에 대한 항체가 처리된 대조선을 순차적으로 포함하는 단일 멤브레인이 구비되고,
상기 멤브레인의 검사선 일측에는 상기 대조선의 항-인간 면역글로불린 G에 대한 항체와 반응하는 토끼 (Rabbit) 항-염소 항체 (Anti-goat antibody) 콘쥬게이트 (Conjugate)가 처리된 단일 접합체 패드가 형성되며, 상기 접합체 패드 위에 혈청 시료를 적하하는 단일 샘플 패드가 중첩되어 구비되며,
상기 멤브레인의 대조선 타측에는 백킹 카드 위에 반응이 종료된 혈청을 흡수하는 단일 흡수 패드가 구비되는 면역크로마토그래피를 이용한 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트.A single membrane is provided on the backing card sequentially comprising two test lines treated with a COVID-19 virus-specific antigen for the purpose of detecting immunoglobulin G and a control line treated with an antibody to anti-human immunoglobulin G,
A single conjugate pad treated with a rabbit anti-goat antibody conjugate that reacts with an antibody against anti-human immunoglobulin G of the control line is formed on one side of the test line of the membrane, , a single sample pad for dropping a serum sample on the conjugate pad is provided overlapping,
Immunoglobulin G rapid detection diagnostic kit for COVID-19 virus using immunochromatography provided on the other side of the control line of the membrane with a single absorbent pad absorbing the serum after the reaction has been completed on a backing card. 제 1항에 있어서, 상기 COVID-19 바이러스 특이 항원은 서열번호 1로 표시되는 COVID-19 spike protein S1 subunit, 서열번호 2로 표시되는 COVID-19 spike protein S1 subunit의 receptor binding domain, 서열번호 3으로 표시되는 COVID-19 spike protein S2 subunit, 또는 서열번호 4로 표시되는 COVID-19 nucleocapsid protein에서 선택되는 어느 하나 이상의 항원인 것을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트. According to claim 1, wherein the COVID-19 virus-specific antigen is a receptor binding domain of the COVID-19 spike protein S1 subunit represented by SEQ ID NO: 1, COVID-19 spike protein S1 subunit represented by SEQ ID NO: 2, SEQ ID NO: 3 Immunoglobulin G rapid detection diagnostic kit for COVID-19 virus, characterized in that it is any one or more antigens selected from the displayed COVID-19 spike protein S2 subunit, or the COVID-19 nucleocapsid protein represented by SEQ ID NO: 4. 제 1항에 있어서, 상기 2개의 검사선은 청구항 2의 COVID-19 바이러스 특이 항원 중 같은 종류의 항원으로 처리될 수 있는 것을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트.The diagnostic kit for rapid detection of immunoglobulin G for COVID-19 virus according to claim 1, wherein the two test lines can be treated with the same type of antigen among the COVID-19 virus-specific antigens of claim 2. 제 1항에 있어서, 상기 2개의 검사선은 청구항 2의 COVID-19 바이러스 특이 항원 중 서로 다른 종류의 항원으로 처리될 수 있는 것을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트.The diagnostic kit for rapid detection of immunoglobulin G for COVID-19 virus according to claim 1, wherein the two test lines can be treated with different types of antigens from among the COVID-19 virus-specific antigens of claim 2. 삭제delete 제 1항에 있어서, 상기 멤브레인은 니트로셀룰로오스 멤브레인인 것을 특징으로 하는 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트.The diagnostic kit for rapid detection of immunoglobulin G for COVID-19 virus according to claim 1, wherein the membrane is a nitrocellulose membrane. 삭제delete 제 1항, 제 2항 및 제 6항 중 어느 하나의 항에 의한 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트를 이용하여 생물학적 시료 내에 COVID-19 바이러스에 대한 면역글로불린 G가 포함되어 있는지 여부를 평가하는 것을 특징으로 하는 면역크로마토그래피법을 이용한 COVID-19 바이러스에 대한 면역글로불린 G 검출에 대한 정보를 제공하는 방법.Whether immunoglobulin G against COVID-19 virus is contained in the biological sample using the diagnostic kit for rapid detection of immunoglobulin G against COVID-19 virus according to any one of claims 1, 2 and 6 A method of providing information on the detection of immunoglobulin G for COVID-19 virus using immunochromatography, characterized in that it evaluates whether or not. 제 8항에 있어서, 상기 생물학적 시료는 전혈, 혈장, 또는 혈청인 것인, COVID-19 바이러스에 대한 면역글로불린 G 검출에 대한 정보를 제공하는 방법.The method of claim 8, wherein the biological sample is whole blood, plasma, or serum. COVID-19 바이러스의 양성 판정 오류 배제를 위한 정보를 제공하는 방법으로서,
(a) 제 3항에 의한 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트의 샘플 패드에 생물학적 시료를 적하하는 단계;
(b) 샘플 패드 및 대조선 사이의 2개의 검사선에 표시되는 발색 여부를 확인하는 단계를 포함하되, 2개의 검사선 모두에서 발색되는 경우를 COVID-19 바이러스의 양성 판정 오류가 배제된 판정으로 결정하는 것을 특징으로 하는, COVID-19 바이러스의 양성 판정 오류 배제를 위한 정보를 제공하는 방법.A method of providing information for excluding false positives for the COVID-19 virus, comprising:
(a) dropping a biological sample onto the sample pad of the diagnostic kit for rapid detection of immunoglobulin G for COVID-19 virus according to claim 3;
(b) determining whether the color is displayed on the two test lines between the sample pad and the control line, wherein the case of color development on both test lines is determined as a determination in which an error in positive determination of the COVID-19 virus is excluded A method of providing information for the exclusion of positive diagnosis errors of the COVID-19 virus, characterized in that it does. COVID-19 바이러스에 대한 항체 형성의 경중도를 판단하기 위한 정보를 제공하는 방법으로서,
(a) 제 3항에 의한 COVID-19 바이러스에 대한 면역글로불린 G 신속검출 진단키트의 샘플 패드에 생물학적 시료를 적하하는 단계;
(b) 샘플 패드 및 대조선 사이의 2개의 검사선에 표시되는 발색 여부를 확인하는 단계를 포함하되, 샘플 패드에서 가까운 검사선에서만 발생되는 경우를 COVID-19 바이러스에 대한 항체 형성이 경도라고 판단하고, 2개의 검사선 모두에서 발색되는 경우를 COVID-19 바이러스에 대한 항체 형성이 중도라고 판단하는 것을 특징으로 하는, COVID-19 바이러스에 대한 항체 형성의 경중도를 판단하기 위한 정보를 제공하는 방법.A method of providing information for judging the severity of antibody formation against COVID-19 virus, comprising:
(a) dropping a biological sample onto the sample pad of the diagnostic kit for rapid detection of immunoglobulin G for COVID-19 virus according to claim 3;
(b) determining whether the color is displayed on the two test lines between the sample pad and the control line, but if it occurs only on the test line close to the sample pad, it is determined that the formation of antibodies to the COVID-19 virus is mild, and , A method of providing information for judging the severity of antibody formation to the COVID-19 virus, characterized in that it is determined that the formation of antibodies to the COVID-19 virus is moderate when color is developed on both test lines.
KR1020200053003A 2020-05-01 2020-05-01 Rapid Detection Method of COVID-19 Immunoglobulin G KR102368133B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020200053003A KR102368133B1 (en) 2020-05-01 2020-05-01 Rapid Detection Method of COVID-19 Immunoglobulin G

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020200053003A KR102368133B1 (en) 2020-05-01 2020-05-01 Rapid Detection Method of COVID-19 Immunoglobulin G

Publications (2)

Publication Number Publication Date
KR20210134452A KR20210134452A (en) 2021-11-10
KR102368133B1 true KR102368133B1 (en) 2022-02-28

Family

ID=78500281

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020200053003A KR102368133B1 (en) 2020-05-01 2020-05-01 Rapid Detection Method of COVID-19 Immunoglobulin G

Country Status (1)

Country Link
KR (1) KR102368133B1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101782862B1 (en) 2016-04-26 2017-09-29 원광대학교산학협력단 Monoclonal antibody for diagnosing MERS virus and immunochromatographic diagnostic kit
CN111024954A (en) * 2020-03-09 2020-04-17 深圳市易瑞生物技术股份有限公司 Colloidal gold immunochromatography device for combined detection of COVID-19 antigen and antibody and use method thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101073984B1 (en) * 2008-07-10 2011-10-21 대한민국 Diagnostic kit and method for the rapid detection of Rabies antibodies using immunochromatography
KR20170139199A (en) * 2016-06-08 2017-12-19 베트올 (주) Diagnostic kits of the Foal Immunoglobulin G using semi-quantitative lateral flow immunoassay

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101782862B1 (en) 2016-04-26 2017-09-29 원광대학교산학협력단 Monoclonal antibody for diagnosing MERS virus and immunochromatographic diagnostic kit
CN111024954A (en) * 2020-03-09 2020-04-17 深圳市易瑞生物技术股份有限公司 Colloidal gold immunochromatography device for combined detection of COVID-19 antigen and antibody and use method thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Xingwang Jia et al, Frontiers in Medicine (2020.04.12.), vol 8, article 569266, pp 1-8.

Also Published As

Publication number Publication date
KR20210134452A (en) 2021-11-10

Similar Documents

Publication Publication Date Title
CN111089962A (en) Colloidal gold kit for joint detection of novel coronavirus IgM/IgG antibody and preparation method thereof
CN103140758B (en) Immunochromatography devices, methods and kits
CN103558400A (en) Procalcitonin latex enhanced immunoturbidimetry detection kit
CN113295865B (en) Immunochromatographic device for detecting SARS-CoV-2 virus neutralizing antibody and its use
JP7175147B2 (en) Immunological detection method and kit for Mycoplasma pneumoniae
CN108152506B (en) Influenza B virus IgA antibody immunofluorescence detection test strip and preparation method, detection method and application thereof
CN112334481A (en) Antibody pairs for rapid influenza B diagnostic testing
WO2021205228A1 (en) Assay device
KR101587645B1 (en) Multi-influenza Detection Kit and Method for Detecting Influenza Using the Same
KR100905066B1 (en) The recombinant proteins for the diagnosis of diseases infected from Mycoplasma pneumoniae and the diagnostic kits comprising the same
KR102368133B1 (en) Rapid Detection Method of COVID-19 Immunoglobulin G
CN112334478A (en) Antibody pairs for rapid influenza a diagnostic tests
Chaya et al. Evaluation of a newly designed sandwich enzyme linked immunosorbent assay for the detection of hydatid antigen in serum, urine and cyst fluid for diagnosis of cystic echinococcosis
KR101032956B1 (en) Rapid diagnostic kit of hemorrhagic fever with renal syndrome detecting specific IgM and IgG using nucleocapsid protein derived from Soochong virus
KR20240026885A (en) Antibodies against nucleocapsid protein of SARS-CoV-2 and their uses
KR102437114B1 (en) A severe acute respiratory syndrome coronavirus specific antigen tagged with streptavidin binding peptide and use thereof
KR20120028603A (en) Antibody for detecting hemagglutinin of swine influenza and use thereof
CN101561438A (en) Colloidal gold detection test paper for abrin, preparation method thereof and application thereof
JP2017133951A (en) Method for detecting specific bacteria in sample
KR102021540B1 (en) Peptides for diagnosis of mosquito-borne viruses and uses thereof
KR102021539B1 (en) Peptides for diagnosis of mosquito-borne viruses and uses thereof
JP6676387B2 (en) Immunochromatographic method for detecting specific bacteria and kit used therefor
KR102021538B1 (en) Peptides for diagnosis of mosquito-borne viruses and uses thereof
KR102467073B1 (en) Kit for detecting of antibodies in blood to Coronavirus
CN115877000A (en) Novel coronavirus neutralizing antibody detection kit

Legal Events

Date Code Title Description
E701 Decision to grant or registration of patent right
GRNT Written decision to grant