KR101849101B1 - Monoclonal antibody specific to hemagglutinin of H5 subtype influenza virus and rapid fluorescence-linked immunochromatographic diagnostic kit using the same - Google Patents
Monoclonal antibody specific to hemagglutinin of H5 subtype influenza virus and rapid fluorescence-linked immunochromatographic diagnostic kit using the same Download PDFInfo
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- KR101849101B1 KR101849101B1 KR1020160096152A KR20160096152A KR101849101B1 KR 101849101 B1 KR101849101 B1 KR 101849101B1 KR 1020160096152 A KR1020160096152 A KR 1020160096152A KR 20160096152 A KR20160096152 A KR 20160096152A KR 101849101 B1 KR101849101 B1 KR 101849101B1
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- monoclonal antibody
- antibody
- influenza virus
- virus
- influenza
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Abstract
본 발명은 H5 아형 인플루엔자 바이러스의 헤마글루티닌에 특이적인 단클론항체 및 이를 이용한 신속 형광면역 진단키트에 관한 것으로, 본 발명에서는 인플루엔자 A H5N1 바이러스 헤마글루티닌1 항원에 특이적으로 결합하는 단클론항체 및 하이브리도마 세포주를 제조하였으며 상기 단클론항체를 활용한 형광면역키트를 통해 H5 아형 인플루엔자 바이러스를 신속하게 검출할 수 있음을 확인하였다. 따라서, 본 발명의 단클론항체는 신속진단이 가능한 신속 키트에 사용될 수 있으며, 이런 항원-항체 진단키트는 국내 뿐 아니라 해외에서 인플루엔자가 발생하고 있거나 발생 가능성이 있는 국가에 보급함으로써 선두 주자로서의 시장 경쟁력을 확보할 수 있을 것으로 기대된다.The present invention relates to a hemagglutinin-specific monoclonal antibody of the H5 subtype influenza virus and a rapid fluorescence immunoassay kit using the same, wherein the monoclonal antibody specifically binding to the influenza A H5N1 virus hemaglutinin 1 antigen And a hybridoma cell line were prepared, and it was confirmed that H5 subtype influenza virus can be rapidly detected through the fluorescent immuno kit using the above monoclonal antibody. Accordingly, the monoclonal antibody of the present invention can be used in a rapid kit capable of rapid diagnosis. Such an antigen-antibody diagnostic kit can be marketed as a leader by spreading it to countries where influenza is present or may occur, It is expected to be secured.
Description
본 발명은 H5 아형 인플루엔자 바이러스의 헤마글루티닌에 특이적인 단클론항체 및 이를 이용한 신속 형광면역 진단키트에 관한 것이다.The present invention relates to a hemagglutinin-specific monoclonal antibody of H5 subtype influenza virus and a rapid fluorescence immunoassay kit using the same.
매년, 계절적 인플루엔자는 U.S. 단독에서 300,000 입원 및 36,000 사망의 원인이 된다. 인플루엔자(influenza)는 인플루엔자 바이러스에 의한 호흡기 질환이다. 원인병원체인 인플루엔자 바이러스는 구형(직경 80~120nm)의 바이러스로 오소믹소바이러스 패밀리(Orthomyxoviridae family)의 구성원이다. 인플루엔자 바이러스 입자는 하기의 단백질을 암호화하는, 부분으로 나누어진 네거티브-센스-RNA 유전체를 포함한다: 헤마글루티닌(Hemagglutinin, HA), 뉴라미니다제(neuraminidase, NA), 매트릭스(Matrix, M1), 양성자 이온-채널 단백질(M2), 뉴클레오단백질(NP), 폴리머라제 염기 단백질 1(PB1), 폴리머라제 염기 단백질 2(PB2), 폴리머라제 산성 단백질(PA) 및 비구조적 단백질 2(NS2). HA, NA, M1 및 M2는 멤브레인과 연관된 반면에, NP, PB1, PB2, PA 및 NS2는 뉴클레오캡시드와 연관된 단백질이다. M1 단백질은 인플루엔자 입자에서 가장 풍부한 단백질이다. HA 및 NA 단백질은 세포로 바이러스 입자의 바이러스 부착 및 침투에 대해 책임지고, 바이러스 중성화 및 면역을 보호하기 위한 주요 면역우성항원결정기의 원천인 외피(envelope) 당단백질이다. HA 및 NA 단백질 모두는 예방 인플루엔자를 위한 가장 중요한 구성요소로 고려된다. Every year, seasonal influenza is U.S. It alone causes 300,000 hospitalizations and 36,000 deaths. Influenza (influenza) is a respiratory disease caused by influenza virus. Influenza virus, a causative agent, is a globular virus (80-120 nm in diameter) and is a member of the family Orthomyxoviridae. Influenza virus particles include a subdivided negative-sense-RNA genome that encodes the following proteins: Hemagglutinin (HA), neuraminidase (NA), Matrix (M1, ), Proton ion channel protein (M2), nucleoprotein (NP), polymerase base protein 1 (PB1), polymerase base protein 2 (PB2), polymerase acidic protein (PA) ). HA, NA, M1 and M2 are associated with membranes, whereas NP, PB1, PB2, PA and NS2 are proteins associated with nucleocapsids. The M1 protein is the most abundant protein in influenza particles. The HA and NA proteins are envelope glycoproteins that are responsible for viral attachment and penetration of viral particles into cells, and are the source of the major immunoassay antigenic determinants for viral neutralization and immunity protection. Both HA and NA proteins are considered to be the most important components for preventive influenza.
인플루엔자 바이러스는 항원의 차이에 기초하여 A형, B형 및 C형으로 분류된다. 인플루엔자 A 바이러스는 사람뿐만 아니라 조류 및 돼지도 감염시킨다. C형 인플루엔자는 사람에서 경미한 질병만 일으킨다. 인플루엔자 A 바이러스는 서브타입 또는 타입, 지리적 기원, 균주 번호 및 단리 연도를 포함하는 명명법에 의하여, 예를 들면 A/베이징/353/89로 기술된다. 또한, 인플루엔자 A 바이러스는 표면항원인 헤마글루타닌(HA) 항원과 뉴라미다아제(NA) 항원에 의해 아형이 결정된다. HA 항원은 H1-H16까지 알려져 있으며, NA 항원은 N1-N9까지 알려져 있어, 이들의 조합에 의해 총 144종의 아형 발생이 이론적으로 가능하다. 인플루엔자 A 바이러스의 유전자는 8개의 유전자 조각으로 구성되어 있으며, 이러한 이유로 유전자 재조합이 일어날 수 있다. 모든 서브타입은 조류에서 발견되나, H1-H3 및 N1-N2는 인간, 돼지 및 말에서 발견되는 것으로 알려져 있다. Influenza viruses are classified as type A, type B, and type C based on differences in antigen. Influenza A virus infects not only humans but also birds and swine. Influenza C causes only mild illness in humans. Influenza A viruses are described by nomenclature including subtype or type, geographic origin, strain number and isolation date, for example, A / Beijing / 353/89. Influenza A virus is subtyped by the surface antigen hemagglutinin (HA) antigen and the neuramidase (NA) antigen. The HA antigen is known to H1-H16, and the NA antigen is known to N1-N9. Therefore, a total of 144 subtypes are theoretically possible by the combination of these. The gene for influenza A virus is composed of 8 gene fragments, and for this reason, genetic recombination can occur. All subtypes are found in birds, but H1-H3 and N1-N2 are known to be found in humans, pigs and horses.
한편, H5 아형에 속하는 조류 인플루엔자 바이러스는 2003년경으로부터 아시아를 중심으로 인간에의 감염과 그 치사율의 높이에 관해서 많이 보고되어 있고, 이들 바이러스에 대하여 인간은 면역력을 갖지 않기 때문에 폭발적으로 유행이 되는 것이 세계적으로 우려되고 있어, 그 예방 및 유행시의 대책에 대해서 검토되고 있다. H5N1은 치사율이 주로 10-19세의 젊은 세대에게 심각한 호흡기 질환을 일으키고 있고. 대부분 심각한 호흡기 질환 말기에 병원에 입원한 환자에게서 검출되고 있어, 경미하게 아픈 사람의 경우, 진단이 안되어, 보고되지 않을 확률도 존재하여, 치사율이 높은 병원체임에도 불구하고, 실제적으로 발병률이 더 높을 수 있을 것으로 추측되고 있다. 주된 감염은 가축을 취급하는 사람이, 가축으로부터 사람으로 전파되고 있고, 최근에도 베트남, 아프리카 등은 지속적인 가축 발병을 초래하고 있고 최근에 가축에서 H5N9까지 신변종으로 발병하고, 새로운 고병원성 조류인플루엔자로 분류되고 있어 H5 아형 바이러스의 진단법 개발이 시급한 상황이다. On the other hand, the avian influenza virus (H5) belonging to the subtype H5 has been reported to have a high incidence of infection and mortality in humans, mainly in Asia, from around 2003. Since humans do not have immunity against these viruses, It is concerned about the world, and the prevention and the measures at the time of the epidemic are being examined. H5N1 is a serious respiratory illness that affects the younger generation, predominantly in the 10-19 age group. Most people with severe respiratory illness are detected in hospitalized patients, and in the case of a mildly ill person, there is a possibility that the diagnosis is not made and is not reported, so that the incidence of the disease may actually be higher It is estimated that there will be. The main infection is spreading from livestock to people who handle livestock. In recent years, Vietnam and Africa have caused ongoing livestock outbreaks, and recent onset of livestock to H5N9 has led to the development of new species, and classified as new highly pathogenic avian influenza And it is urgent to develop a diagnostic method for H5 subtype virus.
사람에 대한 H5N1을 특이적으로 진단하는 조류 인플루엔자 진단법은 가장 민감한 방법으로 바이러스 분리 및 배양법이 있으나 2-3주 걸리고, 혹은 유전자 진단법으로 RNA 분리 후 rRT-PCR를 수행하게 되지만, RNA의 불안정성, 고가의 장비 필요, 숙련공이 필요하여 현장에서 진단을 신속하게 수행하기에 단점이 있다. 또한, 현재 인플루엔자 신속 진단법은 약 80% 민감도밖에 되지 않으므로 바이러스 검출율이 높고, 고병원성 H5 특이적으로 진단하는 고민감한 항체 및 이를 활용한 신속진단시스템 개발이 필요하다.Although avian influenza detection methods that specifically diagnose H5N1 in humans are the most sensitive method, virus isolation and culture methods exist, but it takes 2-3 weeks, or rRT-PCR is performed after RNA isolation by genetic diagnosis method, but RNA instability, Equipment needs, skilled workers need to be able to quickly perform the diagnosis on the spot. In addition, since the rapid diagnosis method of influenza is currently only about 80% sensitive, it is necessary to develop a highly sensitive antibody which has a high virus detection rate, highly pathogenic H5 specific diagnosis and a rapid diagnosis system utilizing the antibody.
한편, 한국등록특허 제1628331호에서는 '인플루엔자 A 바이러스 특이적 단클론 항체 및 이를 이용한 인플루엔자 감염 치료 및 진단 방법'이 개시되어 있고, 한국등록특허 제1625979호에서는 '항 Ｈ5 아형 Ａ형 인플루엔자 바이러스 헤마글루티닌 모노클로날 항체'가 개시되어 있으나, 본 발명에서와 같이 'H5 아형 인플루엔자 바이러스의 헤마글루티닌에 특이적인 단클론항체 및 이를 이용한 신속형광면역 진단키트'에 대해서는 개시된 바가 없다.Korean Patent No. 1628331 discloses a method of treating and diagnosing influenza infection using influenza A virus-specific monoclonal antibodies, and Korean Patent No. 1625979 discloses an anti-H5 subtype influenza virus hemagglutinin Nin monoclonal antibody 'has been disclosed. However, as described in the present invention, there is no disclosure of a' mAb specific for hemaglutinin of H5 subtype influenza virus and a rapid fluorescence immunoassay kit using the same.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명에서는 인플루엔자 A H5N1 바이러스 헤마글루티닌1 항원에 특이적으로 결합하는 단클론항체 및 하이브리도마 세포주를 제조하였다. 상기 단클론항체를 활용한 형광면역키트를 통해 H5 아형 인플루엔자 바이러스를 신속하게 검출할 수 있음을 확인함으로써, 본 발명을 완성하였다.The present invention has been made in view of the above-mentioned needs. In the present invention, monoclonal antibodies and hybridoma cell lines specifically binding to the influenza A
상기 과제를 해결하기 위해, 본 발명은 기탁번호가 KCTC 13068BP인 하이브리도마 세포주에 의하여 생산되고, 서열번호 1의 아미노산 서열로 이루어진 인플루엔자 A H5N1 바이러스 헤마글루티닌(hemagglutinin)에 특이적으로 결합하는 단클론항체를 제공한다.In order to solve the above problem, the present invention relates to a method for producing an influenza A H5N1 virus hemagglutinin, which is produced by a hybridoma cell line with accession number KCTC 13068BP and which comprises the amino acid sequence of SEQ ID NO: 1 Monoclonal antibody.
또한, 본 발명은 기탁번호가 KCTC 13068BP인 하이브리도마 세포주를 제공한다.In addition, the present invention provides a hybridoma cell line wherein the accession number is KCTC 13068BP.
또한, 본 발명은 상기 단클론항체를 포함하는 H5 아형 인플루엔자 바이러스 검출용 조성물을 제공한다.In addition, the present invention provides a composition for detecting H5 subtype influenza virus comprising the above monoclonal antibody.
또한, 본 발명은 상기 단클론항체를 시료 샘플과 접촉시켜 형성된 항원-항체 복합체를 검출하는 단계를 포함하는 H5 아형 인플루엔자 바이러스 검출방법을 제공한다.In addition, the present invention provides a method for detecting H5 subtype influenza virus comprising the step of detecting an antigen-antibody complex formed by contacting a monoclonal antibody with a sample.
또한, 본 발명은 상기 단클론항체를 포함하는 H5 아형 인플루엔자 바이러스 검출용 키트를 제공한다.The present invention also provides a kit for detecting H5 subtype influenza virus comprising the above monoclonal antibody.
본 발명에서 개발된 단클론항체는 신속진단이 가능한 신속 키트에 사용될 수 있으며, 이런 항원-항체 진단키트는 국내 뿐 아니라 해외에서 인플루엔자가 발생하고 있거나 발생 가능성이 있는 국가에 보급함으로써 선두 주자로서의 시장 경쟁력을 확보할 수 있을 것으로 기대된다.The monoclonal antibody developed in the present invention can be used in a rapid kit capable of rapid diagnosis. Such an antigen-antibody diagnostic kit can be marketed as a leader by spreading it to countries where influenza is present or may occur, It is expected to be secured.
도 1은 인플루엔자 A H5N1 바이러스의 HA1 재조합항원으로 면역된 마우스(M)의 혈청내 항체가를 ELISA법으로 측정한 결과이다. 음성대조군으로는 PBS(인산완충액, pH 7.4)와 건강한 마우스의 혈청(Normal, 1:200 희석)을 사용하였다.
도 2는 세포융합 후 96-웰 플레이트와 75T 배양 플라스크에서 배양된 하이브리도마 세포(hybridoma cell)(1E12)로부터 분비한 항체가를 ELISA법으로 측정한 결과이다. 양성대조군(positive)으로 면역된 마우스의 혈청(1:200으로 희석)을 사용하였으며, 음성대조군으로 PBS(인산완충액, pH 7.4)와 건강한 마우스의 혈청(Normal, 1:200 희석)을 사용하였다.
도 3은 제한개수희석법(limiting dilution)을 수행한 후 96-웰 플레이트와 75T 배양 플라스크에서 배양된 단클론세포주(1E12)로부터 분비한 항체가 측정 결과이다. 양성대조군으로 면역된 마우스의 혈청(1:200으로 희석)을 사용하였으며, 음성대조군으로 PBS(인산완충액, pH 7.4)와 건강한 마우스의 혈청(Normal, 1:200 희석)을 사용하였다.
도 4는 본 발명에 따른 단클론항체 1E12의 중쇄(heavy chain) 및 경쇄(light chain) 양상을 SDS-PAGE(전기영동법)으로 확인한 결과이다. M은 단백질 마커를 나타낸다.
도 5는 본 발명에 따른 단클론항체 1E12 및 상용화 항체 1C5의 인플루엔자 H5N3 바이러스에 대한 반응성을 웨스턴 블롯으로 확인한 결과이다. M은 단백질 마커를 나타낸다.
도 6은 본 발명에 따른 단클론항체 1E12 및 상용화 항체 1C5의 인플루엔자 H5N3 바이러스에 대한 반응성을 indirect ELISA를 통해 확인한 결과이다.
도 7은 본 발명에 따른 항체를 이용한 H5 아형 인플루엔자 바이러스 검출용 신속 형광면역진단시스템(형광면역크로마토그라피법 스트립 형태)의 구성 모식도를 나타낸다.
도 8은 본 발명에 따른 신속 형광면역진단시스템의 서로 다른 아형(H5N3, H1N1, H7N1) 바이러스에 대한 검출 성능을 확인한 결과이다.Fig. 1 shows the result of ELISA assay for antibody titers in the serum of mouse (M) immunized with HA1 recombinant antigen of influenza A H5N1 virus. As a negative control, PBS (phosphate buffer, pH 7.4) and healthy mouse serum (Normal, 1: 200 dilution) were used.
FIG. 2 shows the result of ELISA measurement of the antibody released from hybridoma cells (1E12) cultured in a 96-well plate and a 75T culture flask after cell fusion. Serum (1: 200 dilution) of positive control immunized mice was used and PBS (phosphate buffer, pH 7.4) and healthy mouse serum (Normal, 1: 200 dilution) were used as negative controls.
FIG. 3 shows the results of measurement of antibody secreted from a monoclonal cell line (1E12) cultured in a 96-well plate and a 75T culture flask after performing limiting dilution. Serum (1: 200 dilution) of mice immunized with a positive control was used and PBS (phosphate buffer, pH 7.4) and healthy mouse serum (Normal, 1: 200 dilution) were used as negative controls.
FIG. 4 shows the result of SDS-PAGE (electrophoresis) of the heavy chain and light chain of the monoclonal antibody 1E12 according to the present invention. M represents a protein marker.
FIG. 5 shows the result of Western blotting the reactivity of the monoclonal antibody 1E12 and the compatibilizing antibody 1C5 according to the present invention to the influenza H5N3 virus. M represents a protein marker.
FIG. 6 shows the results of confirming the reactivity of the monoclonal antibody 1E12 and the compatibilizing antibody 1C5 according to the present invention to the influenza H5N3 virus through an indirect ELISA.
FIG. 7 is a schematic diagram showing the composition of a rapid fluorescence immunoassay system (in the form of a fluorescent immunoassay method strip) for detection of H5 subtype influenza virus using an antibody according to the present invention.
FIG. 8 shows the results of confirming the detection performance of different rapid subtypes (H5N3, H1N1 and H7N1) of the rapid fluorescence immunoassay system according to the present invention.
본 발명의 목적을 달성하기 위하여, 본 발명은 기탁번호가 KCTC 13068BP인 하이브리도마 세포주에 의하여 생산되고, 서열번호 1의 아미노산 서열로 이루어진 인플루엔자 A H5N1 바이러스 헤마글루티닌1(hemagglutinin1, HA1)에 특이적으로 결합하는 단클론항체를 제공한다.In order to accomplish the object of the present invention, the present invention relates to an influenza A H5N1 virus hemagglutinin 1 (HA1), which is produced by a hybridoma cell line having a deposit number of KCTC 13068BP and is composed of the amino acid sequence of SEQ ID NO: 1 Thereby providing a specifically binding monoclonal antibody.
헤마글루티닌(hemagglutinin, HA)는 인플루엔자 바이러스의 표면에서 발견되는 항원성 당단백질이다. HA은 인플루엔자 바이러스 표면에 있는 주요 단백질로 3개의 단량체로 구성된 삼량체 형태이다. 각 단량체는 이황화결합으로 연결된 HA1과 HA2의 두 개의 도메인으로 구성되어 있으며, 구조적으로 둥근 머리 부분과 줄기 부분으로 나누어 볼 수 있다. HA1은 시알릭산(sialic acid)을 포함하는 수용체와 결합하여 숙주 세포를 인식하고, HA2는 수용체를 포함하는 숙주세포 막과 HA1을 포함하는 바이러스 막의 융합을 일으켜 숙주 세포에 바이러스 게놈을 삽입하는 역할을 수행한다.Hemagglutinin (HA) is an antigenic glycoprotein found on the surface of influenza viruses. HA is a major protein on the surface of influenza virus and is in the form of a trimer consisting of three monomers. Each monomer is composed of two domains, HA1 and HA2, linked by disulfide bonds, and can be structurally divided into a round head and a stem. HA1 binds to receptors containing sialic acid to recognize host cells and HA2 plays a role of inserting a viral genome into a host cell by causing the fusion of a host cell membrane containing a receptor and a HA1 containing virus membrane .
상기 단클론항체는 H5 아형 인플루엔자 바이러스의 HA1에 특이적으로 결합할 수 있으며, 바람직하게는 서열번호 1의 아미노산 서열로 이루어진 인플루엔자 A H5N1 바이러스의 HA1에 특이적으로 결합할 수 있다.The monoclonal antibody can specifically bind to HA1 of the H5 subclass influenza virus, and can specifically bind HA1 of the influenza A H5N1 virus comprising the amino acid sequence of SEQ ID NO: 1.
본 발명에서 용어 '단클론항체'란 당해 분야에 공지된 용어로서 단일 항원성 부위에 대해서 지시되는 고도의 특이적인 항체를 의미한다. 단클론항체는 단세포군 항체라고 불리기도 한다. 통상적으로, 상이한 에피토프(항원결정기)들에 대해 지시되는 상이한 항체들을 포함하는 다클론항체와는 다르게, 단클론항체는 항원상의 단일 결정기에 대해서 지시된다. 단클론항체는 항원-항체 결합을 이용하는 진단 및 분석학적 분석법의 선택성과 특이성을 개선시키는 장점이 있으며, 또한 하이브리도마 배양에 의해 합성되기 때문에 다른 면역글로불린에 의해 오염되지 않는 또 다른 장점을 갖는다.As used herein, the term " monoclonal antibody " refers to a highly specific antibody directed against a single antigenic site as is known in the art. Monoclonal antibodies are sometimes referred to as monoclonal antibody. Typically, unlike polyclonal antibodies, which contain different antibodies directed against different epitopes (antigenic determinants), monoclonal antibodies are directed against a single determinant on the antigenic surface. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays that utilize antigen-antibody binding and have another advantage that they are not contaminated by other immunoglobulins because they are synthesized by hybridoma cultures.
상기한 하이브리도마가 생산하는 단클론항체는 정제하지 않고 사용할 수도 있으나, 최선의 결과를 얻기 위해서는 본 발명이 속하는 기술분야에 잘 알려져 있는 방법에 따라 고순도(예컨대, 95% 이상)로 정제하여 사용하는 것이 바람직하다. 이러한 정제 기술로는, 예를 들어 겔 전기영동, 투석, 염 침전, 이온교환 크로마토 그래피, 친화성 크로마토그래피 등의 정제 방법을 이용하여 배양 배지 또는 복수액으로부터 분리될 수 있다.The monoclonal antibody produced by the hybridoma may be used without purification. However, in order to obtain the best results, the monoclonal antibody produced by the hybridoma may be purified and used in high purity (for example, 95% or more) according to a method well known in the technical field of the present invention . Such a purification technique can be separated from the culture medium or the plural liquids using a purification method such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography and the like.
본 발명의 일 구현예에서, 본 발명의 단클론항체는 인플루엔자 A H5N1 바이러스의 HA1 항원을 사용하여 면역된 마우스 비장세포로부터 세포융합기술에 의해 획득한 하이브리도마 세포로부터 생산되는 항체를 제작하였으며, 이를 1E12로 명명하였다.In one embodiment of the present invention, the monoclonal antibody of the present invention produced an antibody produced from hybridoma cells obtained by cell fusion technique from immunized mouse spleen cells using HA1 antigen of influenza A H5N1 virus, 1E12.
상기 단클론항체는 IgG2a-카파의 면역글로불린 아이소타입(isotype)일 수 있다.The monoclonal antibody may be an immunoglobulin isotype of IgG2a-kappa.
또한, 본 발명은 상기 단클론항체(1E12)를 생산하는 기탁번호가 KCTC 13068BP인 하이브리도마 세포주를 제공한다.In addition, the present invention provides a hybridoma cell line wherein the accession number for producing said monoclonal antibody (1E12) is KCTC 13068BP.
본 발명의 단클론항체(1E12)는 한국생명공학연구원에 2016년 7월 22일자로 기탁된 KCTC 13068BP인 하이브리도마 세포주로부터 제조된 것이다.The monoclonal antibody (1E12) of the present invention was prepared from a hybridoma cell line, KCTC 13068BP deposited on July 22, 2016 at the Korea Biotechnology Research Institute.
또한, 본 발명은 상기 단클론항체를 포함하는 H5 아형 인플루엔자 바이러스 검출용 조성물을 제공한다.In addition, the present invention provides a composition for detecting H5 subtype influenza virus comprising the above monoclonal antibody.
상기 조성물은 유효성분으로 기탁번호가 KCTC 13068BP인 하이브리도마 세포주에 의하여 생산되고, 서열번호 1의 아미노산 서열로 이루어진 인플루엔자 A H5N1 바이러스 헤마글루티닌1(hemagglutinin1, HA1)에 특이적으로 결합하는 단클론항체를 포함하며, 상기 항체를 이용하여 인플루엔자 바이러스 HA1 항원에 대해 형성된 항원-항체 복합체를 검출함으로써 H5 아형 인플루엔자 바이러스를 면역학적으로 검출할 수 있는 것이다.The composition is produced by a hybridoma cell line having an accession number of KCTC 13068BP as an active ingredient, and is a monoclonal antibody that specifically binds to influenza A H5N1 virus hemagglutinin 1 (HA1) consisting of the amino acid sequence of SEQ ID NO: 1 And detecting the H5 subtype influenza virus by detecting the antigen-antibody complex formed against the influenza virus HA1 antigen using the antibody.
또한, 본 발명은 상기 단클론항체를 시료 샘플과 접촉시켜 형성된 항원-항체 복합체를 검출하는 단계를 포함하는 H5 아형 인플루엔자 바이러스 검출방법을 제공한다.In addition, the present invention provides a method for detecting H5 subtype influenza virus comprising the step of detecting an antigen-antibody complex formed by contacting a monoclonal antibody with a sample.
본 발명에서 용어 "항원-항체 복합체"란, 시료 중의 H5 아형 인플루엔자 바이러스 HA1 항원과 이를 인지하는 본 발명에 따른 단일클론 항체 또는 이의 절편의 결합물을 의미하며, 이러한 항원-항체 복합체는 비색법(colormetric method), 전기화학법(electrochemical method), 형광법(fluorimetric method), 발광법(luminometry), 입자계수법(particle counting method), 육안측정법(visual assessment) 및 섬광계수법(scintillation counting method)으로 이루어진 군에서 선택되는 임의의 방법으로 검출할 수 있다. 그러나 반드시 이들로만 제한되지 않고 다양한 응용과 적용이 가능하다.The term "antigen-antibody complex " in the present invention refers to a combination of the H5 subclass influenza virus HA1 antigen in the sample and the monoclonal antibody or a fragment thereof according to the present invention. The antigen- a method selected from the group consisting of electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment and scintillation counting method Or the like. However, various applications and applications are possible without being limited thereto.
본 발명에서는 항원-항체 복합체를 검출하기 위한 것으로 여러가지 표지체를 사용할 수 있다. 구체적인 예로는 효소, 형광물, 리간드, 발광물, 미소입자, 방사성 동위원소로 이루어진 그룹 중에서 선택될 수 있으며, 반드시 이들로만 한정되는 것은 아니다.In the present invention, various markers can be used for detecting an antigen-antibody complex. Specific examples include, but are not limited to, enzymes, chromophores, ligands, luminescent materials, microparticles, and radioactive isotopes.
검출 표지체로서 사용되는 바람직한 효소로는 아세틸콜린에스테라제, 알칼라인 포스파타제, β-D-갈락토시다제, 호스래디쉬 퍼옥시다제 또는 β-락타마제가 있으며, 바람직한 형광물로는 양자점, 플루오레세인, Eu3 +, Eu3 + 킬레이트 또는 크립테이트가 있으며, 바람직한 리간드로는 바이오틴 유도체가 있고, 바람직한 발광물로는 아크리디늄 에스테르 또는 이소루미놀 유도체가 있으며, 바람직한 미소입자로는 콜로이드 금 또는 착색된 라텍스가 있고, 바람직한 방사성 동위원소로는 57Co, 3H, 125I 또는 125I-볼톤(Bolton) 헌터(Hunter) 시약이 있으나, 이에 한정하는 것은 아니다.Preferable enzymes to be used as the detection label include acetylcholinesterase, alkaline phosphatase,? -D-galactosidase, horseradish peroxidase or? -Lactamase, and preferable examples of the minerals include quantum dots, fluorescein, Eu 3 +, Eu 3 +, and a chelating or creep Tate, preferred ligands may have biotin derivatives, preferred as luminescent substances are acridinium and the ester or isobutyl luminol derivatives, preferred microparticles include colloidal gold or There are colored latexes, and preferred radioisotopes include, but are not limited to, 57 Co, 3 H, 125 I or 125 I Bolton Hunter reagents.
항원-항체 복합체를 검출하는 방법은 바람직하게는 효소면역흡착법(ELISA)을 이용하여 검출할 수 있으나, 이에 한정하는 것은 아니다. 효소면역흡착법은 고체 지지체에 부착된 항원을 인지하는 항체를 이용하는 직접적 ELISA, 고체 지지체에 부착된 항원을 인지하는 항체의 복합체에서 포획 항체를 인지하는 표지된 이차 항체를 이용하는 간접적 ELISA, 고체 지지체에 부착된 항체와 항원의 복합체에서 항원을 인지하는 표지된 또 다른 항체를 이용하는 직접적 샌드위치 ELISA, 고체 지지체에 부착된 항체와 항원의 복합체에서 항원을 인지하는 또 다른 항체와 반응시킨 후 이 항체를 인지하는 표지된 이차 항체를 이용하는 간접적 샌드위치 ELISA 등 다양한 ELISA 방법을 포함한다.The method for detecting the antigen-antibody complex can be preferably detected using enzyme immunoassay (ELISA), but is not limited thereto. Enzyme immunosorbent assays include direct ELISA using an antibody recognizing an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody recognizing the capture antibody in a complex of antibodies recognizing an antigen attached to a solid support, attachment to a solid support A direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of the antibody and the antigen, a label which recognizes the antibody after reacting with another antibody recognizing the antigen in the complex of the antibody and the antigen attached to the solid support And indirect sandwich ELISA using secondary antibodies.
상기 단클론항체 또는 이의 절편은 검출 표지체를 가질 수 있으며, 검출 표지체를 가지지 않을 경우는 이들 단일클론 항체 또는 이의 절편을 포획할 수 있고, 검출 표지체를 가지는 또 다른 항체를 처리하여 확인할 수 있다.The monoclonal antibody or a fragment thereof may have a detectable label, and when it does not have a detectable label, the monoclonal antibody or a fragment thereof can be captured and identified by treating another antibody having the detectable label .
또한, 본 발명은 상기 단클론항체를 포함하는 H5 아형 인플루엔자 바이러스 검출용 키트를 제공한다.The present invention also provides a kit for detecting H5 subtype influenza virus comprising the above monoclonal antibody.
본 발명의 H5 아형 인플루엔자 바이러스 검출용 키트는 시료를 주입하는 시료 주입부; 상기 시료 주입부로부터 일정 간격이 이격된 지점에 위치하는 H5 아형 인플루엔자 바이러스 HA1에 특이적으로 결합할 수 있는 단클론항체(1E12)가 결합된 형광체-항체 복합체를 포함하는 결합부; 상기 결합부로부터 일정 간격이 이격된 위치에 A형 인플루엔자 바이러스 헤마글루티닌에 특이적으로 결합하는 단클론항체가 고정된 검사선(test line); 및 항-마우스 IgG가 고정된 대조선(control line)이 순차적으로 구비되는 것이 바람직하지만 이에 한정되는 것은 아니다.The kit for detecting H5 subtype influenza virus according to the present invention comprises: a sample injection unit for injecting a sample; A binding unit comprising a fluorescent-antibody conjugate conjugated with a monoclonal antibody (1E12) capable of binding specifically to H5 subtype influenza virus HA1 located at a position spaced apart from the sample injecting unit; A test line in which a monoclonal antibody that specifically binds to type A influenza virus hemagglutinin is immobilized at a position spaced apart from the binding portion; And a control line to which anti-mouse IgG is immobilized are sequentially provided. However, the present invention is not limited thereto.
상기 키트에서 검사선에 고정된 항체는 A형 인플루엔자 바이러스 헤마글루티닌에 특이적으로 결합하는 단클론항체이면 특별히 제한 없이 사용 가능하며, 예를 들면, A형 인플루엔자 바이러스 헤마글루티닌에 특이적으로 결합하는 1C5 항체를 사용할 수 있다. 상기 1C5 항체는 젠바디(한국)로부터 제공된 단클론항체이다.The antibody immobilized on the test line in the above kit can be used without particular limitation as long as it is a monoclonal antibody that specifically binds to influenza A virus hemagglutinin. For example, the antibody specifically affects influenza A virus hemagglutinin Binding < / RTI > 1C5 antibody can be used. The 1C5 antibody is a monoclonal antibody provided from Zenbodia (Korea).
상기 형광체-항체 복합체는 라텍스 비드에 친수성 화합물이 코팅된 양자점이 결합되고, 상기 라텍스 비드 표면에 항체가 결합된 양자점-라텍스 비드-항체 복합체일 수 있다. The fluorescent substance-antibody complex may be a quantum dot-latex bead-antibody complex in which quantum dots coated with a hydrophilic compound are bonded to a latex bead, and an antibody is bound to the surface of the latex bead.
본 발명에서 사용되는 라텍스 비드는 양자점 및 항체가 결합할 수 있는 매개체로서 사용할 수 있는 한 특별히 이에 제한되지 않으나, 양자점 및 항체가 결합할 수 있도록 다수의 반응성 아미노기가 존재하는 라텍스 비드인 것이 바람직하다. 상기 라텍스 비드의 직경은 10~2,000nm인 것이 바람직하며, 더 바람직하게는 50~1,000nm인 것이며, 더욱더 바람직하게는 100nm인 것이지만, 이에 한정하는 것은 아니다.The latex beads used in the present invention are not particularly limited as long as they can be used as mediators capable of binding to quantum dots and antibodies, but are preferably latex beads in which a large number of reactive amino groups exist so that quantum dots and antibodies can be bound. The diameter of the latex bead is preferably 10 to 2,000 nm, more preferably 50 to 1,000 nm, and even more preferably 100 nm, but is not limited thereto.
상기 라텍스 비드 및 친수성 화합물이 코팅된 양자점은 1:40~1,000의 몰비로 결합된 것이 바람직하며, 더 바람직하게는 1:70~800의 몰비로 결합된 것이며, 더욱더 바람직하게는 1:90의 몰비로 결합한 것이지만, 이에 한정하는 것은 아니다.The latex beads and the quantum dots coated with the hydrophilic compound are preferably bound in a molar ratio of 1:40 to 1,000, more preferably in a molar ratio of 1:70 to 800, still more preferably in a molar ratio of 1:90 But is not limited thereto.
상기 라텍스 비드 및 인플루엔자 바이러스 항체는 1:50~500의 몰비로 결합된 것이 바람직하며, 더 바람직하게는 1:100~470의 몰비로 결합된 것이고, 더욱더 바람직하게는 1:455의 몰비로 결합된 것이지만 이에 한정하는 것은 아니다. The latex bead and influenza virus antibody are preferably bound in a molar ratio of 1:50 to 500, more preferably in a molar ratio of 1: 100 to 470, and still more preferably in a molar ratio of 1:455 But is not limited thereto.
상기 라텍스 비드와 친수성 화합물이 코팅된 양자점의 결합은 라텍스 비드 표면에 있는 아미노기와 양자점에 포함된 카르복실기 사이의 아마이드 결합인 것이 특징이며, 상기 라텍스 비드와 인플루엔자 바이러스 항체의 결합은 라텍스 비드 표면에 있는 카르복실기와 인플루엔자 바이러스 항체에 포함된 아미노기 사이의 아마이드 결합인 것이 특징이다. The bonding of the latex beads and the quantum dots coated with the hydrophilic compound is characterized by being an amide bond between an amino group on the surface of the latex beads and a carboxyl group contained in the quantum dots. The binding between the latex beads and the influenza virus antibody is caused by a carboxyl group And the amide bond between the amino group contained in the influenza virus antibody.
상기 양자점은 카드뮴셀레나이드(CdSe), 카드뮴설파이드(CdS), 카드뮴텔로리움(CdTe), 징크텔로리움(ZnTe), 징크셀레나이드(ZnSe), 징크설파이드(ZnS), 징크옥사이드(ZnO), 인듐 인화물(InP), 인듐 비화물(InAs), 머큐리텔로리움(HgTe) 및 머큐리셀레나이드(HgSe) 중에서 선택된 하나 이상인 것이 바람직하지만 이에 한정하는 것은 아니다. The quantum dots include at least one selected from the group consisting of CdSe, CdS, CdTe, ZnTe, ZnSe, ZnS, ZnO, , Indium phosphide (InP), indium paraffin (InAs), mercuritolium (HgTe), and mercury selenide (HgSe).
상기 양자점의 직경은 1~30nm인 것이 바람직하지만, 더 바람직하게는 2~20nm이지만, 이에 한정하는 것은 아니다. 상기 양자점의 발광파장은 450~700nm인 것이 바람직하지만, 이에 한정하는 것은 아니다.The diameter of the quantum dot is preferably 1 to 30 nm, more preferably 2 to 20 nm, but is not limited thereto. The emission wavelength of the quantum dot is preferably 450 to 700 nm, but is not limited thereto.
상기 양자점-라텍스 비드-인플루엔자 바이러스 항체 복합체를 유효성분으로 포함하는 인플루엔자 바이러스 검출용 조성물을 적용한 키트는 안정하여 장기간 보관하는 것이 가능하다.The kit to which the composition for detecting influenza virus containing the quantum dot-latex bead-influenza virus antibody complex as an active ingredient is applied can be stably stored for a long period of time.
본 발명의 일 구현예에서, 상기 검출용 키트는 스트립 형태의 나이트로셀룰로오스 멤브레인에 유리섬유(glass fiber), 코튼(cotton) 또는 셀룰로오스 재질의 패드를 결합시켜서 시료를 투입할 수 있는 시료주입부가 구비되고, 상기 시료 주입부로부터 일정 간격을 유지하면서 양자점-라텍스 비드-H5 아형 인플루엔자 바이러스 특이 항체(1E12)가 축합된 복합체가 건조된 축합 패드, A형 인플루엔자 바이러스를 검출할 수 있는 다른 항체(1C5)가 고정된 검사선 및 상기 축합 패드에 존재하는 단클론항체를 검출할 수 있는 이차 항체가 고정된 대조선이 순차적으로 구비된 면역스트립일 수 있다.In one embodiment of the present invention, the detection kit may include a sample injection unit capable of binding a sample by joining a glass fiber, cotton or cellulose pad to a nitrocellulose membrane in strip form (1C5) which is able to detect influenza A virus type influenza virus, a condensed pad in which the complex in which the quantum dot-latex bead-H5 subtype influenza virus specific antibody (1E12) is condensed while maintaining a constant distance from the sample injection part, And a control line to which a secondary antibody capable of detecting a monoclonal antibody present in the condensation pad is immobilized.
상기 인플루엔자 바이러스 검출용 키트에 H5 아형 인플루엔자 바이러스를 함유하는 것으로 의심되는 시료를 투입하여 상기 테스트 스트립의 검사선과 대조선에 형광빛띠가 나타나면 H5 아형 인플루엔자 바이러스 감염을 양성으로 판정하고, 대조선에만 형광빛띠가 나타나면 H5 아형 인플루엔자 바이러스 감염을 음성으로 판정할 수 있다.A specimen suspected of containing H5 subclass influenza virus is added to the kit for detecting influenza virus, and when a fluorescent light band appears on the test line and the control line of the test strip, the H5 subtype influenza virus infection is judged as positive and a fluorescent light band appears on the control line only The H5 subclass influenza virus infection can be judged negative.
본 발명의 일 구현 예에서, 상기 키트는 H5N3 바이러스에 대하여 2.5HAU/㎖에서도 반응성을 나타내었으며, 그에 반해 H1N1 또는 H7N1 바이러스에 대해서는 1000HAU/㎖에서도 반응하지 않음을 확인할 수 있다. 이는 본 발명의 키트가 H5 아형 바이러스를 신속하게 진단할 수 있는 키트임을 알 수 있다.
In one embodiment of the present invention, the kit showed reactivity at 2.5 HAU / ml against the H5N3 virus, while it did not react at 1000 HAU / ml for the H1N1 or H7N1 virus. This shows that the kit of the present invention is a kit for rapidly diagnosing H5 subtype virus.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.
Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
실시예 1. 인플루엔자 A 바이러스 HA1 항원의 마우스 면역Example 1. Mouse immunization of influenza A virus HA1 antigen
인플루엔자 A H5N1 (A/VietNam/1203/2004) 헤마글루티닌 단백질 서브유니트(HA1 Subunit)(Sino Biological Inc, Catalog:10003-V06H1; 서열번호 1)를 면역원으로 사용하였다. 상기 재조합항원(50㎕ 및 25㎕)을 마우스에 2주 간격으로 3회 면역시켜 마우스 혈청 내에서 높은 항체가를 ELISA(면역효소검사법)로 확인하였다(도 1). 음성대조군으로는 PBS(인산완충액, pH 7.4)와 건강한 마우스의 혈청(Normal, 1:200 희석)을 사용하였다.
Influenza A H5N1 (A / VietNam / 1203/2004) Hemagglutinin protein subunit (Sino Biological Inc, Catalog: 10003-V06H1; SEQ ID NO: 1) was used as an immunogen. The recombinant antigen (50 μl and 25 μl) was immunized with the mouse three times at intervals of 2 weeks, and high antibody titer in the mouse serum was confirmed by ELISA (immunoenzymatic assay) (FIG. 1). As a negative control, PBS (phosphate buffer, pH 7.4) and healthy mouse serum (Normal, 1: 200 dilution) were used.
실시예Example 2. 세포융합기술 시행 및 잡종세포 확보 2. Execute cell fusion technology and secure hybrid cells
면역된 마우스의 비장세포(splenocytes)를 얻은 다음, 암세포의 일종인 F/O 세포들과 세포융합기술(cell fusion technique)을 수행하여 여러 하이브리도마 세포(hybridoma cell) 중 1E12 클론(clone)을 택하여 96-웰 플레이트로부터 75T 배양 플라스크로 점차 수를 늘려 배양하였고 항체를 분비하고 있음을 확인하였다(도 2). 양성 대조군으로는 면역된 마우스의 혈청(1:200으로 희석)을 사용하였으며, 음성대조군으로는 PBS(인산완충액, pH 7.4)와 건강한 마우스의 혈청(Normal, 1:200 희석)을 사용하였다.
After obtaining splenocytes from immunized mice, 1E12 clones of hybridoma cells were subjected to cell fusion technique with F / O cells, which are cancer cells, And cultured by increasing the number of cells from a 96-well plate to a 75T culture flask and confirming that the antibody was secreted (FIG. 2). Serum (1: 200 dilution) of immunized mice was used as a positive control and PBS (phosphate buffer, pH 7.4) and healthy mouse serum (Normal, 1: 200 dilution) were used as negative controls.
실시예Example 3. 단클론항체를 분비하는 단클론세포주 확보 3. Securing monoclonal cell line secreting monoclonal antibody
그 중 높은 항체를 분비하는 1개의 클론 즉, 1E12 세포를 택하여 각각 96-웰 플레이트에서 제한개수희석법(limiting dilution)을 수행하였으며, 각 웰에서 항체가가 가장 높은 단클론세포주인 1E12로 명명하고 이를 선택하였다(도 3). Among them, one clone secreting high antibody, i.e., 1E12 cells, was subjected to limiting dilution in each of 96-well plates, and 1E12, which is the highest monoclonal cell line, was named in each well. (Fig. 3).
단클론세포주(monoclone) 1E12를 75T 배양 플라스크에서 대량으로 배양한 후 항체를 분비함을 확인하였는데, 1E12 단클론세포주가 분비하는 단클론항체(monoclonal antibody)의 항체가(ELISA OD value)는 1.445였다(도 3).
The monoclone 1E12 was cultured in a 75T culture flask and the antibody was secreted. The antibody (ELISA OD value) of the monoclonal antibody secreted by the 1E12 monoclonal cell line was 1.445 (Fig. 3 ).
실시예 4. 단클론항체의 대량생산과 정제 Example 4. Mass Production and Purification of Monoclonal Antibodies
단클론항체의 대량생산을 위해 1E12 단클론세포주를 마우스 복강내로 주입하여 복수가 생성되면 채취하였고, 단백질 A 컬럼(protein A column)을 이용하여 단클론항체를 정제하였다. SDS-PAGE 전기영동법으로 정제된 단클론항체 1E12의 중쇄(heavy chain) 및 경쇄(light chain)를 확인하였다(도 4).
For mass production of monoclonal antibodies, 1E12 monoclonal cell line was injected into the mouse abdominal cavity to collect the ascites, and the monoclonal antibody was purified using protein A column. The heavy chain and the light chain of the monoclonal antibody 1E12 purified by SDS-PAGE electrophoresis were confirmed (FIG. 4).
실시예 5. 단클론항체의 바이러스 검출Example 5. Virus detection of monoclonal antibodies
본 발명의 단클론항체가 H5 아형 인플루엔자 바이러스를 검출하는지 확인하기 위하여 웨스턴 블롯을 수행하였다. 또 다른 H5 아형 인플루엔자 특이 단클론항체 1C5은 젠바디(한국)로부터 제공받아 같이 수행하였다. 1000HAU(hemagglutination units)/㎖의 H5N3에 1C5 및 1E12로 바이러스에 대한 반응력을 확인하고자 SDS-PAGE로 바이러스 단백질을 우선 확인하고, 1E12 및 1C5를 1차 항체 (1㎍/㎖)로 사용한 웨스턴 블롯을 수행하여 바이러스 단백질에 대한 반응력을 확인하였다(도 5).Western blotting was performed to confirm whether the monoclonal antibody of the present invention detected the H5 subtype influenza virus. Another H5 subtype influenza monoclonal antibody 1C5 was obtained from GenBody (Korea). In order to confirm the reactivity against 1S5 and 1E12 of H5N3 in 1000 HAU (hemagglutination units) / ml, the virus protein was first identified by SDS-PAGE and Western blotting using 1E12 and 1C5 as the primary antibody (1 μg / ml) And the reaction force against the virus protein was confirmed (FIG. 5).
본 발명의 단클론항체의 바이러스 검출을 확인하기 위한 또 다른 방법으로, indirect ELISA 방법을 수행하였다. 1000HAU/㎖ 역가의 H5N3을 96웰 플레이트에 코팅하고, indirect ELISA로 확인한 결과, 1E12 및 1C5는 각각 1차 항체(1㎍/㎖)로 사용한 결과, 모두 H5N3 바이러스에 반응하였다(도 6).
As an alternative method for confirming virus detection of the monoclonal antibody of the present invention, an indirect ELISA method was performed. H5N3 at a concentration of 1000 HAU / ml was coated on a 96-well plate and confirmed by indirect ELISA. As a result, 1E12 and 1C5 were each used as a primary antibody (1 μg / ml), and all reacted with the H5N3 virus (FIG.
실시예 6. H5 아형 특이 인플루엔자 바이러스 검출용 신속형광면역진단키트 제작Example 6. Production of a rapid fluorescence immunoassay kit for detection of H5 subtypes of influenza virus
6.1. 양자점-라텍스 비드-항체 복합체 제조6.1. Quantum dot-latex bead-antibody complex preparation
항체로는 H5 아형 특이 항체 2종, 1E12 및 1C5 항체를 사용하였다. 형광물질로는 최대발광 파장이 580nm인 양자점을 사용하였다. As the antibody, two H5 subspecific antibodies, 1E12 and 1C5 antibodies were used. As a fluorescent material, a quantum dot having a maximum emission wavelength of 580 nm was used.
먼저 양자점에 라텍스를 가하여, 양자점과 라텍스가 결합된 결합체를 제작하였다. 구체적으로, 약 100nm 크기의 0.044μM 아민 라텍스(amine latex, Life Technology사) 50㎕에 세척완충액(8.77g/ℓ NaCl이 포함된 0.1M Phosphate Buffered Saline, pH 8)을 가하여 1회 세척하였다. 이어, 0.1μM 양자점을 1:90의 몰비로 혼합하고, 라텍스와 양자점간의 아미드 결합을 만들기 위한 커플링 시약으로 0.01M EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) 200㎕ 및 0.01M Sulfo-NHS(N-hydroxysulfosuccinimide) 300㎕을 넣고 실온에서 1시간 동안 반응시켰다. 상기 반응이 종료된 후, 반응물을 원심분리(17000rpm, 5분)를 통해 결합하지 않은 양자점을 제거하고, 침전물을 상기 세척완충용액을 가하여 1회 세척하였다. 상기 양자점-라텍스 비드 복합체에서 양자점이 결합되지 않은 라텍스 비드 표면의 아미노기에 숙신산 무수물(succinic anhydride)을 결합하여 라텍스 비드 표면을 카르복실기로 치환하였다. 이 후 실온에서 1시간 반응시키고 1회 세척하였다.First, latex was added to the quantum dots to prepare a conjugate in which the quantum dots and the latex were combined. Specifically, washing buffer (0.1 M Phosphate Buffered Saline containing 8.77 g / L NaCl, pH 8) was added to 50 μl of 0.044 μM amine latex (amine latex, Life Technology) of about 100 nm size and washed once. Subsequently, 0.1 μM quantum dots were mixed at a molar ratio of 1:90, and 200 μl of 0.01 M EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) and 0.01 M of a coupling reagent for forming an amide bond between the latex and the quantum dots 300 μl of sulfo-NHS (N-hydroxysulfosuccinimide) was added and reacted at room temperature for 1 hour. After completion of the reaction, the reaction mass was removed through centrifugation (17000 rpm, 5 minutes) to remove unbound quantum dots, and the precipitate was washed once with the washing buffer solution. In the quantum dot-latex bead composite, succinic anhydride was bonded to the amino group on the latex bead surface to which the quantum dots were not bonded, thereby replacing the surface of the latex bead with a carboxyl group. Thereafter, the reaction was allowed to proceed at room temperature for 1 hour and washed once.
상기 양자점-라텍스 비드 복합체에 200㎕의 0.1M 소듐 포스페이트 용액(pH 8.0)을 첨가하고 0.01M EDC 100㎕ 및 0.01M Sulfo-NHS 150㎕을 넣어 소니케이션(sonication)한 후, 1E12 항체(1㎎/㎖)를 150㎕를 첨가하고(최종적으로 라텍스에 대한 항체 비가 1:455 몰비가 되게 함) 실온에서 2시간 동안 반응시켰다. 반응 후 원심분리하여 상징액을 제거하였다. 비특이반응을 제거하기 위해, 0.1%의 BSA, 1%의 자당(sucrose), 1%의 젤라틴(gelatin) 및 0.01%의 카제인(casein)을 포함하는 블로킹용액에서 30분 동안 반응시켰다. 상기 반응이 종료된 후, 반응물을 원심분리(15,000rpm, 5분)를 통해 결합하지 않은 인플루엔자 바이러스 항체를 제거하고, 침전물을 상기 세척완충용액(pH7.4)을 가하여 1회 세척한 후, 다시 원심분리하여 상기 세척완충용액(pH7.4) 500㎕에 분산하여 양자점-라텍스 비드-항체 1E12 복합체를 제조하였다.
200 μl of a 0.1 M sodium phosphate solution (pH 8.0) was added to the quantum dot-latex bead complex, sonicated with 100 μl of 0.01 M EDC and 150 μl of 0.01 M Sulfo-NHS, and then 1E12 antibody / Ml) was added (final ratio of antibody to latex was 1: 455 molar ratio) and reacted at room temperature for 2 hours. After the reaction, the supernatant was removed by centrifugation. To remove the nonspecific reaction, the reaction was carried out for 30 minutes in a blocking solution containing 0.1% BSA, 1% sucrose, 1% gelatin and 0.01% casein. After the reaction was completed, the influenza virus antibody not bound through the centrifugation (15,000 rpm, 5 minutes) was removed, and the precipitate was washed once with the washing buffer solution (pH 7.4) Centrifuged and dispersed in 500 μl of the washing buffer solution (pH 7.4) to prepare a quantum dot-latex bead-antibody 1E12 complex.
6.2. 바이러스 진단용 형광면역 진단키트 제작6.2. Production of fluorescence immunoassay kit for virus diagnosis
상기 실시예 6.1.에서 제조한 양자점-라텍스 비드-항체 1E12 복합체를 포함하는 H5 아형 특이 인플루엔자 바이러스 검출용 키트를 제작하였다(도 7). 이때 1C5 단클론항체는 검사선에 점적하는(coating) 항체이고, 1E12 단클론항체는 양자점-라텍스 비드와 축합된 항체 복합체로서의 검출(detection)용으로 사용한 항체로써, 두 항체를 한 셋트로 만든 래피드 키트이다.A kit for detecting H5 subtype specific influenza virus comprising the quantum dot-latex bead-antibody 1E12 complex prepared in Example 6.1 was prepared (Fig. 7). The 1C5 monoclonal antibody is a coating antibody on the test line and the 1E12 monoclonal antibody is an antibody used for detection as an antibody complex condensed with QD-latex beads. The kit is a rapid kit comprising two sets of antibodies .
구체적으로는 스트립 형태의 나이트로셀룰로오스 멤브레인의 일 말단에 유리섬유(glass fiber), 코튼(cotton) 또는 셀룰로오스 재질의 검체 패드를 결합시켜서 비후강 검체 시료를 투입할 수 있는 시료 주입부를 구비시켰다. 상기 검체 패드와 일정 간격을 두고, 상기 양자점-라텍스 비드-인플루엔자 A 바이러스 항체 1E12 복합체 2㎕를 점적하고 건조시키고(축합 패드) 일정 간격을 두고 검사선(Test line; T)을 설정한 다음, 상기 검사선에 H5 아형 특이 항체(1C5) 2.5㎍을 가하여 고정시켰다. 상기 검사선에 고정한 항체는 인플루엔자 A H5N1 바이러스의 HA에 대한 항체이다.Specifically, a sample injecting unit is provided, which is capable of injecting a specimen of thickened specimen by bonding a sample pad made of glass fiber, cotton or cellulose to one end of a strip-shaped nitrocellulose membrane. After 2 μl of the quantum dot-latex bead-influenza A virus antibody 1E12 complex was spotted, dried (condensation pad) at predetermined intervals from the sample pad, test lines (T) were set at regular intervals, H5 subtype specific antibody (1C5) Was added and fixed. The antibody immobilized on the test line is an antibody against HA of influenza A H5N1 virus.
상기 검사선으로부터 일정 간격을 두고, 대조선(Control line; C)을 설정한 다음, 상기 대조선에 인플루엔자 바이러스와 특이적으로 결합하는 항체에 대한 이차 항체(마우스 IgG에 대한 항체) 2㎍을 가하여 고정시켰다.A control line (C) was set at a predetermined interval from the above-mentioned inspecting line, and 2 μg of a secondary antibody (antibody against mouse IgG) against an antibody specifically binding to influenza virus was fixed to the control line .
그 결과로서, 스트립 형태의 나이트로셀룰로오스 멤브레인 위에 검체패드, 형광체-항체 복합체, 검사선 및 대조선이 순차적으로 정렬된 형태의 형광 검출 키트를 제작하였다.
As a result, a fluorescence detection kit in which a sample pad, a fluorescent substance-antibody complex, an inspection line and a control line were sequentially arranged on a nitrocellulose membrane in the form of a strip was prepared.
실시예 7. 진단시스템의 H5 아형 인플루엔자 바이러스 검출 성능 확인Example 7. Confirmation of Detection Performance of H5 Subtype Influenza Virus in Diagnostic System
상기 실시예 6에서 제작한 인플루엔자 바이러스 검출용 키트를 사용하고, H5N3, H1N1 및 H7N1 인플루엔자 바이러스를 각각 시료로서 준비하였다.Using the kit for detecting influenza virus prepared in Example 6, H5N3, H1N1 and H7N1 influenza viruses were prepared as samples, respectively.
상기 시료에 포함된 인플루엔자 바이러스의 양을 H5N3는 적게는 5 HAU/㎖ 많게는 320 HAU/㎖이 되도록 설정하여 인플루엔자 바이러스 검출용 키트의 검출한도를 측정하였다. H1N1 및 H7N1 인플루엔자 바이러스는 1000 HAU/㎖ 역가에서의 반응력을 측정하였다. 이 때, 대조군으로는 바이러스를 처리하지 않은 것을 사용하였다.The amount of influenza virus contained in the sample was H5N3 of less than 5 HAU / ml < / RTI > HAU / ml, and the detection limit of the kit for detecting influenza virus was measured. RTI ID = 0.0 > H1N1 < / RTI > and H7N1 influenza viruses The reaction force at the HAU / ml potency was measured. At this time, as the control group, those not treated with virus were used.
반응 후의 검출용 키트를 375nm 파장에서 여기시켜 검사선과 대조선의 형광세기를 형광리더기(aGcare TRF, Medisensor)로 측정하였다. 인플루엔자 바이러스 검출용 조성물 양자점의 발광파장은 580nm으로 하였다.After the reaction, the detection kit was excited at a wavelength of 375 nm and the fluorescence intensity of the test line and the control line was measured with a fluorescent reader (aGcare TRF, Medisensor). Composition for detecting influenza virus The emission wavelength of the quantum dot was 580 nm.
서로 다른 아형(H5N3, H1N1, H7N1) 바이러스를 검사한 결과 H5N3에만 반응하고 2.5HAU/㎖까지도 바이러스가 검출되었다(도 8). 반면, H1N1나 H7N1에 대해서는 높은 역가(1000 HAU/mL)에서도 전혀 반응하지 않았다. H5N3, H1N1, and H7N1 viruses were tested for H5N3 alone and viruses were detected up to 2.5HAU / ml (FIG. 8). On the other hand, H1N1 and H7N1 did not react at all even at high titers (1000 HAU / mL).
<110> Wonkwang University Center for Industry-Academy Cooperation <120> Monoclonal antibody specific to hemagglutinin of H5 subtype influenza virus and rapid fluorescence-linked immunochromatographic diagnostic kit using the same <130> PN16296 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 342 <212> PRT <213> Artificial Sequence <220> <223> HA1 <400> 1 Met Glu Lys Ile Val Leu Leu Phe Ala Ile Val Ser Leu Val Lys Ser 1 5 10 15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val 20 25 30 Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 35 40 45 Leu Glu Lys Lys His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50 55 60 Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 65 70 75 80 Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val 85 90 95 Glu Lys Ala Asn Pro Val Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn 100 105 110 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu 115 120 125 Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Ser His Glu Ala Ser 130 135 140 Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Lys Ser Ser Phe Phe 145 150 155 160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile 165 170 175 Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180 185 190 Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr Gln 195 200 205 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg 210 215 220 Leu Val Pro Arg Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 225 230 235 240 Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn 245 250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile 260 265 270 Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly 275 280 285 Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser 290 295 300 Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310 315 320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser 325 330 335 Pro Gln Arg Glu Arg Arg 340 <110> Wonkwang University Center for Industry-Academy Cooperation <120> Monoclonal antibody specific to hemagglutinin of H5 subtype influenza virus and rapid fluorescence-linked immunochromatographic diagnostic kit using the same <130> PN16296 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 342 <212> PRT <213> Artificial Sequence <220> <223> HA1 <400> 1 Met Glu Lys Ile Val Leu Leu Phe Ala Ile Val Ser Leu Val Lys Ser 1 5 10 15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val 20 25 30 Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 35 40 45 Leu Glu Lys Lys His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50 55 60 Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 65 70 75 80 Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val 85 90 95 Glu Lys Ala Asn Pro Val Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn 100 105 110 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu 115 120 125 Lys Ile Gln Ile Ile Pro Lys Ser Ser Serp Ser Ser His Glu Ala Ser 130 135 140 Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Lys Ser Ser Phe Phe 145 150 155 160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile 165 170 175 Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180 185 190 Gly Ile His His Pro Asn Asp Ala Glu Gln Thr Lys Leu Tyr Gln 195 200 205 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg 210 215 220 Leu Val Pro Arg Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 225 230 235 240 Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn 245 250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile 260 265 270 Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly 275 280 285 Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser 290 295 300 Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310 315 320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser 325 330 335 Pro Gln Arg Glu Arg Arg 340
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