KR102695056B1 - Comosition for Detecting Koi Herpes Virus Contiaining Monoclonal Antibody Specific for Koi Herpes Virus - Google Patents
Comosition for Detecting Koi Herpes Virus Contiaining Monoclonal Antibody Specific for Koi Herpes Virus Download PDFInfo
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Abstract
본 발명은 잉어허피스바이러스(KHV)에 특이적으로 결합하는 단클론 항체를 포함하는 잉어허피스바이러스 검출용 조성물에 관한 것으로, 본 발명에 따른 KHV 항체를 포함하는 진단 키트를 사용하면, 많은 개체의 감염이 발생하기 전 신속하게 KHV를 진단할 수 있어, 양식어민의 피해를 최소화하고, 방역기관의 초기대응을 신속하게 진행할 수 있다.The present invention relates to a composition for detecting carp herpesvirus (KHV), comprising a monoclonal antibody that specifically binds to KHV. By using a diagnostic kit comprising the KHV antibody according to the present invention, KHV can be rapidly diagnosed before infection of a large number of individuals occurs, thereby minimizing damage to fish farms and enabling a rapid initial response by quarantine agencies.
Description
본 발명은 잉어허피스 바이러스에 특이적으로 결합하는 단클론 항체를 포함하는 잉어허피스 바이러스 검출용 조성물에 관한 것으로 더욱 자세하게는 잉어허피스 바이러스(KHV)에 대하여 특이적인 면역반응성을 나타내는 단클론항체를 생산하는 하이브리도마 세포주 및 상기 하이브리도마 세포주가 생산하는 잉어허피스 바이러스에 특이적으로 결합하는 단클론 항체, 상기 단클론 항체를 포함하는 잉어허피스 바이러스 검출용 조성물에 관한 것이다.The present invention relates to a composition for detecting carp herpes virus, comprising a monoclonal antibody that specifically binds to carp herpes virus, and more specifically, to a hybridoma cell line producing a monoclonal antibody exhibiting specific immunoreactivity against carp herpes virus (KHV), a monoclonal antibody that specifically binds to carp herpes virus produced by the hybridoma cell line, and a composition for detecting carp herpes virus comprising the monoclonal antibody.
잉어허피스바이러스병(KHVD, koi herpes viral disease)은 잉어과 어종에서 전염성이고 급성으로 발병하는 바이러스감염증으로 헤르페스바이러스 감염을 말한다. 일반적으로 KHV 감염은 잉어(Cyprinus carpio carpio), 비단잉어(Cyprinus carpio koi)와 ghost carp (Cyprinus carpio goi) 및 이들의 교잡종에서 발생한다고 알려져 있다.Koi herpes viral disease (KHVD) is a contagious and acute viral infection in cyprinid fish species, which refers to herpes virus infection. KHV infection is generally known to occur in common carp (Cyprinus carpio carpio), koi (Cyprinus carpio koi), ghost carp (Cyprinus carpio goi), and their hybrids.
잉어허피스바이러스는 다른 cyprinid 헤르페스바이러스의 명명법에 따라 cyprinid herpesvirus-3(CyHV-3)이라고도 불린다. KHV의 게놈 크기 295kbp 이며, 바이러스 뉴클레오캡시드는 직경이 100-110nm로 외피에 둘러싸여 있다(Ilouze et al., Ecol. Res., 26:885, 2011).Carp herpesvirus is also called cyprinid herpesvirus-3 (CyHV-3) according to the nomenclature of other cyprinid herpesviruses. The genome size of KHV is 295 kbp, and the viral nucleocapsid is enveloped with a diameter of 100-110 nm (Ilouze et al ., Ecol. Res ., 26:885, 2011).
KHVD는 온도에 영향을 많이 받으며, 수온 16~25℃ 사이에서 발생한다. 모든 연령대의 물고기가 KHV에 취약한 것으로 보이지만, 2.5~6g의 물고기가 230g의 물고기보다 더 감수성이 높게 나타났다. 아가미, 신장 및 비장은 감염 과정동안 KHV가 가장 많이 존재하는 기관들이다. 이 질병이 발병하면 사망률이 눈에 띄게 증가하며, 아가미 출혈, 점액과도 후 아가미 조직 괴사, 거의 대부분의 병어에서 안구 함몰이 관찰되며, 폐사직전 병어는 무기력하며, 평형감각이 상실되며, 아가미나 피부에 퇴색된 부분이 생긴다. 특이하게 피부에 충혈이나 궤양이 형성되지는 않으며, 방향감각을 잃어 과민증상을 보이기도 한다. 또한, 병어가 무리에서 분리되고 주수구에 모여들거나 못 가장자리나 수면에서 입질을 한다. 개체별로 면밀히 관찰하면, 색깔이 창백해지거나 표피가 붉어지며 체표가 거칠어지고, 국소적이거나 전반적인 상피 소실, 피부나 아가미의 점액이 과잉되거나 부족한 증상을 나타낸다. 이 바이러스는 자외선 조사 또는 50℃ 이상에서 1분간 열처리하면 불활성화 된다.KHVD is highly temperature-dependent, occurring between 16 and 25°C. Fish of all ages appear to be susceptible to KHV, but fish weighing 2.5 to 6 g are more susceptible than those weighing 230 g. The gills, kidneys, and spleen are the organs in which KHV is most abundant during the course of infection. Mortality is markedly increased when the disease develops, and gill hemorrhage, mucus overgrowth, gill tissue necrosis, and sunken eyes are observed in almost all diseased fish. Before death, diseased fish become lethargic, lose their sense of balance, and develop discolored areas on the gills and skin. No unusual skin congestion or ulceration occurs, and they may lose their sense of direction and show signs of hypersensitivity. In addition, diseased fish may separate from the school, gather at the water outlet, or bite at the edge of the pond or on the surface of the water. Close observation of each individual reveals symptoms such as paleness or redness of the epidermis, roughness of the body surface, localized or general loss of epithelium, and excessive or insufficient mucus in the skin or gills. The virus is inactivated by ultraviolet irradiation or heat treatment at 50℃ or higher for 1 minute.
임상적으로 감염된 어류의 KHVD 진단은 여러 가지 방법이 있으나, 가장 일반적으로 사용되는 방법은 KHV에 특정한 PCR 기반 분석법이다. KHVD은 OIE 지정 전염병 중 하나로 국내는 제3종 수산생물법정전염병으로 인간에게는 영향이 없지만, 잉어 및 비단잉어에 감염되어 피부 출혈 및 아가미 괴사 등의 증상을 보이며 폐사로 이어져 큰 피해를 야기한다고 알려져 있다.There are several methods for diagnosing KHVD in clinically infected fish, but the most commonly used method is a PCR-based assay specific for KHV. KHVD is one of the OIE-designated infectious diseases, and in Korea, it is a Class 3 legally designated infectious disease of aquatic organisms. Although it does not affect humans, it is known to infect common carp and koi, causing symptoms such as skin hemorrhage and gill necrosis, and leading to death, causing great damage.
세계동물보건기구(World Organization for Animal Health, OIE) 수생동물위생규약(Aquatic Animal Health Code)의 기준에 따른 일반적인 종래 PCR(conventional PCR)법을 수행하여 PCR 증폭산물을 전기영동으로 확인한 다음, 증폭산물을 클로닝하고 다시 정제된 플라스미드 DNA의 염기서열을 확인하여 진단하고 있다. OIE 기준에 따라서 PCR 증폭산물의 전기영동 확인 시 객관적인 분석이 불가능하거나(gray zone), 희미한 PCR 증폭산물을 클로닝 벡터를 이용하여 클로닝한 후 시퀀싱을 통하여 상기 증폭산물의 염기서열을 재확인하는 등의 검증 단계를 거친다. 이러한 검증 단계는 비특이적 산물(non-specific PCR band)의 서열을 확인하여 검증하는 방식으로 분석 절차가 많고 많은 시간을 소모하는 등의 문제점을 가지고 있다. 특히, 종래 PCR법에 의해 증폭산물이 양성으로 확인되면(시퀀싱 판정 이전), 수산생물전염병의 감염이 우려되는 경우에 해당되어 방역조치를 취하게 됨에 따라, 이후 시퀀싱에 따라 위양성으로 판정되는 경우 검사기관의 신뢰성 하락 및 어업인의 피해발생 사례도 있으므로, 신속, 정확하게 법정 전염병을 진단하는 방법의 개선이 절실하다. 이처럼 결과분석에 장시간 소요되는 PCR 방법의 문제점을 개선하고 현장에서 신속예찰이 가능한 진단키트의 도입이 요구된다. According to the standards of the Aquatic Animal Health Code of the World Organization for Animal Health, the conventional PCR method is performed, the PCR amplified product is confirmed by electrophoresis, the amplified product is cloned, and the base sequence of the purified plasmid DNA is confirmed again for diagnosis. According to the OIE standards, objective analysis is impossible (gray zone) when confirming the electrophoresis of the PCR amplified product, or a verification step is performed such as cloning a faint PCR amplified product using a cloning vector and then reconfirming the base sequence of the amplified product. This verification step has problems such as many analysis procedures and taking up a lot of time because it is a method of verifying by confirming the sequence of a non-specific product (non-specific PCR band). In particular, since there is a concern about aquatic organism infectious disease infection when the amplified product is confirmed as positive by the conventional PCR method (before sequencing judgment), and quarantine measures are taken, there are cases where the reliability of the testing agency decreases and damage occurs to fishermen if the sequencing result is determined to be false positive, so there is an urgent need to improve the method for diagnosing legal infectious diseases quickly and accurately. As such, there is a need to improve the problem of the PCR method that takes a long time to analyze the results and introduce a diagnostic kit that enables rapid prediction in the field.
이에, 본 발명자들은 현장에서 전문가 또는 전문장비의 도움없이 KHV를 신속하게 진단하는 기술을 개발하고자 예의 노력한 결과, KHV에 특이적으로 결합하는 단클론 항체 및 이를 함유하는 KHV 진단 키트를 제작하고, 상기 키트를 이용하여, 많은 개체의 감염이 발생하기 전 신속하게 KHV를 진단할 수 있다는 것을 확인하고, 본 발명을 완성하게 되었다.Accordingly, the inventors of the present invention have made efforts to develop a technology for rapidly diagnosing KHV in the field without the assistance of experts or specialized equipment, and as a result, have produced a monoclonal antibody that specifically binds to KHV and a KHV diagnostic kit containing the same, and have confirmed that KHV can be rapidly diagnosed using the kit before infection of a large number of individuals occurs, thereby completing the present invention.
본 발명의 목적은 잉어허피스 바이러스(KHV)의 검출용 조성물을 제공하는데 있다. The purpose of the present invention is to provide a composition for detecting carp herpes virus (KHV).
본 발명의 다른 목적은 잉어허피스 바이러스(KHV)의 검출용 키트를 제공하는데 있다.Another object of the present invention is to provide a kit for detecting carp herpes virus (KHV).
본 발명의 또 다른 목적은 잉어허피스 바이러스(KHV)에 대하여 특이적인 면역반응성을 나타내는 단클론항체를 생산하는 하이브리도마 세포주를 제공하는데 있다.Another object of the present invention is to provide a hybridoma cell line that produces a monoclonal antibody exhibiting specific immunoreactivity against carp herpes virus (KHV).
본 발명의 또 다른 목적은 잉어허피스 바이러스(KHV)에 대하여 특이적인 면역반응성을 나타내는 단클론 항체를 제공하는데 있다.Another object of the present invention is to provide a monoclonal antibody exhibiting specific immunoreactivity against carp herpes virus (KHV).
상기 목적을 달성하기 위하여, 본 발명은 잉어허피스 바이러스(KHV: Infectious myonecrosis virus) 항원 포획을 위한 1차 항체, 검출표지, 상기 검출표지가 결합되는 2차 항체 및 상기 검출표지의 활성 측정용 시약을 함유하는 잉어허피스 바이러스(KHV) 검출용 조성물에 있어서, In order to achieve the above object, the present invention provides a composition for detecting Carp Herpes Virus (KHV), comprising a primary antibody for capturing an antigen of Carp Herpes Virus (KHV), a detection label, a secondary antibody to which the detection label is bound, and a reagent for measuring the activity of the detection label.
상기 잉어허피스 바이러스 항원 포획을 위한 1차 항체는 기탁번호 KCTC15651BP로 기탁된 하이브리도마 세포주에 의해서 생산되고, 잉어허피스 바이러스에 특이적인 면역반응성을 나타내는 단클론항체 6C7인 것을 특징으로 하는, 잉어허피스 바이러스(KHV) 검출용 조성물을 제공한다.The present invention provides a composition for detecting carp herpes virus (KHV), characterized in that the primary antibody for capturing the above-mentioned carp herpes virus antigen is a monoclonal antibody 6C7 produced by a hybridoma cell line deposited under the deposit number KCTC15651BP and exhibiting immunoreactivity specific to carp herpes virus.
본 발명은 또한, 상기 검출용 조성물을 포함하는 잉어허피스 바이러스(KHV) 검출용 키트를 제공한다.The present invention also provides a kit for detecting carp herpes virus (KHV) comprising the above-detected composition.
본 발명은 또한, 잉어허피스 바이러스(KHV)에 대하여 특이적인 면역반응성을 나타내는 단클론항체 6C7를 생산하는 하이브리도마 세포주(KCTC15651BP)를 제공한다.The present invention also provides a hybridoma cell line (KCTC15651BP) producing monoclonal antibody 6C7 exhibiting specific immunoreactivity against carp herpes virus (KHV).
본 발명은 또한, 상기 하이브리도마 세포주(KCTC15651BP)에 의해 생산되고 잉어허피스 바이러스(KHV)에 대하여 특이적인 면역반응성을 나타내는 단클론 항체 6C7를 제공한다.The present invention also provides a monoclonal antibody 6C7 produced by the hybridoma cell line (KCTC15651BP) and exhibiting specific immunoreactivity against carp herpes virus (KHV).
본 발명은 또한, 잉어허피스 바이러스(KHV)에 대하여 특이적인 면역반응성을 나타내는 단클론항체 6G8을 생산하는 하이브리도마 세포주(KCTC15648BP)를 제공한다.The present invention also provides a hybridoma cell line (KCTC15648BP) producing monoclonal antibody 6G8 exhibiting specific immunoreactivity against carp herpes virus (KHV).
본 발명에 따른 KHV 항체를 포함하는 진단 키트를 사용하면, 많은 개체의 감염이 발생하기 전 신속하게 KHV를 진단할 수 있어, 양식어민의 피해를 최소화하고, 방역기관의 초기대응을 신속하게 진행할 수 있다.By using a diagnostic kit containing a KHV antibody according to the present invention, KHV can be rapidly diagnosed before infection occurs in a large number of individuals, thereby minimizing damage to aquaculture farmers and enabling rapid initial response by quarantine authorities.
도 1은 재조합 항원 단백질 정제 결과를 SDS-PAGE로 확인한 결과를 나타낸 것이다(레인 1: ORF68 레인 2: ORF72).
도 2는 KHV 바이러스 2종을 슈크로오스 구배(sucrose gradient)법으로 초원심분리에 의해 분리하는 방법을 나타낸 것이다.
도 3은 분리된 KHV Tp4 바이러스를 SDS-PAGE(A)와 웨스턴 블럿(B)으로 확인한 결과를 나타낸 것이다.
도 4는 재조합 항원 단백질 2종을 투여한 마우스의 항체 역가를 확인한 결과를 나타낸 것이다.
도 5는 선정된 단클론 항체 10종의 KHV 배양액에 대한 반응성을 측정한 결과를 나타낸 것이다.
도 6은 선정된 단클론 항체 중 6종의 웨스턴 블럿 결과를 나타낸 것이다. Figure 1 shows the results of recombinant antigen protein purification confirmed by SDS-PAGE (lane 1: ORF68 lane 2: ORF72).
Figure 2 illustrates a method for separating two types of KHV viruses by ultracentrifugation using a sucrose gradient method.
Figure 3 shows the results of SDS-PAGE (A) and Western blot (B) of the isolated KHV Tp4 virus.
Figure 4 shows the results of confirming the antibody titer of mice administered two types of recombinant antigen proteins.
Figure 5 shows the results of measuring the reactivity of 10 selected monoclonal antibodies to KHV culture medium.
Figure 6 shows the Western blot results of six of the selected monoclonal antibodies.
본 발명에서는 잉어과 어종에서 급성 전염증을 유발하는 잉어허피스 바이러스(KHV)를 효과적으로 진단할 수 있는 키트를 제작하기 위하여, 2종 유전자(ORF68 및ORF72)가 코딩하는 단백질, 6종의 KHV 펩타이드 및 2종의 바이러스(KHV TP4 및 KHV F03/16)을 KHV 항원으로 사용하여, 면역화시킨 마우스 비장세포와 마우스 골수종 유래 세포를 피더세포 존재하에서 융합시켜, KHV 단클론 항체를 생산하는 하이브리도마 세포를 제작하고, 상기 하이브리도마 세포 중 KHV에 반응성 있는 클론 40종을 1차 선별하였다. 이후 Isotype 및 민감도 테스트를 통해 10종의 단클론 항체 2차 선별하고, 이들 항체 중 페어링 테스트를 통하여 캡처 항체 6C7과 CGC 2차 항체 6G8을 선별하였다. 상기 항체에 대한 하이브로도마 세포를 마우스에 주사하여 KHV 단클론 항체를 제작하였다. 상기 제작된 KHV 단클론항체를 이용하여 측방유동면역분석법(LFIA: Leteral Flow Immuno Assay)을 이용한 신속진단키트를 제작하였으며, 상기 제작한 진단 키트는 KHV를 높은 정확도로 검출할 수 있다는 것을 확인하였다. In the present invention, in order to produce a kit capable of effectively diagnosing carp herpes virus (KHV) that causes acute infectious disease in cyprinid species, proteins encoded by two genes (ORF68 and ORF72), six KHV peptides, and two viruses (KHV TP4 and KHV F03/16) were used as KHV antigens, and mouse splenocytes immunized with the fusion of mouse myeloma-derived cells in the presence of feeder cells were produced to produce hybridoma cells that produce KHV monoclonal antibodies, and 40 clones reactive to KHV were first screened among the hybridoma cells. Thereafter, 10 monoclonal antibodies were secondarily screened through isotype and sensitivity tests, and among these antibodies, capture antibody 6C7 and CGC secondary antibody 6G8 were selected through a pairing test. Hybridoma cells for the antibodies were injected into mice to produce KHV monoclonal antibodies. A rapid diagnostic kit using the lateral flow immunoassay (LFIA) was produced using the above-mentioned KHV monoclonal antibody, and it was confirmed that the produced diagnostic kit can detect KHV with high accuracy.
따라서, 본 발명은 일 관점에서, 잉어허피스 바이러스(KHV: Infectious myonecrosis virus) 항원 포획을 위한 1차 항체, 검출표지, 상기 검출표지가 결합되는 2차 항체 및 상기 검출표지의 활성 측정용 시약을 함유하는 잉어허피스 바이러스(KHV) 검출용 조성물에 있어서, Therefore, the present invention, from one aspect, provides a composition for detecting infectious myonecrosis virus (KHV), comprising a primary antibody for capturing an antigen of KHV, a detection label, a secondary antibody to which the detection label is bound, and a reagent for measuring the activity of the detection label.
상기 잉어허피스 바이러스 항원 포획을 위한 1차 항체는 기탁번호 KCTC15651BP로 기탁된 하이브리도마 세포주에 의해서 생산되고, 잉어허피스 바이러스에 특이적인 면역반응성을 나타내는 단클론항체 6C7인 것을 특징으로 하는, 잉어허피스 바이러스(KHV) 검출용 조성물에 관한 것이다.The present invention relates to a composition for detecting carp herpes virus (KHV), characterized in that the primary antibody for capturing the above-mentioned carp herpes virus antigen is a monoclonal antibody 6C7 produced by a hybridoma cell line deposited under the deposit number KCTC15651BP and exhibiting immunoreactivity specific to carp herpes virus.
본 발명에 있어서, 상기 2차 항체는 기탁번호 KCTC15648BP로 기탁된 하이브리도마 세포주에 의해서 생산되고, 잉어허피스 바이러스에 특이적인 면역반응성을 나타내는 단클론항체 6G8인 것을 특징으로 할 수 있다.In the present invention, the secondary antibody may be characterized as being a monoclonal antibody 6G8 produced by a hybridoma cell line deposited under the deposit number KCTC15648BP and exhibiting specific immunoreactivity to the carp herpes virus.
본 발명에 있어서, 검출표지는 비오틴(biotin), HRP(horseradish peroxidase), 염기성탈인산화효소 (alkaline phosphatase), 콜로이드 골드(coloid gold), FITC(poly L-lysine-fluorescein isothiocya nate) 및 RITC(rhodamine-B-isothiocyanate)로 구성된 군에서 선택되는 것을 특징으로 할 수 있다.In the present invention, the detection label may be characterized by being selected from the group consisting of biotin, horseradish peroxidase (HRP), alkaline phosphatase, colloid gold, poly L-lysine-fluorescein isothiocya nate (FITC), and rhodamine-B-isothiocyanate (RITC).
다른 관점에서, 본 발명은 잉어허피스 바이러스(KHV)에 대하여 특이적인 면역반응성을 나타내는 단클론항체 6C7를 생산하는 하이브리도마 세포주(KCTC15651BP)에 관한 것이다. From another perspective, the present invention relates to a hybridoma cell line (KCTC15651BP) producing monoclonal antibody 6C7 exhibiting specific immunoreactivity against carp herpes virus (KHV).
또 다른 관점에서, 본 발명은 상기 하이브리도마 세포주(KCTC15651BP)에 의해 생산되고 잉어허피스 바이러스(KHV)에 대하여 특이적인 면역반응성을 나타내는 단클론 항체 6C7에 관한 것이다. In another aspect, the present invention relates to a monoclonal antibody 6C7 produced by the hybridoma cell line (KCTC15651BP) and exhibiting specific immunoreactivity against carp herpes virus (KHV).
또 다른 관점에서, 본 발명은 잉어허피스 바이러스(KHV)에 대하여 특이적인 면역반응성을 나타내는 단클론항체 6G8을 생산하는 하이브리도마 세포주(KCTC15648BP)에 관한 것이다. In another aspect, the present invention relates to a hybridoma cell line (KCTC15648BP) producing monoclonal antibody 6G8 exhibiting specific immunoreactivity against carp herpes virus (KHV).
또 다른 관점에서, 본 발명은 상기 하이브리도마 세포주(KCTC15648BP)에 의해 생산되고 잉어허피스 바이러스(KHV)에 대하여 특이적인 면역반응성을 나타내는 단클론 항체 6G8에 관한 것이다. In another aspect, the present invention relates to a monoclonal antibody 6G8 produced by the hybridoma cell line (KCTC15648BP) and exhibiting specific immunoreactivity against carp herpes virus (KHV).
또 다른 관점에서, 본 발명은 상기 잉어허피스 바이러스(KHV) 검출용 조성물을 포함하는 잉어허피스 바이러스(KHV) 검출용 키트에 관한 것이다.In another aspect, the present invention relates to a kit for detecting carp herpes virus (KHV) comprising the composition for detecting carp herpes virus (KHV).
본 발명에 있어서, 상기 검출표지와 상기 검출표지의 활성 측정용 시약은 반응에 의해 발색, 형광, 발광 특성 중 어느 하나를 나타내어 이를 측정하기 위함으로 일반적인 면역측정법에 사용되는 각종 검출표지와 각 검출표지에 따른 활성 측정용 시약을 사용할 수 있다.In the present invention, the detection label and the activity measuring reagent of the detection label exhibit one of the color development, fluorescence, and luminescence characteristics by reaction, and thus, various detection labels used in general immunoassay methods and activity measuring reagents according to each detection label can be used to measure the same.
본 발명의 KHV 진단 시약은 상기 진단용 항원, 상기 진단용 항원 포획을 위한 단클론항체, 검출표지, 상기 검출표지가 결합되는 단클론항체 및 상기 검출표지의 활성 측정용 시약 외에 일반적인 면역측정법에 사용되는 시약이 추가로 포함될 수 있다. 보다 구체적으로는, 항체 고정용액, 항원 고정용액, 블로킹 용액, 가검시료 희석제, 혈청 반응 및 항체의 비특이 반응을 제거하기 위한 세척액, 테스트의 유효성을 검증하기 위한 양성 및 음성 대조시약, 상기 검출표지의 활성 측정을 위한 반응을 정지시키기 위한 정지액 등이 포함될 수 있다. 또한, 일반적인 면역측정법에 사용되는 각종 고체상 지지체, 즉, 플레이트, 마이크로플레이트 웰, 플라스틱 시험관, 유리 비드, 플라스틱 비드 등도 포함될 수 있다.The KHV diagnostic reagent of the present invention may further include reagents used in general immunoassays in addition to the diagnostic antigen, the monoclonal antibody for capturing the diagnostic antigen, the detection label, the monoclonal antibody to which the detection label is bound, and the reagent for measuring the activity of the detection label. More specifically, the reagent may include an antibody fixing solution, an antigen fixing solution, a blocking solution, a test sample diluent, a washing solution for removing serum reaction and non-specific antibody reaction, positive and negative control reagents for verifying the validity of the test, a stop solution for stopping the reaction for measuring the activity of the detection label, and the like. In addition, various solid-phase supports used in general immunoassays, i.e., plates, microplate wells, plastic test tubes, glass beads, plastic beads, and the like, may also be included.
본 발명의 KHV 진단 키트는 상기 본 발명의 KHV 진단 시약을 포함하여 이루어지는 키트일 수 있다.The KHV diagnostic kit of the present invention may be a kit comprising the KHV diagnostic reagent of the present invention.
본 발명의 KHV 진단 또는 KHV 바이러스 중화항체 검출방법은 효소면역분석법(Enzyme immunoassay, EIA) 또는 방사선면역분석법(Radio immunoassay, RIA)을 사용하는 것이 바람직하다. 보다 바람직하게는 효소면역항체 분석법을 사용한다. 그러나, 상기 검출방법에 의해 한정되는 것은 아니며, 본 발명의 KHV 바이러스 중화항체 검출용 단클론항체, 진단용 항원 포획용 단클론항체와 진단용 KHV 바이러스 항원의 복합체 및 피검체로부터 채취한 가검시료를 반응시켜 항원-항체 반응을 검출할 수 있는 방법이라면 어느 방법도 사용가능하다.The KHV diagnosis or KHV virus neutralizing antibody detection method of the present invention preferably uses an enzyme immunoassay (EIA) or a radioimmunoassay (RIA). More preferably, an enzyme immunoassay is used. However, it is not limited to the above detection method, and any method can be used as long as it is a method capable of detecting an antigen-antibody reaction by reacting a complex of a monoclonal antibody for detecting a KHV virus neutralizing antibody of the present invention, a monoclonal antibody for capturing a diagnostic antigen, and a diagnostic KHV virus antigen, and a test sample collected from a subject.
상기 가검시료는 피검체로부터 채취한 생물학적인 시료를 의미한다. 세포간 체액이나 혈청을 사용하는 것이 바람직하고, KHV 바이러스의 항원 또는 상기 KHV 바이러스에 대한 자가 항체가 정제되도록 처리한 시료를 채취하여 사용한다.The above test sample refers to a biological sample collected from a subject. It is preferable to use interstitial fluid or serum, and a sample that has been processed to purify the antigen of the KHV virus or the autoantibody to the KHV virus is collected and used.
보다 구체적인 본 발명의 KHV 진단 방법은 고체상 지지체에 항원 포획을 위한 단클론항체를 고정하는 단계; 상기 고체상 지지체에 부착되지 않은 항체들을 세척하여 제거하는 단계; 상기 고체상 지지체에 KHV 바이러스 진단용 항원을 반응시키는 단계; 상기 고체상 지지체에 부착되지 않은 항원들을 세척하여 제거하는 단계; 및 검출표지가 결합된 단클론항체를 고체상 지지체에 고정된 상기 KHV 바이러스 진단용 항원과 반응시키는 단계를 포함하고, 상기 진단용 항원 포획을 위한 단클론항체는 하이브리도마 세포주 KHV 6C7에 의해 생산되고, KHV에 면역반응성을 나타내는 단클론항체를 사용한다.A more specific KHV diagnostic method of the present invention comprises the steps of: fixing a monoclonal antibody for antigen capture to a solid-phase support; washing away antibodies not attached to the solid-phase support; reacting a KHV virus diagnostic antigen to the solid-phase support; washing away antigens not attached to the solid-phase support; and reacting a monoclonal antibody bound to a detection label with the KHV virus diagnostic antigen fixed to the solid-phase support, wherein the monoclonal antibody for diagnostic antigen capture is a hybridoma cell line. It uses a monoclonal antibody produced by KHV 6C7 and showing immunoreactivity to KHV.
본 발명의 검출용 키트는 바람직하게는 항원-항체 반응을 이용한 측방유동면역분석법(LFIA: Leteral Flow Immuno Assay)을 이용한 신속진단키트일 수 있다. The detection kit of the present invention may preferably be a rapid diagnostic kit using lateral flow immunoassay (LFIA) using an antigen-antibody reaction.
측방유동면역분석법(LFIA: Leteral Flow Immuno Assay)은 지지대인 백킹카드(backing card)위에 샘플패드(sample pad), 접합패드(conjugate pad), 멤브레인(membrane) 및 흡수패드(absorbent pad)가 부착된 형태로 구성되어 있다. The lateral flow immunoassay (LFIA) consists of a sample pad, conjugate pad, membrane, and absorbent pad attached to a backing card, which is a support.
측방유동면역분석법의 원리는 멤브레인과 접합패드에 목표물질인 항원(KHV)에 대한 특이항체가 고정화 및 분주되어 있으며, 샘플패드로 주입된 시료내 항원(KHV)이 모세관현상을 통한 측방유동 과정에서 접합패드의 “항체-나노금입자 접합체“와 1차 결합하여 ”항원-항체 접합체“를 형성한 다음 멤브레인의 T-line(test line)에서 고정된 항체와 2차 결합하면서 “항체-항원-항체 접합체“를 형성한다. 항원(KHV)의 농도가 높을수록 T-line에 결합하는 ”항체-항원-항체 접합체”의 수가 증가하고, 항체 접합체의 나노금입자의 수가 증가하면서 분홍색 또는 보라색의 육안으로 판별 가능하게 된다. 판정 방법은 테스트 결과 C-line(Control line)만 발색한 경우 음성으로 판정하며, C-line과 T-line 모두 발색한 경우 양성으로 판정한다.The principle of lateral flow immunoassay is that a specific antibody against the target substance (antigen) (KHV) is immobilized and dispensed onto the membrane and the conjugate pad, and the antigen (KHV) in the sample injected into the sample pad first binds to the “antibody-nanoparticle conjugate” of the conjugate pad through lateral flow through capillary phenomenon to form an “antigen-antibody conjugate,” and then binds a second time to the immobilized antibody at the T-line (test line) of the membrane to form an “antibody-antigen-antibody conjugate.” As the concentration of the antigen (KHV) increases, the number of “antibody-antigen-antibody conjugates” binding to the T-line increases, and as the number of nanoparticles in the antibody conjugate increases, the color becomes pink or purple, which can be visually distinguished. The judgment method is that if only the C-line (control line) develops color as a result of the test, it is judged as negative, and if both the C-line and T-line develop color, it is judged as positive.
KHV 신속진단키트의 장점은 기존의 높은 비용과 고가의 장비를 활용한 real-time PCR기법보다 저렴한 비용과 5~10분내의 짧은 결과 판독시간이 소요되며, 비숙련자가 실험하여도 숙련자가 실험한 기법과 동일한 검사결과를 활용할 수 있어 현장적용에 있어 여러면에서 장점을 갖는다. The advantages of the KHV rapid diagnostic kit are that it is cheaper than the existing high-cost and expensive equipment-based real-time PCR technique, takes a short result interpretation time of 5 to 10 minutes, and can be used by inexperienced people to obtain the same test results as experienced people, so it has many advantages in field application.
본 발명의 진단키트는 현장 사용의 편의성을 위하여 키트 패키지 타입으로 구성될 수 있으며, 구성 물품으로는 KHV 신속진단키트, 완충용액, 간이 분쇄기, 간이 스포이트를 포함할 수 있다. The diagnostic kit of the present invention may be configured as a kit package type for convenience of field use, and its components may include a KHV rapid diagnostic kit, a buffer solution, a simple grinder, and a simple dropper.
본 발명은 수산생물질병의 상시 예찰 및 법정전염병 모니터링을 위한 진단시스템 개발을 통하여 수산양식 산업의 피해를 최소화하고 바이오 신소재를 활용한 대응체계 마련에 방향을 제시하는 등 효과적인 수산방역체계 강화에 기여할 것으로 기대된다.The present invention is expected to contribute to strengthening the effective fisheries quarantine system by minimizing damage to the aquaculture industry through the development of a diagnostic system for constant surveillance of aquatic animal diseases and monitoring of legal infectious diseases, and by suggesting directions for establishing a response system utilizing new bio materials.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those skilled in the art that these examples are intended only to illustrate the present invention, and the scope of the present invention is not to be construed as being limited by these examples.
실시예 1: KHV 바이러스에 대한 재조합 항원 제작Example 1: Production of recombinant antigen for KHV virus
재조합 항원단백질 선정Selection of recombinant antigen proteins
KHV 관련논문(J Gen virol. 91:452-462, 2010; J Fish Dis., 00:1-9, 2020)을 참조하여 ORF자리 156개 중에서 ORF68와 ORF72, 총 2종 염기서열을 선정하였으며, KHV 비리온에서 많이 존재하는 단백질 3종에 대한 펩타이드 6종을 선정하였다. With reference to KHV-related papers ( J Gen virol . 91:452-462, 2010; J Fish Dis ., 00:1-9, 2020), two base sequences, ORF68 and ORF72, were selected from 156 ORF sites, and six peptides for three proteins abundant in KHV virions were selected.
선정된 재조합 항원 2종 염기서열에 대하여 코돈 최적화를 진행 후 유전자 합성을 진행하였고, 상기 선정된 펩타이드 6종에 대한 유전자 합성을 진행하였다.After codon optimization for the two selected recombinant antigen base sequences, gene synthesis was performed, and gene synthesis was performed for the six selected peptides.
(가) ORF68 (서열번호 1)(a) ORF68 (sequence number 1)
Gene size : 1548 bpGene size: 1548 bp
GGATCCGATCAGTTCAAGCAGACCACGGCCGAGCTGGCCGGCATCAAGGCCGACGCTCAGAGCGTCGCCGACGCCATCAAGGCGGCGAACGACGAGCACCAGAAGACCAAGGATGCCCTGGCCCACACGCAGAGCGCCTACGACCTGACCATGGAGCAGGTCAACGAGTACCAGGCCAAGCTCCAGGCCAGCGAGGCCGCGCTCAAGGAGGCGCACGACACGGCCAAGGCCAACATGACCGTCAAGGCCCAGTCGCGCATCTCCGAGCTCGAGGCCGAGGCCGTGCTGGGCCGCGACACCATGAAGGTCAAGGACGCCGAGGTGCGGGACCTCAAGACCAAGCTGGCCAGCGCCAAGAACGAGACGGCCGCAGCGCTCAGCCGCCTCGCGGACAAGGAAGACGAGATCGAGACGCTGGCCAAGACGCTCGCCGACGCCGTCGAGAGCGCCAAGCAGCAGAACGAGACCATCGGCCGCAACGCCGCAGAGTCCATCAAGGACCTGGCGCTCAGGCTCAAGCGCCAGGAGGAGCTCGCCAAGGACGCCGAGGCCAACCTCGCGAACGTGACCCGGGACCTCGAGCGGCGCATGAAGGAGCAGGCCGAGGAGTACAACAACAACGAGCTCAACTACGCCGCCGCCCTCACCGCCTTGCAGCAGTCGTCCAAGGAACTGGAGCAACAGCTGGCCGACGCGCGACGCCAGCTCGCCCAGGCCCAGGCCCAGGCCCAGGCCCAGGCCGCTCAACCTCAACCCCAGCCACCCGCTCAACCCCAGGCGGCCGCTCAGCCACCCGCTCAACCCCAGGCCGCTCAGGCACCCGCTCAACCCCAGCCACCCGCTCAAGCTCAACCCCAGGCCGAGCAGCCACCCGCCGCTCAACCCCAAGCGCAGGCGGCGGCGGTCATCGCGCAAGGCGTCACTGAGGCCGCCTCCGTCGCGGACGATGGCGAGACGCCCATGGAGGAGATTGAGCCGCCCGAGTACGAGCCTCCGGCCGGCGGCGTCGAGTACGAGCCCGACATGGAGATCGAGATGCCGCCCGCCGCCTTCGACCCCGAAGAGTACCGCAAGTCCCAGGAGCGCAAGAAGCTCGAGGAGAGCGCCCGCGCCAACCCCAACCTCGTGCGCAAGCCGACTCCCAAGGTCAACATGTTCGACTTTATGCAGTCCATCAAGAAGCCGGTGCCCGAGACGGTGGAGGCCCTCAAGGCCCGGGTGAAGGAGCTGGAGATGATGCTGAAACTGGTGTACGATGAGCTCACGCGGGTCAACGCCGAGCGCTACCAGGAGGCGGCCACCCGCTGCATGGCGGCCGAGAACTTCAAGCGCCAGATCAAGGACCTGCGCTCCAAGAACGTCGCGCTCAGGGACAAGCTCGAGGAGGCCCGGCTCCGCTTCGAGCTGGACAAGCTCACCTACGAGAACGACTACGCCAAGCAGATGTACAAGTTCCAGGAGAACATCAAGGTCGAGTACAAGGGCAGCGTCGACATGGTGGACCAGCTGAAGCGCGAGAACTCCAGGCTCGAGTCGCAGTGAGAATTC GGATCC GAATTC
(나) ORF72 (서열번호 2)(I) ORF72 (SEQ ID NO: 2)
Gene size : 1122 bp Gene size: 1122 bp
GGATCCATGTATGGCCTGAACAGCGCGAGCGGCTTTCTGGATACCGAATGGGTGGAAAAACAAGGCATGGTGACCCCGCCGGAAGAAGTGGCGAAAATGCTGAACCCGCATCTGGCGACCATGGGCAAACCGGTGGATGTGACGAGCCTGATGCTGCCGAACGGCATTTTTGTGCCGTATACCCCGACCGGCCCGGAACCGAACATTGCGATTGGCGGCGGCTATTTTTATCGCCCGAGCTATCATGGCTATGGCAGCGTGGCGGAAGATAGCGATGATCTGCAGAAACTGCATAAAAACGTGCTGTGCCGCAACGAAGGTCAGTGGACCCGCACCGCGCTGATTGGCGTGCCGACCACCGGCAACTTTCCGGTGGATCTGTTTGATTATAAAAATCAGAAATATCAGATTGTGAACACCGGCCCGGATGCGCTGCATAGCGGCGATGCGTTTATGGTGGAACCGCCGAGCGTGGAAGCGAGCAAAGCGCAGATTGATAGCTTTAAACGTATTCGCGCGCAAGGCTCTTTTCGTCAGCCGACCCTGTTAGGCGGCCTGAGCGCGACCAACCGCGTGCCGAGCGAACTGACCCATCGCATGAAACATATTATGGCGCGCGATATTGGCCTGTGCATGGATATTACCACCCCGGGGCAAGGCGGCGTACAAGCGGGCCTGGCGCGACTTTATCAAGCGCCGACCGAACTGGGCCAAGCGGTGAAAAACAGCGTGATGGCGACCACCCTGGATTGCATGAACAGCCTGGATGTGATTTTTAGCATGATGCGCCGCATTGCGGCGCTGCCGCGCGGCGTGCTGGAAACCCTGGCGGAATTTCATAACTTTAAACATGGCGGCCCGAACGCGACCATGCCTGATCCGGCGATTCTGATTGAAATTTGGCGCACCCCGGCGGTGGCGGATCTGTTTATGGAAAGCATGGATATGGGCTTTCAGAGCATTGATCGCCTGCGCCATTGCACCTATGGCACCGTGATTAAAAGCACCGAAGCGAACCCGACGAGCACCCGCGGCGAAATTATTTATAACGCGTATAGCGGTCAGTATGATACCGGCGATACCTTTGAAGGCGCGGTGCTGAGCTTTAACGGCTATCTCGAG GGATCC CTCGAG
상기 합성된 2종의 항원에 대한 유전자를 플라스미드 pET28a(Novagen)에 삽입하고, 대장균 BL21 plyss 또는 대장균 BL21 Rosetta1에 형질전환시켰다. 재조합 플라스미드는 형질전환된 대장균을 배양하여, 전기영동을 통해 형질전환 결과를 확인하였다. The genes for the two synthesized antigens were inserted into the plasmid pET28a (Novagen) and transformed into E. coli BL21 plyss or E. coli BL21 Rosetta1. The recombinant plasmid was cultured in the transformed E. coli, and the transformation results were confirmed through electrophoresis.
재조합 항원 단백질 유전자가 형질전환된 발현 균주 BL21 plyss 또는 BL21 Rosetta1를 LB-kanamycin 배지 1L에 접종 후 37℃에서 O.D값이 0.5-0.6이 될 때 까지 배양 후, 1mM IPTG를 이용하여 재조합 항원 단백질의 발현을 유도한 후, 37℃에서 하룻밤 배양하였다. 배양된 균주를 수득한 후, 용해 버퍼(6M Urea, 20 mM Tris, 500 mM NaCl, 5 mM Imidazole, 1mM 2-mercapoethanol)에 현탁하여 초음파 파쇄를 실시하고, 17,000 rpm에서 20분간 원심분리한 후 미리 결합버퍼로 평형화시켜 놓은 Ni-NTA His-Tag Affinity ㅋ크클크로마토그래피 칼럼에 적용하여 결합시킨 후 세척버퍼로 세척하고, 용출버퍼를 이용하여 재조합 항원 단백질을 용출시켰다. 그 다음 20mM Sodium Carbonate 버퍼에서 하룻밤 투석한 후 농축시켰다. The recombinant antigen protein gene-transformed expression strain BL21 plyss or BL21 Rosetta1 was inoculated into 1 L of LB-kanamycin medium and cultured at 37°C until the OD value became 0.5-0.6. The expression of the recombinant antigen protein was induced using 1 mM IPTG, and then cultured overnight at 37°C. The cultured strain was obtained, suspended in lysis buffer (6 M Urea, 20 mM Tris, 500 mM NaCl, 5 mM Imidazole, 1 mM 2-mercapoethanol), disrupted by sonication, centrifuged at 17,000 rpm for 20 minutes, applied to a Ni-NTA His-Tag Affinity chromatography column that had been equilibrated with binding buffer in advance, bound, washed with wash buffer, and eluted with elution buffer. It was then dialyzed overnight in 20 mM Sodium Carbonate buffer and concentrated.
정제한 각 재조합 항원 단백질은 SDS-PAGE를 통하여 순도와 분자량을 확인하였다(도 1 및 표 3).The purity and molecular weight of each purified recombinant antigen protein were confirmed through SDS-PAGE (Figure 1 and Table 3).
정제 KHV 바이러스 확보Purified KHV virus obtained
배양 후 정제한 2종의 KHV Tp4(국립수산물품질관리원) 및 KHV F03/16(국립수산물품질관리원)바이러스 배양액 2종을 초원심분리기를 사용하여 sucrose gradient법으로 바이러스를 분리하였다. 분리된 KHV Tp4 바이러스를 SDS-PAGE(도 3A)와 웨스턴 블럿(도 3B)으로 확인하였다.After culturing, two types of purified KHV Tp4 (National Fisheries Products Quality Management Service) and KHV F03/16 (National Fisheries Products Quality Management Service) viruses were separated by the sucrose gradient method using an ultracentrifuge. The separated KHV Tp4 virus was confirmed by SDS-PAGE (Fig. 3A) and Western blot (Fig. 3B).
실시예 2: 항-KHV 단클론 항체 제작Example 2: Production of anti-KHV monoclonal antibodies
실시예 1에서 제작한 재조합 항원 2종, 펩타이드 항원 6종을 8주령 Balb/c mouse에 표 4와 같은 스케줄 및 용량으로 투여하여 면역을 진행하였다. Immunization was performed by administering two types of recombinant antigens and six types of peptide antigens produced in Example 1 to 8-week-old Balb/c mice according to the schedule and dose shown in Table 4.
ImmunizationImmunization
DaysDays
(I.P.)Intraperitoneal injection
(IP)
(I.P.)Intraperitoneal injection
(IP)
(I.P.)Intraperitoneal administration
(IP)
(I.P.)Intraperitoneal injection
(IP)
1st Boosting1st boost
1st Boosting
(I.V.)Intravenous injection
(IV)
2nd Boosting2nd boosting
2nd Boosting
(I.V.)Intravenous injection
(IV)
면역화 후 4일 뒤에 꼬리 미정맥에서 채혈하여 혈청을 얻은 뒤 효소면역측정법 (ELISA, Enzyme Linked Immuno-Sorbent Assay)으로 항체가를 결정하였다. 측정된 흡광도 수치가 음성대조군의 마우스(항원을 주입하지 않은 쥐의 혈청)과 비교하였다. 그 결과를 표 5, 도 4에 나타내었다. 펩타이드 면역된 마우스 혈청엔 KHV 바이러스에 대한 역가가 6종 펩타이드 모두에서 검출되지 않아 결과에서 제외하였다.Four days after immunization, blood was collected from the tail vein to obtain serum, and antibody titers were determined by enzyme-linked immunosorbent assay (ELISA). The measured absorbance values were compared with those of the negative control group (sera from mice not injected with antigen). The results are shown in Table 5 and Fig. 4. Since the serum from peptide-immunized mice did not detect any titers for KHV virus in all six peptides, it was excluded from the results.
양호한 결과가 얻어진 경우 면역화가 성공한 것으로 보고 세포 간 융합 준비를 하였다. If good results were obtained, immunization was considered successful and preparation for cell-to-cell fusion was performed.
*High: Serum 희석배수 1:12800에서 O.D값 1.0 이상 (충분한 항체역가)*High: O.D value 1.0 or higher at serum dilution ratio 1:12800 (sufficient antibody titer)
*Low: Serum 희석배수 1:12800에서 O.D값 1.0 이하*Low: O.D value 1.0 or less at serum dilution ratio 1:12800
피더세포(feeder cell) 준비Feeder cell preparation
골수종세포 (myeloma)와 비장 세포의 융합 18시간 이전에, 12주령 이상의 마우스(Balb/c 암컷)의 복부 피부를 벗겨 낸 후 복근 속 복강 내로 8㎖의 4℃, 11.3% sucrose (Sigma) 용액을 주입한 후 복강을 마사지하여, 복강 내 대식세포들의 유리를 촉진한 후, 이를 회수하여 1300 rpm으로 원심분리하여, 펠렛을 PBS 혹은 RPMI-1640(Sigma) 배지를 이용한 세척한 후, 1X HAT (hypoxanthine aminopterine tymidine, Sigma), 20 % FBS (fetal bovine serum, Sigma), RPMI-1640에 현탁시키고, 세포배양용 96 well plate (Nunc)에 분주한 후 37℃ CO2 incubator (Binder)에서 배양하였다. Eighteen hours before the fusion of myeloma cells and spleen cells, the abdominal skin of mice older than 12 weeks (Balb/c female) was peeled off, 8 ml of 4°C, 11.3% sucrose (Sigma) solution was injected into the abdominal cavity through the abdominal muscles, and the abdominal cavity was massaged to promote the release of macrophages in the peritoneal cavity. These were collected, centrifuged at 1300 rpm, and the pellet was washed with PBS or RPMI-1640 (Sigma) medium, suspended in 1X HAT (hypoxanthine aminopterine tymidine, Sigma), 20% FBS (fetal bovine serum, Sigma), RPMI-1640, dispensed into a 96-well plate for cell culture (Nunc), and cultured in a 37°C CO 2 incubator (Binder).
피더 세포를 미리 준비하는 것은 융합과정 중 여러 단계를 거치면서 손상을 입은 융합세포들이 피더세포들이 분비하는 다양한 사이토카인에 의한 치유를 촉진하고, 지나친 세포희석으로 인한 성장 저해를 억제하기 위해서이다.Preparing feeder cells in advance is to promote healing of damaged fused cells through various cytokines secreted by feeder cells during the fusion process and to suppress growth inhibition due to excessive cell dilution.
비장세포와 골수종세포의 융합 (Cell fusion)Fusion of spleen cells and myeloma cells (Cell fusion)
세포융합 방법은 PEG(polyethylene glycol, Sigma)를 fusogen으로 이용하여Khler와 Milstein 방법에 따라 수행하였다. 세포융합 2주 전에 계대한 골수종세포 (myeloma, Sp2/0-Ag14, HGPRT-, 한국세포주은행)와 항체가(antibody titer)가 높게 나타난 마우스의 비장세포(splenocyte)를 미리 준비한 후 이들을 혼합하여 FBS가 첨가되지 않은 세포배양용 배지로 세척한 후, 37℃의 1㎖의 PEG를 이용하여 1분간에 걸쳐 서서히 저어주면서 융합시켰다. 융합 후 수분에 걸쳐 천천히 1X HAT 배지 (20% FBS, RPMI-1640)를 첨가해 주면서 융합과정을 마쳤다. 이러한 전과정은 37℃ 항온수조(water bath)를 이용하여 시행하였다. Cell fusion was performed using PEG (polyethylene glycol, Sigma) as a fusogen according to the method of Khler and Milstein. Myeloma cells (myeloma, Sp2/0-Ag14, HGPRT-, Korea Cell Line Bank) passaged 2 weeks before cell fusion and mouse spleen cells (splenocytes) with high antibody titer were prepared in advance, mixed, washed with cell culture medium without FBS, and fused using 1㎖ of PEG at 37℃ with gentle stirring for 1 minute. After fusion, 1X HAT medium (20% FBS, RPMI-1640) was slowly added over several minutes to complete the fusion process. This entire process was performed using a 37℃ water bath.
전날 준비한 피더세포가 깔린 96 well plate에 well 당 1개의 colony가 생길 정도의 농도로 세포를 HAT가 첨가된 배지로 희석하여 분주하고, 분주된 96well plate들은 37℃ CO2 배양기에서 7일간 배양하며 스크리닝 단계에 접어들 때까지 배양기를 열지 않고 배양하였다. The cells were diluted with HAT-supplemented medium to a concentration that produced one colony per well and seeded into 96-well plates equipped with feeder cells prepared the day before. The seeded 96-well plates were cultured in a 37°C CO2 incubator for 7 days, and the incubator was kept unopened until the screening stage.
비장세포와 골수종세포의 융합 (Cell fusion)Fusion of spleen cells and myeloma cells (Cell fusion)
세포융합 방법은 PEG(polyethylene glycol, Sigma)를 fusogen으로 이용하여Khler와 Milstein 방법에 따라 수행하였다. 세포융합 2주 전에 계대한 골수종세포 (myeloma, Sp2/0-Ag14, HGPRT-, 한국세포주은행)와 항체가(antibody titer)가 높게 나타난 마우스의 비장세포 (splenocyte)를 미리 준비한 후 이들을 혼합하여 FBS가 첨가되지 않은 세포배양용 배지로 세척한 후, 37℃의 1㎖의 PEG를 이용하여 1분간에 걸쳐 서서히 저어주면서 융합시켰다. 융합 후 수분에 걸쳐 천천히 1X HAT 배지 (20% FBS, RPMI-1640)를 첨가해 주면서 융합과정을 마쳤다. 이러한 전과정은 37℃ 항온수조(water bath)를 이용하여 시행하였다. Cell fusion was performed using PEG (polyethylene glycol, Sigma) as a fusogen according to the method of Khler and Milstein. Myeloma cells (myeloma, Sp2/0-Ag14, HGPRT-, Korea Cell Line Bank) passaged 2 weeks before cell fusion and mouse spleen cells (splenocytes) with high antibody titer were prepared in advance, mixed, washed with cell culture medium without FBS, and fused using 1㎖ of PEG at 37℃ with gentle stirring for 1 minute. After fusion, 1X HAT medium (20% FBS, RPMI-1640) was slowly added over several minutes to complete the fusion process. This entire process was performed using a 37℃ water bath.
전날 준비한 피더세포가 깔린 96 well plate에 well 당 1개의 colony가 생길 정도의 농도로 세포를 HAT가 첨가된 배지로 희석하여 분주하고, 분주된 96well plate들은 37℃ CO2 배양기에서 7일간 배양하며 스크리닝 단계에 접어들 때까지 배양기를 열지 않고 배양하였다. The cells were diluted with HAT-supplemented medium to a concentration that produced one colony per well and seeded into 96-well plates equipped with feeder cells prepared the day before. The seeded 96-well plates were cultured in a 37°C CO2 incubator for 7 days, and the incubator was kept unopened until the screening stage.
특이성 검사 대상 well의 선별Selection of target wells for specificity testing
충분히 성장한 하이브리도마 세포의 배양 상층액을 사용하여 KHV TP4 바이러스 배양액에 반응성이 있는 64개 클론을 대상으로 스크리닝 ELISA를 실시하고, CCB 세포에 반응성이 없으면서, KHV TP4에 반응성 있는 클론 40종을 1차 선별하였다(표 7).A screening ELISA was performed on 64 clones reactive to KHV TP4 virus culture using culture supernatants from sufficiently grown hybridoma cells, and 40 clones reactive to KHV TP4 but not to CCB cells were initially selected (Table 7).
이후, 선정된 40개 클론에 대하여, Isotype 테스트를 통해 IgM, IgA 클론들을 제외하고, KHV TP4에 대하여 반응성이 낮은 클론들을 제외하여 최종 10종의 단클론 항체를 선정하였다. Afterwards, for the 40 selected clones, IgM and IgA clones were excluded through isotype testing, and clones with low reactivity to KHV TP4 were excluded to select the final 10 monoclonal antibodies.
확보한 항-KHV 단클론항체의 특이성 검사 Specificity testing of acquired anti-KHV monoclonal antibodies
효소면역측정법(ELISA)을 이용하여, 선별된 10개 클론에 대한 항원 특이성을 확인하였다.The antigen specificity of 10 selected clones was confirmed using enzyme-linked immunosorbent assay (ELISA).
KHV 항원 용액을 10㎍/㎖의 농도로 100㎕씩 96 well plate에 분주하고 37℃, 60분간 반응시켰다. 희석액으로는 0.05M sodium bicarbonate 용액을 사용하였으며, 반응액을 제거하고 세척액 (인산염완충액, 0.05% Tween 20) well당 200㎕ 씩 첨가한 후 다시 제거하는 과정을 3회 반복하였다. 세척 후 각 well에 차단액(blocking solution, 1% BSA가 함유된 인산염 완충액)을 200㎕ 첨가하고 37℃, 30분간 반응시켰다. 차단액 반응액을 제거하고 상기 세척액을 well당 200㎕씩 첨가한 후 다시 제거하는 과정을 3회 반복하였다. 단클론항체 (MAb)용액을 well당 100㎕씩 분주하고 음성대조군용 well에 희석액을 100㎕ 분주한 다음 37℃에서 60분간 반응시켰다. 반응액을 제거하고 상기 세척액을 well당 200㎕씩 첨가한 후 다시 제거하는 과정을 3회 반복함. 항-마우스 IgG에 HRP (Horse reddish peroxidase)가 융합된 시약을 well당 100㎕씩 분주하고 37℃에서 60분간 반응시킴. 반응액을 제거하고 상기 세척액을 well당 200㎕씩 첨가한 후 다시 제거하는 과정을 3회 반복하였다. KHV antigen solution was dispensed into 96-well plates at a concentration of 10 μg/㎖ (100 μl) and reacted at 37°C for 60 minutes. 0.05 M sodium bicarbonate solution was used as the diluent, and the reaction solution was removed, added 200 μl of washing solution (phosphate buffer, 0.05% Tween 20) per well, and removed again. This process was repeated three times. After washing, 200 μl of blocking solution (phosphate buffer containing 1% BSA) was added to each well and reacted at 37°C for 30 minutes. The blocking reaction solution was removed, and the washing solution was added 200 μl per well, and removed again. 100 μl of monoclonal antibody (MAb) solution was dispensed per well, and 100 μl of the dilution solution was dispensed to the negative control wells, and then reacted at 37°C for 60 minutes. The process of removing the reaction solution, adding 200 ㎕ of the washing solution per well, and removing it again was repeated three times. 100 ㎕ of a reagent in which anti-mouse IgG is fused with HRP (Horse reddish peroxidase) was dispensed per well and reacted at 37°C for 60 minutes. The process of removing the reaction solution, adding 200 ㎕ of the washing solution per well, and removing it again was repeated three times.
효소 기질 용액 (o-phenylenediamine 0.4㎎/㎖ in phosphate citrate buffer pH 5.0))을 100㎕씩 분주하고 암실의 상온에서 30분간 반응시킨 후, 492 nm (yellow)에서 흡광도값을 확인하여 음성대조군의 흡광도의 3배 이상 되는 흡광도 값을 양성 반응의 최소치로 기준하여 음성, 양성으로 판정하였다. 100 ㎕ of enzyme substrate solution (o-phenylenediamine 0.4 mg/㎖ in phosphate citrate buffer pH 5.0)) was dispensed and reacted for 30 minutes at room temperature in a dark room. The absorbance value at 492 nm (yellow) was checked and an absorbance value that was three times or more the absorbance of the negative control group was used as the minimum value for a positive reaction and was judged as negative or positive.
그 결과, 표 8에 나타난 바와 같이, KHV mAb는 자가 항원에 특이적인 반응을 확인할 수 있었으며, 다른 항원에 대한 비특이반응은 확인되지 않았으며, ELISA를 실시하여, 이소타입을 확인하였으며, 10종 클론 모두 IgG로 확인되었다.As a result, as shown in Table 8, KHV mAb was able to confirm a specific reaction to self-antigen, and no non-specific reaction to other antigens was confirmed. ELISA was performed to confirm the isotype, and all 10 clones were confirmed as IgG.
실시예 3: 단클론 항체 기반 KHV 진단 키트 제작Example 3: Production of a monoclonal antibody-based KHV diagnostic kit
5종 항체의 페어링 테스트(pairing test)Pairing test of 5 types of antibodies
CGC(Colloidal gold conjugate) 용액 100ul당 10종의 KHV 단클론 항체를 각각 적정량으로 혼합하여 골드 패드를 제작하고, LFIA(lateral Flow Immuno Assay) 스트립 페어 테스트를 진행하였다. A gold pad was made by mixing 10 types of KHV monoclonal antibodies in appropriate amounts per 100 μl of CGC (Colloidal gold conjugate) solution, and a LFIA (lateral flow immunoassay) strip pair test was performed.
그 결과, 표 9에 나타난 바와 같이, KHV에 민감도가 높고, 버퍼에 대한 비특이 반응이 낮은 페어로 캡처는 #4F5와 #6C7 단클론 항체를 선정하였고, CGC는 #6F11과 #6G8 단클론을 선정하였다. As a result, as shown in Table 9, the pairs with high sensitivity to KHV and low non-specific reaction to the buffer were selected for capture using monoclonal antibodies #4F5 and #6C7, and the CGC selected monoclonal antibodies #6F11 and #6G8.
진단키트 최적화 조건 선정Selecting diagnostic kit optimization conditions
가) 항체-콜로이달 골드 입자 접합체 제조a) Manufacturing of antibody-colloidal gold particle conjugates
CGC 원료로 선정된 단클론 항체 #6F11과 #6G8을 이용하여 40nm Colloidal Gold Conjugate(CGC) 제작 조건 테스트 진행하였으며, #6F11의 40nm SC CGC 제작을 위한 조건은 pH 6.0, 항체농도 8㎍/ml 이며, #6G8의 40nm SC CGC 제작을 위한 조건은 pH 7.0, 항체농도 14㎍/ml로 선정하였다(표 10 및 표 11). We tested the conditions for producing 40 nm Colloidal Gold Conjugate (CGC) using monoclonal antibodies #6F11 and #6G8 selected as CGC raw materials. The conditions for producing 40 nm SC CGC of #6F11 were pH 6.0 and antibody concentration 8㎍/ml, and the conditions for producing 40 nm SC CGC of #6G8 were pH 7.0 and antibody concentration 14㎍/ml (Tables 10 and 11).
나) 항체-콜로이달 골드 접합체 최적 함량 결정b) Determination of optimal content of antibody-colloidal gold conjugate
단클론 항체 #6G8, #6F11 40nm CGC를 사용하여 컨쥬게이트 패드에 분주하는 CGC의 농도를 O.D 기준으로 2.0, 4.0, 6.0 OD 을 각각 분주하여 테스트 한 결과 #6G8 4.0 O.D로 선정하였다(표 12). Using monoclonal antibodies #6G8, #6F11 40nm CGC, the concentration of CGC dispensed into the conjugate pad was tested by dispensing 2.0, 4.0, and 6.0 OD based on O.D., respectively, and #6G8 4.0 OD was selected (Table 12).
다) Capture 항체 분주 함량 결정D) Determination of Capture Antibody Dispensing Content
니트로셀룰로오즈 막의 테스트 라인에 고정하는 항체 #6C7의 농도를 1.0, 1.5, 2.0 mg/ml을 각각 분주한 다음 스트립을 제작하여 KHV TP4+F03 바이러스 배양액과 CCB 세포 배양액, 버퍼테스트 결과 분주농도가 높아질수록 민감도 유지되는 반면, 위양성은 증가하여 T-line 항체 분주 농도는 1.0 mg/ml으로 선정하였다(표 13). The concentrations of antibody #6C7 fixed to the test line of the nitrocellulose membrane were dispensed at 1.0, 1.5, and 2.0 mg/ml, respectively, and strips were produced and tested with KHV TP4+F03 virus culture medium, CCB cell culture medium, and buffer. As a result of the dispensed concentration increasing, the sensitivity was maintained, but the false positive rate increased, so the T-line antibody dispensed concentration was selected as 1.0 mg/ml (Table 13).
라) 샘플패드 종류 및 첨가제 테스트a) Sample pad types and additive testing
샘플패드 2종 Cytosep 1662와 Fusion 5에 버퍼 및 첨가제 함량에 따른 테스트를 실시한 결과 Cytosep 1662 / 200mM Tris-Hcl (pH 8.0) / 1% Tween 20 / 0.2% casein / 0.1% NaN3로 선정하였다(표 14). As a result of testing the buffer and additive contents on two sample pads, Cytosep 1662 and Fusion 5, Cytosep 1662 / 200 mM Tris-Hcl (pH 8.0) / 1% Tween 20 / 0.2% casein / 0.1% NaN3 was selected (Table 14).
마) 비특이 반응 제거를 위한 capture 첨가제 테스트b) Test of capture additives to remove non-specific reactions
버퍼 및 음성검체 비특이 반응 제거를 위해 캡쳐 항체 분주 시 0.5% 카제인, 0.5% BSA, 0.5% protein saver를 첨가한 결과 민감도 유지되면서 비특이 반응 제거되는 0.5% BSA 조건을 선정하였다. To remove non-specific reactions from buffer and negative samples, 0.5% casein, 0.5% BSA, and 0.5% protein saver were added during capture antibody dispensing, and the 0.5% BSA condition was selected to remove non-specific reactions while maintaining sensitivity.
바) 타사키트 비교 테스트b) Third-party kit comparison test
최종 선정된 항체 쌍 #6C7(캡처) #6G8(CGC) 조건으로 키트를 제작(SFI032301)하고, 타사키트와 비교실험을 진행하였다. A kit was produced (SFI032301) under the conditions of the final selected antibody pair #6C7 (capture) and #6G8 (CGC), and a comparative experiment was conducted with third-party kits.
바) 타사키트 비교 테스트b) Third-party kit comparison test
최종 선정된 항체 쌍 #6C7(캡처) #6G8(CGC) 조건으로 키트를 제작(SFI032301)하고, 타사키트와 비교실험을 진행하였다. A kit was produced (SFI032301) under the conditions of the final selected antibody pair #6C7 (capture) and #6G8 (CGC), and a comparative experiment was conducted with third-party kits.
국립수산물품질관리원에서 정제한 KHV TP4 sucrose gradient fraction 3과 fraction 7을 1/100 희석배수부터 1/3200 희석배수까지 1/2 serial dilution 한 후 KHV 타사키트와 비교 테스트 실시하였다. KHV TP4 sucrose gradient fraction 3 and fraction 7 purified by the National Fisheries Products Quality Management Service were serially diluted by 1/2 from a dilution ratio of 1/100 to 1/3200, and then a comparative test was conducted with a KHV third-party kit.
그 결과, 표 16에 나타난 바와 같이, 타사키트는 1/800 희석배수까지 T-line band를 육안으로 확인할 수 있는 반면, 자사 키트(SFI032301)는 1/3200 희석배수까지 육안으로 확인 가능하여 타사키트 대비 높은 민감도를 보였다. 또한, 같은 희석배수의 검체를 사용 시 타사키트에 비해 시 T-line의 강도가 높아 육안으로 판별이 용이하였다. As a result, as shown in Table 16, while the third-party kit could visually confirm the T-line band up to a dilution ratio of 1/800, our kit (SFI032301) could visually confirm up to a dilution ratio of 1/3200, showing higher sensitivity than the third-party kit. In addition, when using a sample with the same dilution ratio, the intensity of the T-line was higher than that of the third-party kit, making it easier to discern with the naked eye.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.While the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and that the scope of the present invention is not limited thereby. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
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Claims (10)
상기잉어허피스 바이러스 항원 포획을 위한 1차 항체는 기탁번호 KCTC15651BP로 기탁된 하이브리도마 세포주에 의해서 생산되고, 잉어허피스 바이러스 TP4에 특이적인 면역반응성을 나타내는 단클론항체 6C7인 것을 특징으로 하는, 잉어허피스 바이러스(KHV) 검출용 조성물.
A composition for detecting Koi Herpes Virus (KHV), comprising a primary antibody for capturing a Koi Herpes Virus (KHV) antigen, a detection label, a secondary antibody to which the detection label is bound, and a reagent for measuring the activity of the detection label,
A composition for detecting carp herpes virus (KHV), characterized in that the primary antibody for capturing the above-mentioned carp herpes virus antigen is a monoclonal antibody 6C7 produced by a hybridoma cell line deposited under the deposit number KCTC15651BP and exhibiting specific immunoreactivity to carp herpes virus TP4.
A composition for detecting carp herpes virus (KHV), characterized in that in claim 1, the secondary antibody is a monoclonal antibody 6G8 produced by a hybridoma cell line deposited with the deposit number KCTC15648BP and exhibits specific immunoreactivity to carp herpes virus TP4.
A composition for detecting carp herpes virus (KHV), characterized in that in claim 1, the detection label is selected from the group consisting of biotin, horseradish peroxidase (HRP), alkaline phosphatase, colloid gold, poly L-lysine-fluorescein isothiocya nate (FITC), and rhodamine-B-isothiocyanate (RITC).
A kit for detecting carp herpes virus (KHV), comprising the detection composition of claim 1.
A hybridoma cell line (KCTC15651BP) producing monoclonal antibody 6C7 that exhibits specific immunoreactivity against carp herpes virus (KHV).
Monoclonal antibody 6C7 produced by the hybridoma cell line (KCTC15651BP) of Article 5 and exhibiting specific immunoreactivity against carp herpes virus (KHV).
In claim 6, a monoclonal antibody 6C7 characterized by exhibiting specific immunoreactivity against KHV TP4.
A hybridoma cell line (KCTC15648BP) producing monoclonal antibody 6G8 that exhibits specific immunoreactivity against carp herpes virus (KHV).
Monoclonal antibody 6G8 produced by the hybridoma cell line (KCTC15648BP) of Article 8 and exhibiting specific immunoreactivity against carp herpes virus (KHV).
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