KR101990795B1 - Rabies virus-specific antibody and detecting method of rabies virus using thereof - Google Patents
Rabies virus-specific antibody and detecting method of rabies virus using thereof Download PDFInfo
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- KR101990795B1 KR101990795B1 KR1020170167753A KR20170167753A KR101990795B1 KR 101990795 B1 KR101990795 B1 KR 101990795B1 KR 1020170167753 A KR1020170167753 A KR 1020170167753A KR 20170167753 A KR20170167753 A KR 20170167753A KR 101990795 B1 KR101990795 B1 KR 101990795B1
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- rabies virus
- rabies
- antibody
- specific antibody
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- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
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Abstract
본 발명은 광견병바이러스 특이 항체에 관한 것으로서, 더욱 상세하게는 상기 광견병바이러스 특이 항체를 이용한 광견병바이러스 검출방법에 관한 것이다. 본 발명에 따른 광견병바이러스 특이 항체를 이용하여 효소면역분석을 수행할 경우 광견병바이러스에 대한 민감도, 특이도 및 정확도가 우수할 뿐만 아니라 고가의 분석 장비 없이도 광견병을 신속하고, 정확하며, 간편하게 진단할 수 있는바, 광견병 예방, 치료 및 진단 분야에서 다양하게 활용할 수 있다.TECHNICAL FIELD The present invention relates to a rabies-specific antibody, and more particularly, to a rabies-virus detection method using the rabies-specific antibody. When the enzyme immunoassay using the rabies virus specific antibody according to the present invention is performed, the sensitivity, specificity and accuracy of the rabies virus are excellent, and the rabies can be diagnosed quickly, accurately and easily without expensive analytical equipment It can be used in various fields such as bar, rabies prevention, treatment and diagnosis.
Description
본 발명은 광견병바이러스 특이 항체에 관한 것으로서, 더욱 상세하게는 상기 광견병바이러스 특이 항체를 이용한 광견병바이러스 검출방법에 관한 것이다.TECHNICAL FIELD The present invention relates to a rabies-specific antibody, and more particularly, to a rabies-virus detection method using the rabies-specific antibody.
광견병은 사람과 모든 온혈동물에서 가장 치명적인 질병으로, 임상증상이 나타나면 높은 비율로 폐사한다. 한국을 포함한 모든 나라가 광견병을 인수공통전염병으로 인식하고 있으며, 세계동물보건기구(OIE)에서는 광견병이 발생하면 반드시 보고해야하는 질병으로 지정하였다. 또한 광견병은 국내 보건 분야에서는 3 군 법정감염병으로, 수의 분야에서는 2 종 가축전염병으로 규정하고 관리하고 있다. 각 국가들은 수입 또는 수출되는 동물로부터 광견병 전파를 차단하기 위하여 검역을 강화하고 있다.Rabies is the most fatal disease in humans and in all warm-blooded animals, with high rates of mortality when clinical symptoms occur. All countries including Korea recognize rabies as a common infectious disease, and the World Animal Health Organization (OIE) has designated it as a disease that must be reported when rabies occurs. In addition, rabies is regulated and managed as a livestock infectious disease in the field of veterinary medicine, and as a third-party infectious disease in the domestic health field. Each country is strengthening its quarantine to prevent the spread of rabies from imported or exported animals.
광견병의 진단은 광견병바이러스에 감염된 조직을 검사하여 항원인 광견병바이러스를 확인하거나, 광견병바이러스에 감염된 개체의 침에서 항원을 검출하는 방법이 있다. 또한 혈청에 포함된 광견병바이러스에 대한 항체의 농도(serum antibody titer)를 측정하여 광견병 백신 접종여부를 확인할 수 있다. 구체적으로, 상기 혈청에 포함된 광견병바이러스에 대한 항체의 농도를 측정하는 방법에는 바이러스 중화시험(Fluorescent Antibody Virus Neutralization; FAVN, Rapid Focus Fluorescent Inhibition Test; RFFIT)이 있다. 상기 바이러스 중화시험은 항체검사 결과가 정확하다는 장점이 있으나, 실험실 내에서 수행되므로 세포배양과 관련된 장비 및 숙련된 실험자가 필요하고, 실험과정이 복잡하다는 단점이 있다. 외국에서 상용화되어 있는 효소면역반응법을 이용한 광견병바이러스에 대한 항체 검사법은 가격이 비싸고, 국내에서 상용화되어있지 않다.The diagnosis of rabies can be made by examining tissues infected with the rabies virus to identify rabies virus as an antigen, or to detect an antigen in the saliva of an individual infected with a rabies virus. In addition, the serum antibody titer of the rabies virus contained in the serum can be measured to confirm whether or not the rabies vaccination is performed. Specifically, a method for measuring the concentration of an antibody against rabies virus contained in the serum includes a fluorescence antifody neutralization (FAVN, Rapid Focus Fluorescent Inhibition Test; RFFIT). The virus neutralization test has an advantage that the antibody test result is accurate, but it is performed in a laboratory, so that equipment related to cell culture and skilled experimenters are required, and the experiment procedure is complicated. Antibody tests for rabies viruses using enzyme immunoassays, which are commercially available in foreign countries, are expensive and not commercially available in the country.
이에 본 발명자들은 신속하고 정확도, 민감도 및 특이도가 높은 광견병바이러스 검출 방법을 개발하기 위하여 연구를 수행한 결과, 광견병바이러스 특이 항체를 개발하고, 상기 항체를 이용하여 광견병바이러스 검출을 위한 효소면역분석 수행 시 정확도, 민감도 및 특이도가 우수하다는 것을 확인함으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors have conducted studies to develop a rapid, accurate, sensitive and specific rabies virus detection method. As a result, they have developed a rabies-specific antibody, and conducted enzyme immunoassay for detecting rabies virus using the antibody Sensitivity, sensitivity and specificity, and thus completed the present invention.
따라서 본 발명의 목적은, 서열번호 2의 아미노산 서열로 표시되는 단일 사슬 가변 단편(Single-chain variable fragment, scFv)을 포함하는 광견병바이러스 특이 항체를 제공하는 것이다.Accordingly, an object of the present invention is to provide a rabies-specific antibody comprising a single-chain variable fragment (scFv) represented by the amino acid sequence of SEQ ID NO: 2.
본 발명의 다른 목적은, 서열번호 4의 아미노산 서열로 표시되는 경쇄 가변영역 및 서열번호 6의 아미노산 서열로 표시되는 중쇄 가변영역을 포함하는 광견병바이러스 특이항체를 제공하는 것이다.Another object of the present invention is to provide a rabies-specific antibody comprising a light chain variable region represented by the amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 6.
본 발명의 또 다른 목적은, 상기 광견병바이러스 특이 항체를 포함하는 광견병바이러스 검출용 효소면역분석 키트를 제공하는 것이다.It is still another object of the present invention to provide an enzyme immunoassay kit for rabies virus detection comprising the rabies virus specific antibody.
본 발명의 또 다른 목적은, (a) 생물학적 시료를 코팅용 항체와 반응시키는 단계; (b) 상기 단계 (a)의 반응물을 검출용 항체와 반응시키는 단계; 및 (c) 상기 시료에 포함되어 있는 광견병바이러스와 결합된 검출용 항체의 존재를 효소면역분석에 의하여 검출하는 단계;를 포함하는 광견병바이러스 검출방법을 제공하는 것이다.Yet another object of the present invention is to provide a method of screening a biological sample, comprising: (a) reacting a biological sample with an antibody for coating; (b) reacting the reactant of step (a) with an antibody for detection; And (c) detecting the presence of an antibody for detection bound to the rabies virus contained in the sample by enzyme immunoassay.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 2의 아미노산 서열로 표시되는 단일 사슬 가변 단편(Single-chain variable fragment, scFv)을 포함하는 광견병바이러스 특이 항체를 제공한다.In order to achieve the above object, the present invention provides a rabies virus-specific antibody comprising a single-chain variable fragment (scFv) represented by the amino acid sequence of SEQ ID NO: 2.
또한 본 발명은 서열번호 4의 아미노산 서열로 표시되는 경쇄 가변영역 및 서열번호 6의 아미노산 서열로 표시되는 중쇄 가변영역을 포함하는 광견병바이러스 특이 항체를 제공한다.The present invention also provides a rabies virus-specific antibody comprising a light chain variable region represented by the amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 6.
또한 본 발명은 상기 광견병바이러스 특이 항체를 포함하는 광견병바이러스 검출용 효소면역분석 키트를 제공한다.The present invention also provides an enzyme immunoassay kit for rabies virus detection comprising the rabies virus specific antibody.
또한 본 발명은 (a) 생물학적 시료를 코팅용 항체와 반응시키는 단계; (b) 상기 단계 (a)의 반응물을 검출용 항체와 반응시키는 단계; 및 (c) 상기 시료에 포함되어 있는 광견병바이러스와 결합된 검출용 항체의 존재를 효소면역분석에 의하여 검출하는 단계;를 포함하는 광견병바이러스 검출방법을 제공한다.(A) reacting the biological sample with an antibody for coating; (b) reacting the reactant of step (a) with an antibody for detection; And (c) detecting the presence of the antibody for detection bound to the rabies virus contained in the sample by enzyme immunoassay.
본 발명에 따른 광견병바이러스 특이 항체를 이용하여 효소면역분석을 수행할 경우 광견병바이러스에 대한 민감도, 특이도 및 정확도가 우수할 뿐만 아니라 고가의 분석 장비 없이도 광견병을 신속하고, 정확하며, 간편하게 진단할 수 있는바, 광견병 예방, 치료 및 진단 분야에서 다양하게 활용할 수 있다.When the enzyme immunoassay using the rabies virus specific antibody according to the present invention is performed, the sensitivity, specificity and accuracy of the rabies virus are excellent, and the rabies can be diagnosed quickly, accurately and easily without expensive analytical equipment It can be used in various fields such as bar, rabies prevention, treatment and diagnosis.
도 1은 세포 종류에 따른 광견병바이러스 함량 측정 결과를 나타내는 도이다.
도 2는 광견병바이러스가 접종된 베로 세포의 각 세대별 광견병바이러스 함량 측정 결과를 나타내는 도이다.
도 3은 광견병바이러스가 접종된 베로 세포의 배양 시간 별 광견병바이러스의 함량 측정 결과를 나타내는 도이다.
도 4는 광견병바이러스 ERAGS의 G 유전자 염기서열 분석 결과를 나타내는 도이다.
도 5는 광견병바이러스 ERAGS의 전자현미경 사진 및 웨스턴 블롯팅 결과를 나타내는 도이다.
도 6은 본 발명에 따른 광견병 특이 항체의 효소면역분석 결과를 나타내는 도이다.
도 7은 본 발명에 따른 광견병 특이 항체 B2H17의 경쇄 및 중쇄의 상보성결정부위(CDR, complementarity-determining region)를 나타낸 것이다.
도 8은 광견병바이러스 항원의 코팅농도 결정을 위한 효소면역분석 결과를 나타는 도이다.
도 9는 검체 희석 배수 결정을 위한 효소면역분석 결과를 나타내는 도이다.
도 10은 광견병바이러스 특이 항체와 겨자무과산화효소의 접합체의 희석 배수 결정을 위한 효소면역분석 결과를 나타내는 도이다.FIG. 1 is a graph showing the results of measurement of rabies virus content according to cell type.
FIG. 2 is a graph showing the results of rabies virus content measurement for each generation of Bero cells inoculated with rabies virus.
FIG. 3 is a graph showing the results of measurement of the content of rabies virus by culture time of Bero cells inoculated with rabies virus.
Fig. 4 is a diagram showing the result of G gene base sequence analysis of rabies virus ERAGS.
FIG. 5 is an electron micrograph and Western blotting result of rabies virus ERAGS. FIG.
FIG. 6 is a graph showing an enzyme immunoassay result of the rabies-specific antibody according to the present invention. FIG.
FIG. 7 shows the complementarity-determining region (CDR) of light and heavy chains of rabies-specific antibody B2H17 according to the present invention.
Fig. 8 shows the result of enzyme immunoassay for determination of coating concentration of rabies virus antigen.
9 is a diagram showing the result of enzyme immunoassay for determination of sample dilution ratio.
FIG. 10 is a graph showing the result of enzyme immunoassay for determining the dilution factor of a conjugate of a rabbit virus specific antibody and a mustard non-peroxidase. FIG.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 양태에 따르면, 본 발명은 서열번호 2의 아미노산 서열로 표시되는 단일 사슬 가변 단편(Single-chain variable fragment, scFv)을 포함하는 광견병바이러스 특이 항체를 제공한다.According to an aspect of the present invention, there is provided a rabies virus-specific antibody comprising a single-chain variable fragment (scFv) represented by the amino acid sequence of SEQ ID NO: 2.
본 발명에 있어서, “광견병바이러스”는 랍도바이러스군에 속하며, 포유동물에게 감염되어 광견병을 발생시킬 수 있는 바이러스를 제한없이 포함하며, 바람직하게는 광견병바이러스 ERAGS를 포함한다. 상기 광견병바이러스 ERAGS는 광견병바이러스 ERA의 돌연변이체로, 보다 상세하게는 광견병바이러스 ERA의 G 단백질이 194번째 아미노산 아스파라긴에서 세린으로, 333번째 아미노산이 아르기닌에서 글루탐산으로 치환된 것이다. 상기 광견병바이러스 ERAGS의 G 단백질은 서열번호 1의 염기서열로 표시되는 것이 바람직하다.In the present invention, " rabies virus " belongs to the Robodovirus group and includes, without limitation, viruses capable of infecting mammals and causing rabies, preferably including the rabies virus ERAGS. The rabies virus ERAGS is a mutant of the rabies virus ERA. More specifically, the G protein of the rabies virus ERA is serine at the 194th amino acid asparagine, and the 333rd amino acid is replaced by glutamic acid at arginine. The G protein of the rabies virus ERAGS is preferably represented by the nucleotide sequence of SEQ ID NO: 1.
본 발명에 있어서, “단일 사슬 가변 단편(Single-chain variable fragment, scFv)”은 실제로 항체의 단편은 아니지만, 약 10 내지 25 개의 아미노산으로 이루어진 짧은 링커 펩타이드로 연결된 항체의 중쇄 및 경쇄의 가변 영역의 융합단백질을 의미한다.In the present invention, " single-chain variable fragment (scFv) " is not actually a fragment of an antibody, but a variable region of heavy and light chains of an antibody linked by a short linker peptide consisting of about 10 to 25 amino acids Means a fusion protein.
본 발명의 광견병바이러스 특이 항체는 서열번호 2의 아미노산 서열로 표시되는 단일 사슬 가변 단편을 포함하고, 상기 단일 사슬 가변 단편의 기능적 동등물을 포함할 수 있다. 상기 "기능적 동등물"이란 아미노산의 부가, 치환 또는 결실의 결과, 상기 서열번호 2로 표시되는 아미노산 서열과 적어도 70 % 이상, 바람직하게는 80 % 이상, 더욱 바람직하게는 90 % 이상, 더 더욱 바람직하게는 95 % 이상의 서열 상동성을 갖는 것으로, 서열번호 2로 표시되는 아미노산 서열로 이루어진 펩타이드와 실질적으로 동질의 생리활성을 나타내는 펩타이드를 말한다. 또한, 본 발명의 펩타이드는 생체내의 단백질 절단 효소들로부터 보호하고 안정성을 증가시키기 위해서 N 말단 또는 C 말단을 변형하거나 여러 유기단으로 보호한 형태일 수 있다. 즉, 상기 펩타이드의 C 말단은 안정성을 증가시키기 위해서 변형될 수 있는 형태라면, 특별한 제한은 없으나 바람직하게는 히드록시기(-OH) 또는 아미노기(-NH2)로 변형되는 것일 수 있다. 또한 상기 펩타이드의 N 말단은 안정성을 증가시키기 위해서 변형될 수 있는 형태라면, 특별한 제한은 없으나 바람직하게는 아세틸(Acetyl)기, 플루오레닐 메톡시 카르보닐(Fmoc)기, 포르밀(Formyl)기, 팔미토일(Palmitoyl)기, 미리스틸(Myristyl)기, 스테아릴(Stearyl)기 및 폴리에틸렌글리콜(PEG)로 이루어진 군에서 선택되는 기로 변형되는 것일 수 있다.The rabies virus-specific antibody of the present invention comprises a single chain variable fragment represented by the amino acid sequence of SEQ ID NO: 2 and may comprise the functional equivalent of the single chain variable fragment. Is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70%, more preferably at least 90%, more preferably at least 90% Quot; refers to a peptide having substantially the same physiological activity as a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2, having a sequence homology of 95% or more. In addition, the peptide of the present invention may be in the form of N-terminal or C-terminal modified or protected by various organic terminals in order to protect from protein-cleaving enzymes in vivo and increase stability. That is, the C-terminus of the peptide may be modified to a hydroxyl group (-OH) or an amino group (-NH 2), without any particular limitation, provided that the C-terminal of the peptide can be modified to increase stability. The N-terminus of the peptide is not particularly limited as long as it can be modified to increase stability. Preferably, the N-terminus of the peptide is an acetyl group, a fluorenylmethoxycarbonyl (Fmoc) group, a formyl group , A palmitoyl group, a myristyl group, a stearyl group, and a polyethylene glycol (PEG).
본 발명의 일 구체예에서, 서열번호 2의 아미노산 서열로 표시되는 단일 사슬 가변 단편을 포함하는 광견병바이러스 특이 항체를 4G36으로 명명하였다. 상기 광견병바이러스 특이 항체 4G36은 광견병바이러스 ERAGS를 마우스에 접종한 후 마우스의 비장세포를 분리하고, 상기 비장세포와 골수종 세포를 융합하여 제조된 하이브리도마 세포주로부터 생산된 것이다. 효소면역분석을 통해 음성혈청의 평균 흡광도/양성혈청의 평균 흡광도 값(N/P value)을 확인한 결과, 상기 광견병바이러스 특이 항체 4G36은 N/P value가 높아 효소면역분석의 코팅용 항체로 사용하기에 적합함을 확인하였다.In one embodiment of the present invention, the rabies virus specific antibody comprising a single chain variable fragment represented by the amino acid sequence of SEQ ID NO: 2 was named 4G36. The rabies virus specific antibody 4G36 is produced from a hybridoma cell line prepared by inoculating mouse mouse with rabies virus ERAGS, isolating spleen cells from mice, and fusing the spleen cells and myeloma cells. The N / P value of the rabies virus specific antibody 4G36 was high and it was used as an antibody for coating in the enzyme immunoassay, as a result of confirming the average absorbance of the negative serum / the average absorbance value (N / P value) .
본 발명의 다른 양태에 따르면, 서열번호 4의 아미노산 서열로 표시되는 경쇄 가변영역 및 서열번호 6의 아미노산 서열로 표시되는 중쇄 가변영역을 포함하는 광견병바이러스 특이 항체를 제공한다.According to another aspect of the present invention, there is provided a rabies virus-specific antibody comprising a light chain variable region represented by the amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region represented by the amino acid sequence of SEQ ID NO:
본 발명에 있어서, “가변영역”은 항체의 변이 부위로서, 중쇄 및 경쇄 N-말단의 약 100 내지 120 개의 아미노산으로 이루어진 부분을 의미한다. 상기 가변영역은 동일 개체에서도 아미노산에 차이가 크며, 이러한 차이는 서로 다른 항원결합부위를 만듦으로서 병원체에 대한 특이성을 만든다.In the present invention, " variable region " refers to a mutation site of an antibody, which is a portion consisting of about 100 to 120 amino acids of the heavy chain and the light chain N-terminal. The variable region has a large amino acid difference even in the same individual, and this difference creates a specificity for the pathogen by creating different antigen binding sites.
본 발명의 광견병바이러스 특이 항체는 서열번호 4의 아미노산 서열로 표시되는 경쇄 가변영역 및 서열번호 6의 아미노산 서열로 표시되는 중쇄 가변영역을 포함하고, 상기 경쇄 및 중쇄 펩타이드의 기능적 동등물을 포함할 수 있다.The rabies virus-specific antibody of the present invention includes a light chain variable region represented by the amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 6, and may include functional equivalents of the light and heavy chain peptides have.
본 발명의 일 구체예에서, 서열번호 4의 아미노산 서열로 표시되는 경쇄 가변영역 및 서열번호 6의 아미노산 서열로 표시되는 중쇄 가변영역을 포함하는 광견병바이러스 특이 항체를 B2H17로 명명하였다. 상기 광견병바이러스 B2H17은 광견병바이러스 ERAGS를 마우스에 접종한 후 마우스의 비장세포를 분리하고, 상기 비장세포와 골수종 세포를 융합하여 제조된 하이브리도마 세포주로부터 생산된 것이다. 효소면역분석을 통해 음성혈청의 평균 흡광도/양성혈청의 평균 흡광도 값(N/P value)을 확인한 결과, 상기 광견병바이러스 특이항체 B2H17은 N/P value가 낮아 효소면역분석의 검출용 항체로 사용하기에 적합함을 확인하였다.In one embodiment of the present invention, the rabies virus specific antibody comprising a light chain variable region represented by the amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 6 was named B2H17. The rabies virus B2H17 is produced from a hybridoma cell line prepared by inoculating a mouse with the rabies virus ERAGS, isolating the mouse spleen cells, and fusing the splenocytes and myeloma cells. The N / P value of the rabbit virus-specific antibody B2H17 was low and the antibody was used as an antibody for detection of enzyme immunoassay. .
본 발명의 또 다른 양태에 따르면, 상기 광견병바이러스 특이 항체 4G36 또는 B2H17을 포함하는 광견병바이러스 검출용 효소면역분석 키트를 제공한다.According to another aspect of the present invention, there is provided an enzyme immunoassay kit for rabies virus detection comprising the rabies virus specific antibody 4G36 or B2H17.
본 발명에 있어서, “효소면역분석(enzyme linked immunosorvent assay, ELISA)”은 비색법, 화학발광법 및 형광분석법에 기초하는 것을 포함한다. 효소면역분석은 혈장 및 뇨를 포함하는 생물학적 시료 중 소량의 약물 및 기타 항원 성분 측정시에 성공적으로 적용되어 왔으며, 수행이 간편하다는 장점이 있다.In the present invention, " enzyme linked immunosorbent assay (ELISA) " includes those based on colorimetry, chemiluminescence, and fluorescence assay. Enzyme immunoassay has been successfully applied in the measurement of small quantities of drugs and other antigenic components in biological samples including plasma and urine, and has the advantage of being easy to perform.
본 발명의 효소면역분석 키트는 상기 광견병바이러스 특이 항체 뿐만아니라, 광견병바이러스를 선별적으로 인지할 수 있는 한 다클론항체, 키메릭-항체, 인간화-항체, 단일클론항체의 단편과 같은 다른 항체를 더 포함할 수 있다.The enzyme immunoassay kit of the present invention can be used not only for the rabies virus specific antibody but also for other antibodies such as a polyclonal antibody, a chimeric antibody, a humanized antibody or a fragment of a monoclonal antibody that can selectively recognize rabies virus .
또한 본 발명의 효소면역분석 키트는 효소면역분석에 사용되는 도구 및 시약을 더 포함할 수 있다. 상기 도구 및 시약으로는 담체, 검출 가능한 신호를 생성할 수 있는 표지 물질, 용해제, 세정제 등이 포함된다. 상기 표지 물질이 효소인 경우에는 효소 활성을 측정할 수 있는 기질 및 반응 정지제를 함께 포함할 수 있다. 상기 담체로는, 이에 한정되지는 않으나, 가용성 담체, 예를 들어 당 분야에 공지된 생리학적으로 허용되는 완충액 또는 불용성 담체를 일 수 있다. 상기 완충액의 예로는 PBS 등이 있으며, 불용성 담체의 예로는 폴리스티렌, 폴리에틸렌, 폴리프로필렌, 폴리에스테르, 폴리아크릴로니트릴, 불소 수지, 가교 덱스트란, 폴리사카라이드, 라텍스에 금속을 도금한 자성 미립자와 같은 고분자, 기타 종이, 유리, 금속, 아가로오스 및 이들의 조합이 있다.Further, the enzyme immunoassay kit of the present invention may further comprise tools and reagents used for enzyme immunoassay. Such tools and reagents include a carrier, a labeling substance capable of generating a detectable signal, a solubilizer, a cleaning agent, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring the enzyme activity and a reaction terminator. Such carriers include, but are not limited to, soluble carriers, for example, physiologically acceptable buffers or insoluble carriers known in the art. Examples of the buffer include PBS and the like. Examples of the insoluble carrier include polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, magnetic fine particles plated with metal on latex The same polymer, other paper, glass, metal, agarose, and combinations thereof.
본 발명의 또 다른 양태에 따르면, (a) 생물학적 시료를 코팅용 항체와 반응시키는 단계; (b) 상기 단계 (a)의 반응물을 검출용 항체와 반응시키는 단계; 및 (c) 상기 시료에 포함되어 있는 광견병바이러스와 결합된 검출용 항체의 존재를 효소면역분석에 의하여 검출하는 단계;를 포함하는 광견병바이러스 검출 방법을 제공한다.According to another aspect of the present invention, there is provided a method of detecting a biological sample comprising: (a) reacting a biological sample with an antibody for coating; (b) reacting the reactant of step (a) with an antibody for detection; And (c) detecting the presence of the antibody for detection bound to the rabies virus contained in the sample by enzyme immunoassay.
본 발명에 있어서, “생물학적 시료”는 임의의 동물, 예컨대 인간, 가축 및 농장 동물, 및 동물원용, 스포츠용 또는 애완용 동물, 예컨대, 개, 말, 고양이, 양, 돼지, 소 등을 비롯한 포유동물로서 분류되는 임의의 동물로부터 분리된 신체 시료를 의미하며, 바람직하게는 광견병 미끼백신을 섭취한 동물, 광견병 생백신 혹은 불활화백신을 접종 받았거나, 받은 것으로 의심되는 동물 유래의 시료이다.As used herein, the term "biological sample" refers to any animal, including humans, livestock and farm animals, and mammals including zoos, sport or pet animals, such as dogs, horses, cats, sheep, pigs, , Preferably a sample derived from an animal that has received a rabies vaccine vaccine, a rabies live vaccine, or an animal that has been vaccinated or suspected of having received an inactivated vaccine.
상기 시료는 생물학적 체액, 예컨대, 혈청, 혈장, 유리체액, 림프액, 활액, 소포액, 정액, 양수액, 젖, 전혈, 뇨, 뇌-척수액, 타액, 객담, 눈물, 땀, 점액, 종양 용해물, 및 조직 배양 배지뿐만 아니라, 균질화된 조직, 종양 조직, 및 세포 추출물과 같은 조직 추출물을 포함한다. 바람직하게, 시료는 동물로부터 분리된 혈청이다. 본 발명에서 상기 혈청은, 동물로부터 얻어진 혈액으로부터 혈괴를 제거하고 얻어진 상층액을 사용하되 용혈된 혈청은 제외하는 것이 바람직하다.The sample may be a biological fluid such as serum, plasma, vitreous humor, lymphatic fluid, synovial fluid, semen, semen, milk, whole blood, urine, brain-spinal fluid, saliva, sputum, tears, sweat, mucus, , And tissue culture media as well as tissue extracts such as homogenized tissue, tumor tissue, and cell extracts. Preferably, the sample is serum isolated from the animal. In the present invention, it is preferable to use the supernatant obtained by removing blood clots from blood obtained from an animal, but excluding hemolyzed serum.
본 발명의 일 구체예에서는 광견병바이러스 특이 항체 4G36을 코팅용 항체로 사용하였다. 상기 코팅용 항체는 지지체에 코팅(고정화)된 형태인 것이 바람직하며, PBS (pH 9.6)에 0.1 내지 10 μl/ml의 농도로 희석한 후 지지체에 코팅되는 것이 바람직하다. 코팅(고정화)은 통상적으로, 분석 절차에 앞서 수-불용성 매트릭스 또는 표면으로의 흡착에 의해 또는 비-공유 또는 공유 커플링에 기재된 것과 같이 당업계에 공지된 질산 및 환원제로 지지체를 미리 활성화시키거나 활성화시키지 않은 채로, 글루타르알데히드 또는 카르보디이미드 가교를 사용하여 포획 시약들을 불용화시키거나, 또는 분석 절차 후에 예로서, 면역침전법에 의해 포획 시약들을 불용화시킴으로써 달성된다.In one embodiment of the present invention, the rabies virus specific antibody 4G36 was used as an antibody for coating. Preferably, the coating antibody is coated (immobilized) on a support. It is preferable that the coating antibody is coated on a support after diluted to a concentration of 0.1 to 10 μl / ml in PBS (pH 9.6). The coating (immobilization) is typically carried out by adsorption to a water-insoluble matrix or surface prior to the analytical procedure, or by pre-activating the support with nitric and reducing agents known in the art such as described in non-covalent or covalent coupling Without activating, glutaraldehyde or carbodiimide crosslinking is used to insolubilize the capture reagents, or by insolubilizing the capture reagents by immunoprecipitation as an example after the assay procedure.
고정화를 위해 사용되는 고체상은, 예를 들어, 표면, 입자, 다공성 매트릭스 등의 형태의 지지체를 비롯한, 필수적으로 수-불용성이고 면역분석 측정법에서 유용한 임의의 불활성 지지체 또는 담체일 수도 있다. 통상적으로 사용되는 지지체의 예는 작은 쉬트, 세파덱스, 폴리비닐 클로라이드, 플라스틱 비드, 및 96-웰 플레이트를 포함하여 폴리에틸렌, 폴리프로필렌, 폴리스티렌 등으로부터 제조된 분석 플레이트 또는 시험관 뿐만 아니라 여과지, 아가로스, 가교된 덱스트란 및 기타 다당류와 같은 입상 물질을 포함한다.The solid phase used for immobilization may be any inert support or carrier that is essentially water-insoluble and useful in immunoassay assays, including, for example, supports in the form of surfaces, particles, porous matrices, and the like. Examples of commonly used supports include analytical plates or test tubes made from polyethylene, polypropylene, polystyrene and the like, including small sheets, sephadex, polyvinyl chloride, plastic beads, and 96-well plates, as well as filter paper, agarose, Cross-linked dextran, and other polysaccharides.
본 발명의 다른 구체예에서는 상기 단계 (a)를 수행한 후 단계 (a)의 반응물을 블로킹 용액을 생물학적 시료와 반응시키는 단계를 추가적으로 수행하였다. 구체적으로, 생물학적 시료와 코팅용 항체를 반응시킨 후 나머지 부분의 반응을 정지시키기 위하여, 5 % 스킴밀크 또는 5 % 소혈청알부민과 같은 블로킹 용액을 반응시키는 것이 바람직하다.In another embodiment of the present invention, a step of reacting the reactant of step (a) with the biological sample after performing the step (a) is further performed. Specifically, it is preferable to react a blocking solution such as 5% skim milk or 5% bovine serum albumin in order to stop the reaction of the remaining part after reacting the biological sample with the coating antibody.
본 발명의 또 다른 구체예에서는 광견병바이러스 특이 항체 B2H17을 검출용 항체로 사용하였다. 상기 검출용 항체는 코팅용 항체에 결합된 항원(광견병바이러스)에 반응하는 것이 바람직하다. 또한 항원에 결합된 검출용 항체를 효소면역분석을 통해 검출함으로서, 항원인 광견병바이러스의 존재여부를 확인한다.In another embodiment of the present invention, the rabies virus specific antibody B2H17 was used as an antibody for detection. Preferably, the detection antibody reacts with an antigen (rabies virus) bound to an antibody for coating. In addition, the detection antibody bound to the antigen is detected by enzyme immunoassay to confirm the presence of an antigen, rabies virus.
본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 기술분야에서 통상적으로 사용되는 의미를 갖는 것이다.Terms not otherwise defined herein have meanings as commonly used in the art to which the present invention belongs.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
실시예Example 1. 효소면역분석을 위한 항원 제조 1. Antigen preparation for enzyme immunoassay
효소면역분석(Enzyme-linked immunosorbent assay, ELISA)에 사용할 항원을 제조하기 위하여 다량의 광견병바이러스가 필요하다. 또한 배양과정 중 실험자가 광견병바이러스에 노출될 위험이 있어 병원성이 낮거나 없는 광견병바이러스가 필요한바, 하기 과정을 통해 광견병바이러스 항원을 제조하였다.A large amount of rabies virus is required to produce an antigen for use in an enzyme-linked immunosorbent assay (ELISA). Also, rabies virus antigen was prepared through the following procedure, because the rabies virus is required because the experimenter is exposed to the rabies virus during the incubation process and has low or no pathogenicity.
1-1. 1-1. 광견병바이러스Rabies virus 증식을 위한 세포주 선정 Cell line selection for proliferation
광견병바이러스 증식을 위한 세포주를 선정하기 위하여, 각 세포에 광견병바이러스를 감염시킨 후 바이러스의 함량을 측정하였다. 상기 광견병바이러스는 마우스 뇌에 접종하여도 안전한 광견병바이러스 ERAGS를 사용하였고, 세포주는 베로(African green monkey kidney cell), BHK-21(baby hamster kidney cell) 및 NG108-15를 이용하였다. 상기 광견병바이러스 ERAGS는 광견병바이러스 ERA의 돌연변이체로, 보다 상세하게는 광견병바이러스 ERA의 G 단백질이 194 번째 아미노산 아스파라긴에서 세린으로, 333 번째 아미노산이 아르기닌에서 글루탐산으로 치환된 것이다.In order to select a cell line for rabies virus propagation, each cell was infected with rabies virus and then the content of virus was measured. The rabies virus used ERAGS, a rabies virus that is safe even when inoculated into mouse brain, and the cell line used was African green monkey kidney cell, BHK-21 (baby hamster kidney cell) and NG108-15. The rabies virus ERAGS is a mutant of the rabies virus ERA. More specifically, the G protein of the rabies virus ERA is serine at the 194th amino acid asparagine, and the 333rd amino acid is replaced by glutamic acid at arginine.
먼저, 베로 세포를 롤러(roller) 배양기에서 배양하고, 세포밀도가 90 %에 달하였을 때 광견병바이러스 ERAGS를 접종하였다. 접종 96 시간 후 바이러스를 수확하였다. 3 번의 동결 및 융해 과정을 통해 세포 내 바이러스를 세포 외로 유리시킨 후 3500 내지 4000 rpm에서 30 분 동안 원심 분리하여 세포 찌꺼기를 제거한 후 광견병바이러스의 함량을 측정하였다. BHK-21 및 NG108-15 세포도 베로 세포와 같은 방법으로 광견병바이러스의 함량을 측정하였다. 광견병바이러스 함량 측정 결과는 도 1에 나타내었다.First, Vero cells were cultured in a roller incubator and inoculated with the rabies virus ERAGS when the cell density reached 90%. The virus was harvested 96 hours after inoculation. After intracellular virus was liberated out of the cell through three freezing and thawing processes, the cell debris was removed by centrifugation at 3500-4000 rpm for 30 minutes, and the content of rabies virus was measured. BHK-21 and NG108-15 cells were also assayed for rape virus content in the same manner as for Vero cells. The rabies virus content measurement results are shown in Fig.
도 1에 나타낸 바와 같이, 광견병바이러스 함량은 베로 및 NG108-15 세포가 가장 높았다. 마우스 신경세포 유래의 NG108-15 세포는 증식능이 좋아 광견병바이러스의 함량은 높게 측정되었으나, 배양 시 세포를 유지하는 것이 어렵다는 단점이 있다. 상기 결과를 토대로 베로 세포를 광견병바이러스 증식을 위한 세포주로 선정하였다.As shown in Fig. 1, the rabies virus content was highest in Vero and NG108-15 cells. NG108-15 cells derived from mouse neurons were highly proliferative, and the content of rabies virus was high, but it is difficult to maintain the cells in culture. Based on the above results, Vero cells were selected as a cell line for propagation of rabies virus.
1-2. 증식을 위한 1-2. For proliferation 광견병바이러스Rabies virus 선정 selection
증식을 위한 광견병바이러스를 선정하기 위하여, 베로 세포에 광견병바이러스 ERAGS를 적응시켰다. 구체적으로, 광견병바이러스 ERAGS가 접종된 베로 세포를 10 대까지 계대배양 하였으며, 각 세대의 광견병바이러스 함량을 측정하였다. 각 세대의 광견병바이러스 함량은 도 2에 나타내었다.To select the rabies virus for proliferation, we adapted the rabies virus ERAGS to BERO cells. Specifically, BERRO cells infected with the rabies virus ERAGS were subcultured to the 10th generation and the rabies virus content of each generation was measured. The rabies virus content of each generation is shown in Fig.
도 2에 나타낸 바와 같이, 9 세대 이상 계대한 베로 세포가 광견병바이러스의 함량이 108 FAID50/ml로 가장 높았다. 따라서 10 세대의 광견병바이러스 ERAGS를 증식을 위한 광견병바이러스로 선정하였다.As shown in FIG. 2, the content of rabies virus was the highest at 10 8 FAID 50 / ml for Vero cells of 9 generations or more. Therefore, 10th generation rabies virus ERAGS was selected as a rabies virus for propagation.
1-3. 1-3. 광견병바이러스의Rabies virus 최적 수확시간 선정 Optimal harvest time selection
광견병바이러스 ERAGS의 최적 수확시간을 조사하기 위하여, 10 세대의 광견병바이러스 ERAGS를 베로 세포에 접종하여 배양하였다. 각 시간마다 광견병바이러스 ERAGS의 함량을 측정하였다. 각 시간별 광견병바이러스 함량 측정 결과는 도 3에 나타내었다.In order to investigate the optimum harvest time of rabies virus ERAGS, 10th generation rabies virus ERAGS was inoculated into Bero cells and cultured. The content of rabies virus ERAGS was measured at each hour. The results of measurement of rabies virus content per hour are shown in Fig.
도 3에 나타낸 바와 같이, 광견병바이러스 ERAGS의 함량은 접종 24 시간 후 106 FAID50/ml로 측정되었다. 또한 접종 48 시간 후는 107.5 FAID50/ml였으며, 접종 96 시간 후는 108 FAID50/ml로 가장 높았으나, 그 이후에는 광견병바이러스의 함량이 점차 감소하는 것을 확인하였다. 이상의 결과를 통해 최적 수확시간은 광견병바이러스 ERAGS 접종 96 시간 후인 것을 알 수 있다.As shown in Fig. 3, the content of rabies virus ERAGS was measured at 10 6 FAID 50 / ml after 24 hours of inoculation. In addition, the highest value was 10 7.5 FAID 50 / ml after 48 hours of inoculation and the highest value was 10 8 FAID 50 / ml after 96 hours of inoculation. After that, it was confirmed that the content of rabies virus gradually decreased after that. These results suggest that the optimal harvest time is 96 hours after inoculation of rabies virus ERAGS.
1-4. 1-4. 광견병바이러스Rabies virus G 유전자의 염기서열 분석 Sequence analysis of G gene
실시예 1-2에서 선정한 광견병바이러스 ERAGS에서 G 단백질을 발현하는 유전자의 염기서열 분석을 수행하였다. G 유전자의 염기서열 분석 결과는 도 4에 나타내었다.Sequence analysis of the gene expressing G protein in the rabies virus ERAGS selected in Example 1-2 was performed. The result of the nucleotide sequence analysis of the G gene is shown in Fig.
도 4에 나타낸 바와 같이, 광견병바이러스 ERAGS에서 광견병바이러스의 병원성과 관련이 있는 G 단백질의 194 및 333 번째 아미노산을 암호화하는 염기가 병원성을 가진 광견병바이러스 ERA와 차이가 있는 것을 알 수 있다. 상기 염기의 차이로 인하여, 광견병바이러스 ERAGS의 G 단백질의 194번째 아미노산이 아스파라긴에서 세린으로, 333 번째 아미노산이 아르기닌에서 글루탐산으로 치환된다. 따라서 광견병바이러스 ERAGS는 병원성을 나타내는 G 단백질의 변형으로 인하여 병원성 복귀 가능성이 낮다는 것을 알 수 있다.As shown in Fig. 4, it can be seen that the nucleotide encoding the 194th and 333rd amino acids of the G protein related to the pathogenicity of the rabies virus in the rabies virus ERAGS differs from the pathogenic rabies virus ERA. Due to the difference in the base, the 194th amino acid of the G protein of rabies virus ERAGS is replaced by asparagine to serine, and the 333rd amino acid is replaced by glutamic acid from arginine. Therefore, it can be seen that ERAGS, a rabies virus, is less likely to return to pathogenicity due to the modification of pathogenic G protein.
1-5. 1-5. 광견병바이러스의Rabies virus 정제 refine
효소면역분석의 특이도, 민감도 및 정확도에 영향을 미치는 인자들 중 가장 큰 영향을 차지하는 것이 항원의 정제 정도이다. 항원의 정제 순도가 높을수록 효소면역분석의 특이도, 민감도 및 정확도가 높다.The most important factor affecting the specificity, sensitivity and accuracy of enzyme immunoassays is the degree of purification of the antigen. The higher the purity of the antigen, the higher the specificity, sensitivity and accuracy of the enzyme immunoassay.
높은 순도의 항원을 얻기 위하여, 네 단계에 거쳐 농축 및 정제하였다.In order to obtain high purity antigen, it was concentrated and purified through four steps.
(i) 실시예 1-2의 광견병바이러스 ERGAS 배양액에 포함된 50 kDa이하의 물질을 제거하기 위하여, 50 내지 100 kDa의 크기를 여과할 수 있는 필터(vivaspin)를 사용하여 광견병바이러스 ERGAS 배양액 1000 ml를 3,000 rpm에서 30분간 원심 분리하였다. 상기 과정을 통해 광견병바이러스 배양액 1000 ml가 100 ml로 농축되었다.(i) In order to remove the 50 kDa or less substance contained in the rabies virus ERGAS of Example 1-2, 1000 ml of the rabies virus ERGAS culture solution was added using a filter (vivaspin) capable of filtering 50-100 kDa Were centrifuged at 3,000 rpm for 30 minutes. Through the above procedure, 1000 ml of the rabies virus culture solution was concentrated to 100 ml.
(ii) 삼각플라스크에 1차 농축된 광견병바이러스 ERGAS 배양액 100 ml를 넣은 후 얼음 위에 올려두었다. 상기 삼각플라스크에 0.5 M 염화나트륨 (8.76g/100 ml)과 10 % PEG-8000 (10 g/100 ml)을 첨가하여 용해시켰다. 용해시킨 후 광견병바이러스 ERGAS가 PEG-8000에 응집될 수 있도록 4 ℃에서 12 시간 동안 정치하였다. 그 이후, 3600 rpm에서 30 분 동안 원심 분리하여 침전된 광견병바이러스 ERAGS를 수집하였다. 이때 상층액은 피펫을 통해 모두 제거하였다. 상층액 제거 후 TE 버퍼(50 mM Tris, 1 mM EDTA, pH 7.5)를 이용하여 최초 바이러스 용량의 1/50에 해당하는 용액, 즉 2 ml을 첨가하여 광견병바이러스 ERAGS를 부유시켰다. 부유한 용액을 10000 g에서 5 분 동안 원심 분리하고, 상층액을 얻었다. 이때 상층액에 포함된 광견병바이러스 ERAGS 농축액은 바이러스 이외의 성분이 많기 때문에 효소면역분석의 항원으로 사용하기에 적합하지 않은바, 하기 단계를 수행하였다.(ii) 100 ml of the first concentrated rabies virus ERGAS culture was placed in an Erlenmeyer flask and placed on ice. 0.5 M sodium chloride (8.76 g / 100 ml) and 10% PEG-8000 (10 g / 100 ml) were added to the Erlenmeyer flask to dissolve. After dissolution, the rabies virus ERGAS was allowed to stand at 4 캜 for 12 hours so that it could aggregate in PEG-8000. Thereafter, the precipitated rabies virus ERAGS was collected by centrifugation at 3600 rpm for 30 minutes. At this time, the supernatant was removed through a pipette. After removal of the supernatant, a solution corresponding to 1/50 of the original virus volume, ie, 2 ml, was added to the rabies virus ERAGS using TE buffer (50 mM Tris, 1 mM EDTA, pH 7.5). The suspended solution was centrifuged at 10000 g for 5 minutes, and the supernatant was obtained. Since the rabies virus ERAGS concentrate contained in the supernatant was not suitable for use as an antigen in the enzyme immunoassay because the components other than the virus were many, the following steps were performed.
(iii) TE 버퍼를 이용하여 50 %, 40 %, 30 %, 20 % 및 10%의 수크로오스(sucrose) 용액을 제조하였다. 투명한 초원심 튜브에 50 %, 40 %, 30 %, 20 % 및 10%의 수크로오스(sucrose) 용액을 각각 2 ml씩 넣어 농도 구배가 형성되도록 준비하였고, 상기 초원심 튜브에 광견병바이러스 ERAGS 농축액 1 ml을 첨가하였다. 상기 초원심 튜브를 초원심분리기를 이용하여 100000 g에서 3 시간 동안 원심 분리하여 바이러스를 분리하였다. 원심분리한 후 수크로오스 농도 20 %와 30 % 사이에서 보이는 광견병바이러스 ERAGS를 피펫을 이용하여 회수하였다. 회수한 이후에 같은 방법으로 다시 한 번 투석하였다.(iii) 50%, 40%, 30%, 20% and 10% sucrose solutions were prepared using TE buffer. 2 ml of each of 50%, 40%, 30%, 20% and 10% sucrose solution was prepared in a transparent ultracentrifuge tube so as to form a concentration gradient. To the ultracentrifuge tube, 1 ml of the rabies vaccine ERAGS concentrate Was added. The ultracentrifuge tube was centrifuged at 100,000 g for 3 hours using an ultracentrifuge to isolate the virus. After centrifugation, the rabies virus ERAGS, which showed a sucrose concentration between 20% and 30%, was recovered using a pipette. After the recovery, dialysis was repeated once again in the same manner.
(iv) 투석한 광견병바이러스 ERAGS를 아미콘 초원심필터(Amicon ultra centrifugal filter)를 이용하여 여과하였다. 여과된 광견병바이러스 ERAGS의 농도는 1.09 mg/ml 이었다.(iv) Dialyzed rabies virus ERAGS was filtered using an Amicon ultra centrifugal filter. The concentration of the filtered rabies virus ERAGS was 1.09 mg / ml.
상기 단계 (i) 내지 (iv)에 따라 정제된 광견병바이러스 ERAGS를 전자현미경 및 웨스턴 블롯팅으로 확인하였고, 그 결과는 도 5에 나타내었다. 상기 웨스턴 블롯팅은 종래 알려진 방법으로 수행하였다.The purified rabies virus ERAGS according to the above steps (i) to (iv) was confirmed by electron microscopy and Western blotting, and the results are shown in FIG. The western blotting was performed by a conventionally known method.
도 5에 나타낸 바와 같이, 광견병바이러스 ERAGS는 입자의 크기가 80 내지 120 nm임을 확인하였다.As shown in Fig. 5, it was confirmed that the particle size of the rabies virus ERAGS was 80 to 120 nm.
실시예Example 2. 효소면역분석을 위한 광견병 진단용 항체 제조 2. Manufacture of rabies diagnostic antibody for enzyme immunoassay
2-1. 2-1. 광견병바이러스Rabies virus 특이 항체 생산 및 정제 Production and purification of specific antibodies
광견병에 특이적인 항체는 효소면역분석의 특이도, 민감도에 큰 영향을 미친다. 따라서 광견병바이러스에 특이적인 항체를 얻기 위하여, 실시예 1-5에서 정제한 광견병바이러스 ERAGS를 6 주령 blab/c 마우스에 3 차에 걸쳐 접종하였다. 광견병바이러스 ERAGS가 접종된 마우스의 비장세포와 골수종(myeloma) 세포주인 sp2/0을 융합시켜 하이브리도마(hybridoma)를 제조하였다. 제조된 하이브리도마를 선별한 결과, B2H17, 3E6, 4G36 및 7G48의 하이브리도마를 얻었다. 상기 하이브리도마를 blab/c 마우스 복강에 각각 접종하고, 복강으로부터 복수를 채취하여 항체를 생산하였다. B2H17, 3E6, 4G36 및 7G48을 각각 접종하여 수득한 복수를 친화력 크로마토그래피법으로 정제하였다. 상기 친화력 크로마토그래피는 G 단백질이 충진되어 있는 컬럼에 B2H17, 3E6, 4G36 및 7G48을 각각 주입하였고, 바인딩 버퍼로 컬럼을 3 회 이상 세척한 후 용출액으로 용출시켰다. 항체의 농도를 확인하기 위하여, 상기 용출액을 PBS로 투석한 후 280 nm에서 흡광도를 측정하였다.Antibodies specific to rabies have a great effect on the specificity and sensitivity of enzyme immunoassays. Thus, in order to obtain an antibody specific for rabies virus, the rabies virus ERAGS purified in Example 1-5 was inoculated to 6-week-old blab / c mice three times. A hybridoma was prepared by fusing spleen cells of mice inoculated with rabies virus ERAGS and myeloma cell line sp2 / 0. The prepared hybridomas were screened to obtain hybridomas of B2H17, 3E6, 4G36 and 7G48. The hybridomas were individually inoculated into the abdominal cavity of the blab / c mice, and the ascites was collected from the abdominal cavity to produce antibodies. B2H17, 3E6, 4G36, and 7G48 were inoculated respectively, and the obtained plurality was purified by affinity chromatography. The affinity chromatography was performed by injecting B2H17, 3E6, 4G36 and 7G48 into a column filled with G protein, washing the column three times with binding buffer, and then eluting with the eluate. To confirm the concentration of antibody, the eluate was dialyzed against PBS and absorbance was measured at 280 nm.
상기 광견병바이러스 ERAGS에 대한 광견병 특이 항체 B2H17, 3E6, 4G36 및 7G48의 농도는 각각 1.673, 1.056, 1.136 및 0.16 mg/ml이었다.The concentrations of rabies-specific antibodies B2H17, 3E6, 4G36 and 7G48 to the rabies virus ERAGS were 1.673, 1.056, 1.136 and 0.16 mg / ml, respectively.
2-2. 2-2. 광견병바이러스Rabies virus 특이 항체와 Specific antibody 겨자무Mustard radish 과산화효소의 접합체 제조 Preparation of conjugate of peroxidase
실시예 2-1에서 정제된 광견병바이러스 특이 항체 B2H17, 3E6, 4G36 및 7G48을 퍼옥시데이즈(peroxidase) 법을 이용하여, 겨자무 과산화효소(horse radish peroxidase, HRP)와 접합시켰다. 구체적으로, HRP를 증류수에 녹인 후 0.1 M 과산화나트륨을 첨가하여 상온에서 20분 동안 반응시켰다. 반응한 HRP는 1 mM 아세트산나트륨(pH 4.0)으로 투석하였다. 투석된 HRP와 광견병바이러스 특이 항체 B2H17, 3E6, 4G36 및 7G48을 각가 혼합하여 상온에서 2 시간 동안 교반하여 광견병바이러스 특이 항체-HRP 접합체를 제조하였다. 제조된 광견병바이러스 특이 항체-HRP 접합체를 안정한 상태로 환원시키기 위하여 4 mg/ml의 수산화붕소나트륨을 첨가하여 4 ℃에서 2 시간 동안 반응시켰다. 환원된 광견병바이러스 특이 항체-HRP 접합체는 PBS로 투석하였다.The rabies virus specific antibodies B2H17, 3E6, 4G36 and 7G48 purified in Example 2-1 were conjugated with horse radish peroxidase (HRP) using the peroxidase method. Specifically, HRP was dissolved in distilled water, and 0.1 M sodium peroxide was added thereto, followed by reaction at room temperature for 20 minutes. The reacted HRP was dialyzed with 1 mM sodium acetate (pH 4.0). The dialyzed HRP and rabies virus specific antibodies B2H17, 3E6, 4G36 and 7G48 were mixed and stirred at room temperature for 2 hours to prepare a rabies vaccine-specific antibody-HRP conjugate. In order to reduce the rabies virus specific antibody-HRP conjugate to stable state, 4 mg / ml sodium borohydride was added and reacted at 4 ° C for 2 hours. The reduced rabies virus specific antibody-HRP conjugate was dialyzed against PBS.
2-3. 효소면역분석을 위한 코팅용 항체 및 검출용 항체 선별2-3. Antibody for coating and antibody for detection for enzyme immunoassay
실시예 2-1에서 제조된 광견병바이러스 ERAGS에 대한 광견병 특이 항체 중 효소면역분석에 사용할 코팅용 항체 및 검출용 항체를 선별하였다. Among the rabies-specific antibodies against the rabies virus ERAGS prepared in Example 2-1, coating antibodies and detection antibodies to be used for enzyme immunoassay were selected.
먼저, 정제된 광견병 특이 항체 B2H17를 각각 농도 2 μg/ml가 되도록 PBS (pH 9.6)에 희석하였고, 희석된 광견병 특이 항체를 플레이트에 1 차 코팅하여, 22 ℃에서 16 시간 동안 반응시켰다. 반응 후 플레이트 내의 용액을 버린 후 블로킹 용액(blocking solution)(5 % skim milk in PBS) 200 μl를 플레이트에 첨가하여 1 시간 동안 반응시켰다. 반응 후 블로킹 용액을 제거하고, 정제된 광견병바이러스 ERAGS 100 μl를 플레이트에 첨가하여 1차 코팅한 항체와 결합하도록 37 ℃에서 2 시간동안 반응시켰다. 검체 희석액으로는 광견병 음성 혈청 및 양성 혈청을 사용하였다. 상기 검체 희석액을 각각 플레이트에 첨가한 후, 37 ℃에서 1 시간 동안 반응하였다. PBST로 플레이트 표면의 항원과 반응하지 않은 잔여물을 제거하였다. 상기 실시예 2-2에서 제조한 광견병 특이 항체-HRP 접합체를 첨가하고, 37 ℃에서 30 분 동안 반응시켰다. 반응 후 세척을 통해 반응하지 않은 광견병 특이 항체-HRP 접합체를 제거하고, TMB 기질(substrate)을 첨가하여 상온에서 15 분 동안 반응시켰다. 그 후 반응정지액을 주입하여 HRP와 TMB의 반응을 중지시킨 후, 발색 정도를 흡광도 측정을 통해 확인하였다. 상기 흡광도 측정은 측정 파장 450 nm, 참조 파장 620 nm이다. 또한 광견병 특이 항체 3E6, 4G36 및 7G48도 상기와 같은 방법으로 흡광도를 측정하였다. 상기 흡광도 측정 결과는 도 6에 나타내었다.First, the purified rabies-specific antibody B2H17 was diluted in PBS (pH 9.6) to a concentration of 2 μg / ml, and the diluted rabies-specific antibody was first coated on the plate and reacted at 22 ° C for 16 hours. After the reaction, the solution in the plate was discarded, and 200 μl of a blocking solution (5% skim milk in PBS) was added to the plate and allowed to react for 1 hour. After the reaction, the blocking solution was removed, and 100 μl of the purified rabies virus ERAGS was added to the plate and reacted at 37 ° C for 2 hours to bind with the primary coated antibody. Rabbit negative sera and positive sera were used as sample dilutions. Each of the sample dilutions was added to the plate, followed by reaction at 37 占 폚 for 1 hour. PBST was used to remove residues that did not react with the antigen on the plate surface. The rabies-specific antibody-HRP conjugate prepared in Example 2-2 was added and reacted at 37 DEG C for 30 minutes. After the reaction, unreacted rabies-specific antibody-HRP conjugate was removed by washing, and TMB substrate was added, followed by reaction at room temperature for 15 minutes. After stopping the reaction between HRP and TMB by stopping the addition of the reaction stop solution, the degree of color development was confirmed by absorbance measurement. The absorbance was measured at a wavelength of 450 nm and a reference wavelength of 620 nm. In addition, the rabies-specific antibodies 3E6, 4G36 and 7G48 were also measured for absorbance in the same manner as described above. The results of the above absorbance measurement are shown in FIG.
또한 흡광도 측정 결과, 광견병 음성 혈청과 양성 혈청 각각의 평균값을 기준으로, 음성혈청/양성혈청 값(N/P value)이 가장 큰 광견병 특이 항체를 코팅항체로, N/P value가 가장 낮은광견병 특이 항체를 검출용 항체로 선정하였다. 상기 N/P value 계산식은 수학식 1과 같다.In addition, as a result of the absorbance measurement, the rabies-specific antibody with the highest negative / positive serum value (N / P value) as the coating antibody and the lowest N / P value as the rabbit negative serum Antibodies were selected for detection. The N / P value calculation equation is shown in Equation (1).
[수학식 1][Equation 1]
N/P value = 음성 혈청 평균 흡광도/양성 혈청 평균 흡광도N / P value = negative serum average absorbance / positive serum average absorbance
도 6에 나타낸 바와 같이, 코팅 항체는 N/P value가 가장 큰 광견병 특이항체 4G36을 선정하였고, 검출용 항체는 N/P value이 가장 낮은 B2H17을 선정하였다.As shown in Fig. 6, the rabies-specific antibody 4G36 having the highest N / P value was selected as the coating antibody, and B2H17 having the lowest N / P value was selected as the detection antibody.
실시예Example 3. 효소면역분석을 위한 광견병 진단용 항체의 아미노산 서열 분석 3. Amino acid sequence analysis of rabies diagnostic antibody for enzyme immunoassay
3-1. 광견병 특이 항체 3-1. Rabies-specific antibody 4G36의Of 4G36 아미노산 서열 분석 Amino acid sequence analysis
실시예 2에서 코팅항체로 선정된 광견병 특이 항체 4G36의 단일 사슬 가변 단편(Single-chain variable fragment, scFv)의 아미노산 서열 분석을 수행하였다.Amino acid sequence analysis of a single-chain variable fragment (scFv) of the rabies-specific antibody 4G36 selected as a coating antibody in Example 2 was performed.
광견병 특이 항체 4G36의 단일 사슬 가변 단편의 아미노산 서열 분석 결과, 광견병 특이 항체 4G36은 서열번호 2의 아미노산 서열로 표시된다는 것을 확인하였다.As a result of amino acid sequence analysis of the single chain variable fragment of rabies-specific antibody 4G36, it was confirmed that the rabies-specific antibody 4G36 is represented by the amino acid sequence of SEQ ID NO: 2.
3-2. 광견병 특이 항체 3-2. Rabies-specific antibody B2H17의Of B2H17 동형, 염기서열 및 아미노산 서열분석 Isotype, nucleotide and amino acid sequence analysis
실시예 2에서 검출용 항체로 선정된 광견병 특이 항체 B2H17의 동형, 염기서열 및 아미노산 서열을 분석하였다.The homozygous, nucleotide and amino acid sequences of the rabies-specific antibody B2H17 selected as an antibody for detection in Example 2 were analyzed.
먼저, 효소면역분석(ELISA)을 통해 광견병 특이 항체 B2H17의 동형(isotype)을 확인하였다. 구체적으로, 실시예 2-1의 B2H17 하이브리도마 세포의 배양 상층액을 이용하여 효소면역분석을 수행하였고, 상기 효소면역분석은 Thermo Fisher 사의 Rapid ELISA Mouse mAb Isotyping Kit를 사용하였다. 광견병 특이 항체 B2H17의 동형 분석 결과는 표 1에 나타내었다.First, isotype of rabies-specific antibody B2H17 was confirmed by enzyme immunoassay (ELISA). Specifically, enzyme immunoassay was performed using the culture supernatant of B2H17 hybridoma cells of Example 2-1, and the enzyme immunoassay was performed using a Rapid ELISA Mouse mAb Isotyping Kit from Thermo Fisher. The results of homology analysis of rabies-specific antibody B2H17 are shown in Table 1.
표 1에 나타낸 바와 같이, 광견병 특이 항체 B2H17은 마우스 IgG1/ kappa 형인 것을 알 수 있다.As shown in Table 1, the rabies-specific antibody B2H17 is mouse IgG1 / kappa type.
또한 광견병 특이 항체 B2H17의 염기서열 및 아미노산 서열 분석을 수행하였다. 구체적으로, 실시예 2-1의 B2H17 하이브리도마 세포의 배양 상층액으로부터 DNA를 추출하였다. 상기 DNA 추출은 Bioneer 사의 genomic DNA extraction kit를 이용하였고, 제조사의 매뉴얼에 따라 수행하였다. 또한 추출된 DNA를 이용하여 PCR, TA 클로닝 및 시퀀싱을 수행하여 가변영역(variable region)을 분석하였다. 구체적으로, TaKaRa Ex Taq(RR001A, TaKaRa)를 이용하여 합성된 DNA의 PCR을 수행하였고, PCR 산물로 TA 클로닝을 수행하였다. 상기 TA 클로닝은 pGEM-T Easy 벡터(A1360, Promega)를 사용하였다. 또한 TA 클로닝 산물의 시퀀싱을 수행하여 가변영역의 염기서열 및 아미노산 서열을 분석하였다. The nucleotide sequence and amino acid sequence analysis of rabies-specific antibody B2H17 was also performed. Specifically, DNA was extracted from the culture supernatant of B2H17 hybridoma cells of Example 2-1. The DNA extraction was performed using Bioneer's genomic DNA extraction kit according to the manufacturer's manual. In addition, PCR, TA cloning and sequencing were performed using the extracted DNA to analyze the variable region. Specifically, PCR of the synthesized DNA was performed using TaKaRa Ex Taq (RR001A, TaKaRa), and TA cloning was performed with the PCR product. PGEM-T Easy vector (A1360, Promega) was used for the TA cloning. Sequencing of TA cloning products was also performed to analyze the nucleotide sequence and amino acid sequence of the variable region.
광견병 특이 항체 B2H17의 염기서열 및 아미노산 서열 분석 결과에 따르면, 광견병 특이 항체 B2H17의 경쇄(light chain)의 가변영역은 서열번호 3의 염기서열로 표시되며, 상기 서열번호 3으로 표시되는 염기서열은 서열번호 4로 표시되는 아미노산을 암호화한다는 것을 확인하였다. 또한 광견병 특이 항체 B2H17의 중쇄(heavy chain)의 가변영역은 서열번호 5의 염기서열로 표시되고, 상기 서열번호 5로 표시되는 염기서열은 서열번호 6으로 표시되는 아미노산을 암호화한다는 것을 확인하였다. 또한 광견병 특이 항체 B2H17 경쇄 및 중쇄의 상보성결정부위(CDR, complementarity-determining region)는 도 7과 같다.According to the nucleotide sequence and amino acid sequence analysis result of the rabies-specific antibody B2H17, the variable region of the light chain of the rabies-specific antibody B2H17 is represented by the nucleotide sequence of SEQ ID NO: 3, and the nucleotide sequence of SEQ ID NO: It was confirmed that the amino acid represented by SEQ ID NO: 4 was encoded. In addition, it was confirmed that the variable region of the heavy chain of the rabies-specific antibody B2H17 is represented by the nucleotide sequence of SEQ ID NO: 5, and the nucleotide sequence of SEQ ID NO: 5 encodes the amino acid of SEQ ID NO: In addition, the complementarity-determining region (CDR) of the rabies-specific antibody B2H17 light chain and the heavy chain is shown in FIG.
실시예Example 4. 광견병 진단을 위한 효소면역분석의 최적 조건 확립 4. Establishment of optimal conditions for enzyme immunoassay for rabies diagnosis
4-1. 4-1. 광견병바이러스Rabies virus 항원 antigen ERAGS의Of ERAGS 코팅 농도 결정 Determination of coating concentration
광견병바이러스 항원 ERAGS의 최적 코팅 농도를 조사하였다.The optimal coating concentration of rabies virus antigen ERAGS was investigated.
코팅 항체 4G36를 PBS (pH 9.6)을 이용하여 2.0 μg/ml가 되도록 희석하였다. 농도가 2.0 μg/ml인 코팅 항체 4G36을 96 웰 플레이트에 1차 코팅하고, 블로킹 용액으로 블로킹하였다. 블로킹 용액을 세척한 후, 정제된 광견병바이러스 ERAGS의 농도가 탄산염 버퍼(carbonate buffer) 내에서 각각 0.96, 0.74 및 0.62 μg/ml이 되도록 96 웰 플레이트에 첨가하였고, 코팅 항체 4G36과 결합하도록 2 시간 동안 반응시켰다. 반응된 96 웰 플레이트에 광견병 음성 및 양성 혈청을 각각 첨가한 후 검출용 항체 B2H17을 첨가하여 반응시켰다. 반응 종료 후 96 웰 플레이트를 PBST 버퍼로 2 회 세척하여 96 웰 플레이트의 흡광도를 측정하였다. 항원 코팅 농도별 흡광도 측정 결과는 도 8에 나타내었다.Coating antibody 4G36 was diluted to 2.0 μg / ml with PBS (pH 9.6). Coated antibody 4G36 at a concentration of 2.0 [mu] g / ml was first coated onto a 96 well plate and blocked with blocking solution. After washing the blocking solution, the concentration of the purified rabies virus ERAGS was added to 96-well plates to be 0.96, 0.74 and 0.62 μg / ml in carbonate buffer, respectively, and allowed to bind with coating antibody 4G36 for 2 hours Lt; / RTI > The rabbit negative and positive sera were added to the reacted 96 well plate, and the antibody B2H17 for detection was added and reacted. After completion of the reaction, the 96-well plate was washed twice with PBST buffer to measure the absorbance of the 96-well plate. The results of absorbance measurement by the antigen coating concentration are shown in FIG.
도 8에 나타낸 바와 같이, 항원인 정제된 광견병바이러스 ERAGS를 농도 0.92 μg/ml가 되도록 첨가하였을 때, 흡광도가 가장 높았다. 이러한 결과는 항원을 농도 0.92 μg/ml가 되도록 첨가하는 것이 바람직하다는 것을 의미한다.As shown in Fig. 8, when the purified rabies virus ERAGS, which is an antigen, was added to a concentration of 0.92 μg / ml, the absorbance was the highest. These results indicate that it is preferable to add the antigen to a concentration of 0.92 μg / ml.
4-2. 검체 희석 배수 결정4-2. Determination of dilution of sample
효소면역분석에 사용될 검체인 혈청의 최적 희석 배수를 조사하였다.The optimal dilution ratio of serum sample to be used for enzyme immunoassay was examined.
구체적으로, 코팅 항체 4G36를 PBS (pH 9.6)로 2.0 μg/ml가 되도록 희석하였다. 농도가 2.0 μg/ml인 코팅 항체 4G36을 96 웰 플레이트에 1차 코팅하고, 블로킹 용액으로 블로킹하였다. 블로킹 용액을 세척한 후, 정제된 광견병바이러스 ERAGS의 농도가 탄산염 버퍼(carbonate buffer) 내에서 0.96 μg/ml가 되도록 96 웰 플레이트에 첨가하였고, 코팅 항체 4G36과 결합하도록 2 시간 동안 반응시켰다. 광견병 음성 및 양성 혈청을 각각 5, 10, 및 20 배 희석하여 검체 희석액을 제조하였다. 상기 반응된 96 웰 플레이트에 검체 희석액을 각각 첨가하여 1 시간 동안 반응시킨 후 검출용 항체 B2H17을 첨가하여 다시 한 번 반응시켰다. 발색제 및 발색정지 용액은 상기 실시예 4-1과 동일한 것을 이용하였고, 동일한 방법으로 실험을 실시하였다. 반응 종료 후 96 웰 플레이트를 PBST 버퍼로 2 회 세척하여, 96 웰 플레이트의 흡광도를 측정하였다. 검체 희석 배수별 흡광도 측정 결과는 도 9에 나타내었다.Specifically, coating antibody 4G36 was diluted with PBS (pH 9.6) to 2.0 μg / ml. Coated antibody 4G36 at a concentration of 2.0 [mu] g / ml was first coated onto a 96 well plate and blocked with blocking solution. After washing the blocking solution, the concentration of the purified rabies virus ERAGS was added to the 96 well plate to be 0.96 μg / ml in the carbonate buffer, and reacted for 2 hours to bind with the coating antibody 4G36. Rabbit negative and positive sera were diluted 5, 10, and 20 times, respectively. A sample dilution was added to the reacted 96-well plate, and reacted for 1 hour. Then, the antibody B2H17 for detection was added thereto, and reaction was performed once again. The coloring agent and color-stopping solution were the same as those used in Example 4-1, and the same experiment was carried out. After completion of the reaction, the 96 well plate was washed twice with PBST buffer, and the absorbance of the 96 well plate was measured. The results of the absorbance measurement for each sample dilution lot are shown in Fig.
도 9에 나타낸 바와 같이, 광견병 음성 혈청은 희석 배수에 상관 없이 0.2 이하의 흡광도가 측정되었다. 반면에, 광견병 양성 혈청은 5 배로 희석한 검체 희석액을 사용하였을 때 가장 높은 흡광도를 보였다. 이러한 결과는 효소면역분석 시 검체인 혈청을 5 배 희석하는 것이 바람직하다는 것을 의미한다.As shown in Fig. 9, the rabies-negative serum had an absorbance of 0.2 or less regardless of the dilution ratio. On the other hand, the rabies-positive serum showed the highest absorbance when a 5-fold diluted sample diluent was used. These results indicate that it is desirable to dilute 5 times the sera as a specimen in enzyme immunoassay.
4-3. 4-3. 광견병바이러스Rabies virus 특이 항체와 Specific antibody 겨자무과산화효소의Mustard radish peroxidase 접합체의 희석 배수 결정 Determination of the dilution factor of the conjugate
효소면역분석 시 광견병바이러스 ERAGS 특이 항체-HRP 접합체의 최적 희석 배수를 조사하였다.The optimal dilution ratio of ERAGS specific antibody-HRP conjugate was examined in enzyme immunoassay.
코팅 항체 4G36를 PBS (pH 9.6)을 이용하여 2.0 μg/ml가 되도록 희석하였다. 농도가 2.0 μg/ml인 코팅 항체 4G36을 96 웰 플레이트에 1차 코팅하고, 블로킹 용액으로 블로킹하였다. 블로킹 용액을 세척한 후, 정제된 광견병바이러스 ERAGS의 농도가 탄산염 버퍼(carbonate buffer) 내에서 0.96 ug/ml가 되도록 96 웰 플레이트에 첨가하였고, 코팅 항체 4G36과 결합하도록 2 시간 동안 반응시켰다. 광견병 음성 및 양성 혈청을 5 배 희석하여 검체 희석액을 제조하였다. 상기 반응된 96 웰 플레이트에 검체 희석액을 각각 첨가하여 1 시간 동안 반응시킨 후 검출용 항체 B2H17을 첨가하여 다시 한 번 반응시켰다. 반응 종료 후 96 웰 플레이트 PBST 버퍼로 2 회 세척하였고, 항체-HRP 접합체액의 농도가 4, 2 및 1 μg/ml이 되도록 하였다. 발색제 및 발색정지 용액은 상기 실시예 4-1과 동일한 것을 이용하였고, 동일한 방법으로 실험을 실시하였다. 항체-HRP 접합체 희석 배수별 흡광도 측정 결과는 도 10에 나타내었다.Coating antibody 4G36 was diluted to 2.0 μg / ml with PBS (pH 9.6). Coated antibody 4G36 at a concentration of 2.0 [mu] g / ml was first coated onto a 96 well plate and blocked with blocking solution. After washing the blocking solution, the concentration of the purified rabies virus ERAGS was added to the 96 well plate to be 0.96 ug / ml in the carbonate buffer and reacted for 2 hours to bind with the coating antibody 4G36. Rabbit negative and positive sera were diluted five times to prepare a sample diluent. A sample dilution was added to the reacted 96-well plate, and reacted for 1 hour. Then, the antibody B2H17 for detection was added thereto, and reaction was performed once again. After completion of the reaction, the cells were washed twice with 96-well plate PBST buffer, and the concentration of the antibody-HRP conjugate was 4, 2 and 1 μg / ml. The coloring agent and color-stopping solution were the same as those used in Example 4-1, and the same experiment was carried out. The results of the absorbance measurements for each dilution of the antibody-HRP conjugate are shown in FIG.
도 10에 나타낸 바와 같이, 광견병 음성 혈청은 항체-HRP 접합체액의 농도에 상관없이 0.2 이하의 흡광도를 나타냈다. 반면에 광견병 양성 혈청은 음성 혈청보다 흡광도가 높았으며, 특히 항체-HRP 접합체의 농도가 2 μg/ml인 경우 N/P value 값이 19.1로 가장 높게 나타냈다. 이러한 결과는 효소면역분석 시 항체-HRP 접합체액의 농도는 2 μg/ml인 것이 바람직하다는 것을 의미한다.As shown in Fig. 10, the rabies-negative serum showed an absorbance of 0.2 or less irrespective of the concentration of the antibody-HRP conjugate solution. On the other hand, rabies-positive sera had higher absorbance than negative sera, especially when the antibody-HRP conjugate concentration was 2 μg / ml, the N / P value was the highest at 19.1. These results indicate that the concentration of antibody-HRP conjugate in the enzyme immunoassay is preferably 2 μg / ml.
종합적으로, 광견병 진단을 위한 효소면역분석의 최적 조건은 항원의 농도가 0.92 μg/ml, 혈청 희석 배수는 5 배, 항체-HRP 접합체액의 농도는 2 μg/ml임을 확인하였으며, 상기 조건 시 효소면역분석의 민감도 및 정확도가 우수하다.Overall, the optimal conditions for enzyme immunoassay for rabies diagnosis were found to be 0.92 μg / ml for antigen, 5 times for serum dilution, and 2 μg / ml for antibody-HRP conjugate solution. The sensitivity and accuracy of immunoassay are excellent.
실시예Example 5. 효소면역분석을 이용한 광견병 진단의 민감도, 특이도 및 정확도 확인 5. Sensitivity, specificity and accuracy of rabies diagnosis using enzyme immunoassay
5-1. 개 혈청을 이용한 분석5-1. Analysis using dog serum
실시예 4에서 조사한 효소면역분석 최적 조건 하에서 개 혈청으로부터 광견병바이러스를 검출할 수 있는지 확인하였다. 구체적으로, 코팅항체 4G36 2 μg/ml를 96 웰 플레이트에 코팅하고, 정제한 광견병바이러스 항원 0.92 μg/ml를 플레이트에 코팅하였다. 코팅된 96 웰 플레이트에 138개의 개 혈청을 5 배 희석한 후 100 μl 씩 첨가하였다. 다른 실험 조건 및 방법은 상기 실시예 4-1의 방법과 동일하게 실시하였다. 실험에 사용한 개 혈청은 바이러스 중화시험(FAVN)을 통해 양성 및 음성을 이미 판단한 혈청이며, 본 실시예의 효소면역분석의 민감도, 특이도 및 정확도 확인을 위한 비교대상으로 이용되었다. 개 혈청의 효소면역분석 및 바이러스 중화 시험(FAVN)의 비교 결과는 표 2에 나타내었다.Enzyme immunoassay analysis in Example 4 was carried out to determine whether rabies viruses could be detected from dog serum under optimum conditions. Specifically, 2 μg / ml of the coating antibody 4G36 was coated on a 96-well plate, and 0.92 μg / ml of the purified rabies virus antigen was coated on the plate. 138 seeded sera were diluted 5-fold in coated 96-well plates and then added in 100 μl portions. Other experimental conditions and methods were the same as those of Example 4-1. The dog serum used in the experiment was a serum which had already been judged positive and negative through the virus neutralization test (FAVN), and was used as a comparative example for the sensitivity, specificity and accuracy of the enzyme immunoassay in this example. The results of enzyme immunoassay and virus neutralization test (FAVN) of dog serum are shown in Table 2.
표 2의 효소면역분석 및 바이러스 중화 시험 비교 결과를 바탕으로, 효소면역분석의 민감도, 특이도 및 정확도를 계산하였다. 민감도, 특이도 및 정확도의 계산식은 하기와 같다.Sensitivity, specificity and accuracy of the enzyme immunoassay were calculated based on the results of the enzyme immunoassay and virus neutralization test in Table 2. The formula for sensitivity, specificity and accuracy is as follows.
[수학식 2]&Quot; (2) "
민감도 (%) = 
[수학식 3]&Quot; (3) "
특이도 (%) = 
[수학식 4]&Quot; (4) "
정확도 (%) = 
표 2의 결과를 바탕으로 수학식 2 내지 4에 따라 계산한 결과, 개 혈청에 대한 효소면역분석의 민감도, 특이도 및 정확도는 각각 95.8, 96.5 및 96.3 %임을 확인하였다.Based on the results of Table 2, the sensitivity, specificity, and accuracy of the enzyme immunoassay for dog serum were calculated to be 95.8, 96.5, and 96.3%, respectively, according to
5-2. 너구리 혈청을 이용한 분석5-2. Analysis using raccoon serum
실시예 4에서 조사한 효소면역분석 최적 조건 하에서 너구리 혈청으로부터 광견병바이러스를 검출할 수 있는지 확인하였다. 개 혈청 대신 너구리 혈청을 사용한 것을 제외하고, 상기 실시예 5-1과 동일하게 실험을 실시하였다. 실험에 사용한 너구리 혈청은 바이러스 중화시험(FAVN)을 통해 양성 및 음성을 이미 판단한 혈청이며, 본 실시예의 효소면역분석의 민감도, 특이도 및 정확도 확인을 위한 비교대상으로 이용되었다. 너구리 혈청의 효소면역분석 및 바이러스 중화 시험(FAVN)의 비교 결과는 표 3에 나타내었다.Enzyme Immunoassay in Example 4 [0060] The rabies virus was detected from racoon serum under optimal conditions. The experiment was carried out in the same manner as in Example 5-1 except that raccoon serum was used instead of dog serum. The raccoon serum used in the experiment was a serum that had already been judged positive and negative through the virus neutralization test (FAVN), and was used as a comparative example for confirming the sensitivity, specificity and accuracy of the enzyme immunoassay in this example. Table 3 shows the results of enzyme immunoassay and virus neutralization test (FAVN) of raccoon sera.
표 3의 효소면역분석 및 바이러스 중화 시험 비교 결과를 바탕으로, 효소면역분석의 민감도, 특이도 및 정확도를 수학식 2 내지 4에 따라 계산한 결과, 고양이 혈청에 대한 효소면역분석의 민감도, 특이도 및 정확도는 모두 100 %임을 확인하였다.Based on the results of enzyme immunoassay and virus neutralization test in Table 3, the sensitivity, specificity and accuracy of the enzyme immunoassay were calculated in accordance with
5-3. 고양이 혈청을 이용한 분석5-3. Analysis using cat serum
실시예 4에서 조사한 효소면역분석 최적 조건 하에서 고양이 혈청으로부터 광견병바이러스를 검출할 수 있는지 확인하였다. 개 혈청 대신 고양이 혈청을 사용한 것을 제외하고, 상기 실시예 5-1과 동일하게 실험을 실시하였다. 실험에 사용한 고양이 혈청은 바이러스 중화시험(FAVN)을 통해 양성 및 음성을 이미 판단한 혈청이며, 본 실시예의 효소면역분석의 민감도, 특이도 및 정확도 확인을 위한 비교대상으로 이용되었다. 고양이 혈청의 효소면역분석 및 바이러스 중화 시험(FAVN)의 비교 결과는 표 4에 나타내었다.Enzyme Immunoassay in Example 4 [0060] Under the optimal conditions, it was confirmed whether rabies virus could be detected from the feline serum. The experiment was carried out in the same manner as in Example 5-1 except that cat serum was used instead of dog serum. The feline serum used in the experiment was a serum that had already been judged positive and negative through the virus neutralization test (FAVN), and was used as a comparative example for confirming the sensitivity, specificity and accuracy of the enzyme immunoassay in this example. The results of comparison of enzyme immunoassay and virus neutralization test (FAVN) of cat serum are shown in Table 4.
표 4의 효소면역분석 및 바이러스 중화 시험 비교 결과를 바탕으로, 효소면역분석의 민감도, 특이도 및 정확도를 수학식 2 내지 4에 따라 계산한 결과, 고양이 혈청에 대한 효소면역분석의 민감도, 특이도 및 정확도는 각각 90.9, 100 및 96 %임을 확인하였다.Based on the results of the enzyme immunoassay and the virus neutralization test in Table 4, the sensitivity, specificity, and accuracy of the enzyme immunoassay were calculated according to
종합적으로 본 발명자들은 광견병바이러스 ERAGS에 특이적인 항체를 개발하고, 상기 광견병바이러스 ERAGS에 특이적인 항체를 이용한 효소면역분석의 최적 조건을 확인하였다. 이를 이용하여 광견병 신속하고 정확하게 진단할 수 있고, 고가의 분석 장비 없이도 광견병 진단이 가능함을 의미하는 바, 본 발명의 광견병 진단을 위한 효소면역분석은 광견병 예방, 치료, 및 진단 분야에서 다양하게 활용될 수 있다.In general, the present inventors developed an antibody specific to rabies virus ERAGS and confirmed the optimal conditions for enzyme immunoassay using antibodies specific to rabies virus ERAGS. This means that rabies can be diagnosed quickly and accurately and rabies can be diagnosed without expensive analytical equipments. Enzyme immunoassay for rabies diagnosis of the present invention can be utilized variously in the field of rabies prevention, treatment and diagnosis .
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다.Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
<110> REPUBLIC OF KOREA(Animal and Plant Quarantine Agency) <120> Rabies virus-specific antibody and detecting method of rabies virus using thereof <130> 1-90P <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 1575 <212> DNA <213> rabies virus <220> <221> gene <222> (1)..(1575) <223> rabies virus ERAGS G gene <400> 1 atggttcctc aggctctcct gtttgtaccc cttctggttt ttccattgtg ttttgggaaa 60 ttccctattt acacgatacc agacaagctt ggtccctgga gcccgattga catacatcac 120 ctcagctgcc caaacaattt ggtagtggag gacgaaggat gcaccaacct gtcagggttc 180 tcctacatgg aactaaaagt tggatacatc ttagccataa aaatgaacgg gttcacttgc 240 acaggcgttg tgacggaggc tgaaacctac actaacttcg ttggttatgt cacaaccacg 300 ttcaaaagaa agcatttccg cccaacacca gatgcatgta gagccgcgta caactggaag 360 atggccggtg accccagata tgaagagtct ctacaaaatc cgtaccctga ctaccgctgg 420 cttcgaactg taaaaaccac caaggagtct ctcgttatca tatctccaag tgtggcagat 480 ttggacccat atgacagatc ccttcactcg agggtcttcc ctagcgggaa gtgctcggga 540 gtagcggtgt cttctaccta ctgctccact aaccacgatt acaccatttg gatgcccgag 600 aatccgagac tagggatgtc ttgtgacatt tttacctcca gtagagggaa gagagcatcc 660 aaagggagtg agacttgcgg ctttgtagat gaaagaggcc tatataagtc tttaaaagga 720 gcatgcaaac tcaagttatg tggagttcta ggacttagac ttatggatgg aacatgggtc 780 gcgatgcaaa catcaaatga aaccaaatgg tgccctcccg atcagttggt gaacctgcac 840 gactttcact cagacgaaat tgagcacctt gttgtagagg agttggtcag gaagagagag 900 gagtgtctgg atgcactaga gtccatcatg acaaccaagt cagtgagttt cagacgtctc 960 agtcatttaa gaaaacttgt ccctgggttt ggaaaagcat ataccatatt caacaagacc 1020 ttgatggaag ccgatgctca ctacaagtca gtcgaaactt ggaatgagat catcccttca 1080 aaagggtgtt taagagttgg ggggaggtgt catcctcatg tgaacggggt gtttttcaat 1140 ggtataatat taggacctga cggcaatgtc ttaatcccag agatgcaatc atccctcctc 1200 cagcaacata tggagttgtt ggaatcctcg gttatccccc ttgtgcaccc cctggcagac 1260 ccgtctacca ttttcaagga cggtgacgag gccgaggatt ttgttgaagt tcaccttccc 1320 gatgtgcaca atcaggtctc aggagttgac ttgggtctcc cgaactgggg gaagtatgta 1380 ttactgagtg caggggccct gactgccttg atgttgataa ttttcctgat gacatgttgt 1440 agaagagtca atcgatcaga acctacgcaa cacaatctca gagggacagg gagggaggtg 1500 tcagtcactc cccaaagcgg gaagatcata tcttcatggg aatcacacaa gagtggaggt 1560 gagaccagac tgtga 1575 <210> 2 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> Rabies virus-specific antibody 4G36 scFv <400> 2 Met Ala Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro 1 5 10 15 Gly Ala Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ser 20 25 30 Asp Tyr Leu Met Phe Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu 35 40 45 Trp Ile Gly Asn Ile Ser Pro Tyr Tyr Gly Thr Ile Ser Tyr Asn Leu 50 55 60 Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr 65 70 75 80 Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Arg Arg Tyr Asp Gly Asp Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Asp Ile Glu Leu Thr Gln 115 120 125 Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser 130 135 140 Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn 145 150 155 160 Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala 165 170 175 Ala Ser Asn Gln Gly Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly 180 185 190 Ser Gly Thr Asp Phe Arg Leu Asn Ile His Pro Met Glu Glu Asp Asp 195 200 205 Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys Glu Phe Pro Tyr Thr Phe 210 215 220 Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Ala Ala Glu 225 230 235 <210> 3 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Rabies virus-specific antibody B2H17 <400> 3 gacattgtga tgacccagtc tccatcttcc atgtatgcat ctctaggaga gagagtcact 60 atcacttgca aggcgagtca ggacattaat agttatttaa actggttcca gcagaaacca 120 gggaaatctc ctaagaccct gatctatcgt gcaaacagat tggtagatgg ggtcccatca 180 aggttcagtg gcagtggatc tgggcaagat tattctctca ccatcagcag cctggagtat 240 gaagatatgg gaatttatta ttgtctacag tatgatgagt ttccattcac gttcggctcg 300 gggacaaagt tggaaataaa acgggctgat gctgcaccaa ctgtatccaa tcactag 357 <210> 4 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Rabies virus-specific antibody B2H17 <400> 4 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr 65 70 75 80 Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> Rabies virus-specific antibody B2H17 <400> 5 gaggtcaagc tgcagcagtc tggacctgag ctggtgaagc ctggagcttc aatgaagata 60 tcctgcaagg cttctggtta ctcattcact ggctacacca tgaactgggt gaagcagagc 120 catggaaaga accttgagtg gattggactt attaatcctt acaatggtga tactagctac 180 aaccagaagt tcaagggcaa ggccacatta actgtagaca agtcatccaa cacagcctac 240 atggagctcc tcagtctgac atctgaggac tctgcagtct attattgtgc aactaccccc 300 tttgaccact ggggccaagg caccactctc acagtctctt ca 342 <210> 6 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Rabies virus-specific antibody B2H17 <400> 6 Glu Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Pro Phe Asp His Trp Gly Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser <110> REPUBLIC OF KOREA (Animal and Plant Quarantine Agency) <120> Rabies virus-specific antibody and detecting method of rabies virus using thereof <130> 1-90P <160> 6 <170> KoPatentin 3.0 <210> 1 <211> 1575 <212> DNA <213> rabies virus <220> <221> gene ≪ 222 > (1) .. (1575) <223> rabies virus ERAGS G gene <400> 1 atggttcctc aggctctcct gtttgtaccc cttctggttt ttccattgtg ttttgggaaa 60 ttccctattt acacgatacc agacaagctt ggtccctgga gcccgattga catacatcac 120 ctcagctgcc caaacaattt ggtagtggag gacgaaggat gcaccaacct gtcagggttc 180 tcctacatgg aactaaaagt tggatacatc ttagccataa aaatgaacgg gttcacttgc 240 acaggcgttg tgacggaggc tgaaacctac actaacttcg ttggttatgt cacaaccacg 300 ttcaaaagaa agcatttccg cccaacacca gatgcatgta gagccgcgta caactggaag 360 atggccggtg accccagata tgaagagtct ctacaaaatc cgtaccctga ctaccgctgg 420 cttcgaactg taaaaaccac caaggagtct ctcgttatca tatctccaag tgtggcagat 480 ttggacccat atgacagatc ccttcactcg agggtcttcc ctagcgggaa gtgctcggga 540 gtagcggtgt cttctaccta ctgctccact aaccacgatt acaccatttg gatgcccgag 600 aatccgagac tagggatgtc ttgtgacatt tttacctcca gtagaggaa gagagcatcc 660 aaagggagtg agacttgcgg ctttgtagat gaaagaggcc tatataagtc tttaaaagga 720 gcatgcaaac tcaagttatg tggagttcta ggacttagac ttatggatgg aacatgggtc 780 gcgatgcaaa catcaaatga aaccaaatgg tgccctcccg atcagttggt gaacctgcac 840 gactttcact cagacgaaat tgagcacctt gttgtagagg agttggtcag gaagagagag 900 gagtgtctgg atgcactaga gtccatcatg acaaccaagt cagtgagttt cagacgtctc 960 agtcatttaa gaaaacttgt ccctgggttt ggaaaagcat ataccatatt caacaagacc 1020 ttgatggaag ccgatgctca ctacaagtca gtcgaaactt ggaatgagat catcccttca 1080 aaagggtgtt taagagttgg ggggaggtgt catcctcatg tgaacggggt gtttttcaat 1140 ggtataatat taggacctga cggcaatgtc ttaatcccag agatgcaatc atccctcctc 1200 cagcaacata tggagttgtt ggaatcctcg gttatccccc ttgtgcaccc cctggcagac 1260 ccgtctacca ttttcaagga cggtgacgag gccgaggatt ttgttgaagt tcaccttccc 1320 gatgtgcaca atcaggtctc aggagttgac ttgggtctcc cgaactgggg gaagtatgta 1380 ttactgagtg caggggccct gactgccttg atgttgataa ttttcctgat gacatgttgt 1440 agaagagtca atcgatcaga acctacgcaa cacaatctca gagggacagg gagggaggtg 1500 tcagtcactc cccaaagcgg gaagatcata tcttcatggg aatcacacaa gagtggaggt 1560 gagaccagac tgtga 1575 <210> 2 <211> 238 <212> PRT <213> Artificial Sequence <220> Rabies virus-specific antibody 4G36 scFv <400> 2 Met Ala Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro 1 5 10 15 Gly Ala Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ser 20 25 30 Asp Tyr Leu Met Phe Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu 35 40 45 Trp Ile Gly Asn Ile Ser Pro Tyr Tyr Gly Thr Ile Ser Tyr Asn Leu 50 55 60 Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr 65 70 75 80 Ala Tyr Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Arg Arg Tyr Asp Gly Asp Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Asp Ile Glu Leu Thr Gln 115 120 125 Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser 130 135 140 Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Ile Ser Phe Met Asn 145 150 155 160 Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala 165 170 175 Ala Ser Asn Gln Gly Ser Gly Val Ala Arg Phe Ser Gly Ser Gly 180 185 190 Ser Gly Thr Asp Phe Arg Leu Asn Ile His Pro Met Glu Glu Asp Asp 195 200 205 Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys Glu Phe Pro Tyr Thr Phe 210 215 220 Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Ala Ala Glu 225 230 235 <210> 3 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Rabies virus-specific antibody B2H17 <400> 3 gacattgtga tgacccagtc tccatcttcc atgtatgcat ctctaggaga gagagtcact 60 atcacttgca aggcgagtca ggacattaat agttatttaa actggttcca gcagaaacca 120 gggaaatctc ctaagaccct gatctatcgt gcaaacagat tggtagatgg ggtcccatca 180 aggttcagtg gcagtggatc tgggcaagat tattctctca ccatcagcag cctggagtat 240 gaagatatgg gaatttatta ttgtctacag tatgatgagt ttccattcac gttcggctcg 300 gggacaaagt tggaaataaa acgggctgat gctgcaccaa ctgtatccaa tcactag 357 <210> 4 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Rabies virus-specific antibody B2H17 <400> 4 Asp Ile Val Met Thr Gln Ser Ser Ser Ser Met Tyr Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr 65 70 75 80 Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> Rabies virus-specific antibody B2H17 <400> 5 gaggtcaagc tgcagcagtc tggacctgag ctggtgaagc ctggagcttc aatgaagata 60 tcctgcaagg cttctggtta ctcattcact ggctacacca tgaactgggt gaagcagagc 120 catggaaaga accttgagtg gattggactt attaatcctt acaatggtga tactagctac 180 aaccagaagt tcaagggcaa ggccacatta actgtagaca agtcatccaa cacagcctac 240 atggagctcc tcagtctgac atctgaggac tctgcagtct attattgtgc aactaccccc 300 tttgaccact ggggccaagg caccactctc acagtctctt ca 342 <210> 6 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Rabies virus-specific antibody B2H17 <400> 6 Glu Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Pro Phe Asp His Trp Gly Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser
Claims (9)
A rabies virus specific antibody comprising a light chain variable region represented by the amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 6.
상기 광견병바이러스 특이 항체는 광견병바이러스 ERAGS을 항원으로 하는 것을 특징으로 하는 광견병바이러스 특이 항체.
3. The method of claim 2,
Wherein said rabies-specific antibody specifically rabies virus ERAGS is used as an antigen.
상기 광견병바이러스 ERAGS는 G 유전자가 서열번호 1의 염기서열로 표시되는 것을 특징으로 하는 광견병바이러스 특이 항체.
The method of claim 3,
The rabies virus ERAGS is characterized in that the G gene is represented by the nucleotide sequence of SEQ ID NO: 1.
An enzyme immunoassay kit for rabies virus detection comprising the rabies virus specific antibody according to claim 2.
(b) 상기 단계 (a)의 반응물을 서열번호 4의 아미노산 서열로 표시되는 경쇄 가변영역 및 서열번호 6의 아미노산 서열로 표시되는 중쇄 가변영역을 포함하는 검출용 항체와 반응시키는 단계; 및
(c) 상기 시료에 포함되어 있는 광견병바이러스와 결합된 검출용 항체의 존재를 효소면역분석에 의하여 검출하는 단계;를 포함하는 광견병바이러스 검출 방법.
(a) reacting a serum as a biological sample with an antibody for coating comprising a single-chain variable fragment (scFv) represented by the amino acid sequence of SEQ ID NO: 2;
(b) reacting the reactant of step (a) with a detection antibody comprising a light chain variable region represented by the amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 6; And
(c) detecting the presence of an antibody for detection bound to the rabies virus contained in the sample by enzyme immunoassay.
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