KR20200099421A - An antibody specifically binding symmetric dimethylarginine and uses thereof - Google Patents
An antibody specifically binding symmetric dimethylarginine and uses thereof Download PDFInfo
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- KR20200099421A KR20200099421A KR1020190017394A KR20190017394A KR20200099421A KR 20200099421 A KR20200099421 A KR 20200099421A KR 1020190017394 A KR1020190017394 A KR 1020190017394A KR 20190017394 A KR20190017394 A KR 20190017394A KR 20200099421 A KR20200099421 A KR 20200099421A
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- antibody
- antigen
- binding fragment
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Abstract
Description
대칭적 디메틸아르기닌과 특이적으로 결합하는 항체 및 이의 용도에 관한 것이다.It relates to antibodies that specifically bind to symmetric dimethylarginine and uses thereof.
신부전(renal failure)은 혈액 속의 노폐물을 걸러내고 배출하는 신장의 기능에 장애가 있는 상태를 지칭하며, 혈액 속 노폐물의 농도가 높아지고, 수분의 배출이 증가됨에 따라 고혈압 등과 같은 다양한 합병증을 수반하는 질환이다. 심부전은 그 발병 과정에 따라 급성 신부전(acute renal failure)과 만성 신부전(chronic renal failure)으로 나뉘어지고, 이에 따른 증상도 각각 다르게 나타난다. 만성 신부전은 신장 기능의 3/4 이상이 이미 저하된 상태에서 서서히 진행되는 것을 특징으로 하며, 소변의 색이 엷게 보이는 것이외 별다른 초기 증상은 나타나지 않는다. 이후, 점차 다음 다뇨증(polyuria/polydipsia)이 관찰되며, 상태가 더 진행되면, 구토와 식욕 부진, 체중 감소, 빈혈, 및 요독증(uremia)이 관찰될 뿐만 아니라 경련, 및 혼수 등의 신경 증상까지 초래하게 된다. 급성 신부전은 급격하게 저하된 체액의 불균형으로 소변량이 눈에 띄게 감소하거나 전혀 나오지 않는 동시에, 구토, 식욕부진, 배뇨 곤란, 탈수 상태 등의 증상이 짧은 시간 안에 급속히 악화되는 것을 특징으로 한다. 이런 증상들을 간과하게 되면, 만성 신부전과 마찬가지로 체내에 빠르게 쌓이는 노폐물과 독소로 인해 고칼륨혈증(hyperkalemia)이나 요독증의 원인이 되어 생명이 위험해질 수도 있다.Renal failure refers to a condition in which the function of the kidneys to filter and discharge waste products from the blood is impaired, and it is a disease that accompanies various complications such as high blood pressure as the concentration of waste products in the blood increases and the discharge of water increases. . Heart failure is divided into acute renal failure and chronic renal failure according to the onset process, and symptoms accordingly appear differently. Chronic renal failure is characterized by gradually progressing in a state in which more than 3/4 of the kidney function is already degraded, and there are no early symptoms other than that the color of urine appears pale. Then, the following polyuria (polyuria/polydipsia) is gradually observed, and as the condition progresses, vomiting and loss of appetite, weight loss, anemia, and uremia are observed, as well as convulsions and neurological symptoms such as coma. Is done. Acute renal failure is characterized by a marked decrease in urine volume or not at all due to an imbalance of body fluid that has been rapidly decreased, and symptoms such as vomiting, loss of appetite, difficulty urinating, and dehydration rapidly worsen within a short time. If these symptoms are overlooked, like chronic kidney failure, wastes and toxins that quickly accumulate in the body can cause hyperkalemia or uremia, which can endanger life.
현재, 이러한 신장 질환을 진단하기 위한 방법으로는 신장의 기능을 평가하는 방법이 널리 활용되고 있으며, 일례로서 크레아틴, 혈액 요소질소(BUN) 등의 수준을 측정하는 혈액 검사; 소변의 색을 통해 신장에 의한 농축의 수준을 관찰하는 소변 검사; 및 신장의 크기와 모양을 통해 그 기능성을 예측하는 X-ray, 및 초음파 검사 등이 활용되고 있다(한국 등록특허 10-1782768). Currently, as a method for diagnosing such kidney disease, a method of evaluating the function of the kidney is widely used, and as an example, a blood test for measuring the level of creatine and blood urea nitrogen (BUN); A urine test that observes the level of thickening by the kidneys through the color of the urine; And X-rays for predicting its functionality through the size and shape of the kidney, and ultrasound are being used (Korean Patent Registration No. 10-1782768).
한편, 대칭적 디메틸아르기닌은 비대칭적 디메틸아르기닌의 외인성 이성질체로서, 신장의 기능성을 평가할 수 있는 바이오 마커 중 하나로 알려져 있으나, 이와 유사한 구조를 공유하는 이성질체 또는 물질과의 교차 반응성은 그 검출 효율을 저하시키는 요인으로 작용하고 있다. On the other hand, symmetric dimethylarginine is an exogenous isomer of asymmetric dimethylarginine, and is known as one of the biomarkers that can evaluate the function of the kidney, but cross-reactivity with an isomer or substance that shares a similar structure reduces the detection efficiency. It is acting as a factor.
이러한 기술적 배경 하에서, 본 발명자들은 대칭적 디메틸아르기닌의 아민기를 변형시킨 면역용 항원을 사용하여 제조된 항체가 시료 내 유리 대칭적 디메틸아르기닌과 특이적으로 결합하고 구조적으로 유사한 다른 물질 혹은 비대칭적 이성체에 대해서는 실질적으로 교차 반응성을 나타내지 않는 것을확인하고, 이에 기초하여 본 발명을 완성하였다. Under this technical background, the present inventors believe that an antibody prepared using an antigen for immunization modified with the amine group of symmetric dimethylarginine specifically binds to free symmetric dimethylarginine in the sample and is structurally similar to another substance or asymmetric isomer. It was confirmed that it does not substantially exhibit cross-reactivity, and the present invention was completed based on this.
일 양상은 대칭적 디메틸아르기닌과 특이적으로 결합하는 항체 또는 이의 항원-결합 단편을 제공하는 것이다.One aspect is to provide an antibody or antigen-binding fragment thereof that specifically binds symmetric dimethylarginine.
다른 양상은 대칭적 디메틸아르기닌과 특이적으로 결합하는 항체 또는 이의 항원-결합 단편을 생산하는, 하이브리도마 세포를 제공하는 것이다. Another aspect is to provide hybridoma cells that produce antibodies or antigen-binding fragments thereof that specifically bind symmetric dimethylarginine.
또 다른 양상은 상기의 항체 또는 이의 항원-결합 단편을 포함하는 시료 내 대칭적 디메틸아르기닌 검출용 조성물을 제공하는 것이다. Another aspect is to provide a composition for detecting symmetric dimethylarginine in a sample comprising the antibody or antigen-binding fragment thereof.
또 다른 양상은 상기의 항체 또는 이의 항원-결합 단편을 포함하는 시료 내 대칭적 디메틸아르기닌 검출용 키트를 제공하는 것이다. Another aspect is to provide a kit for detecting symmetric dimethylarginine in a sample comprising the antibody or antigen-binding fragment thereof.
또 다른 양상은 대칭적 디메틸아르기닌과 특이적으로 결합하는 항체 또는 이의 항원-결합 단편을 사용하는 시료 내 대칭적 디메틸아르기닌을 검출하는 방법을 제공하는 것이다.Another aspect is to provide a method of detecting symmetric dimethylarginine in a sample using an antibody or antigen-binding fragment thereof that specifically binds symmetric dimethylarginine.
또 다른 양상은 시료 내에 존재하는 단백질을 제거하는 전단계를 포함하는, 대칭적 디메틸아르기닌을 보다 효과적으로 검출하는 방법을 제공하는 것이다. 일 구체예에 따르면, 상기 전단계는 산 처리에 의한 단백질 침전을 포함할 수 있다.Another aspect is to provide a method for more effectively detecting symmetric dimethylarginine, comprising a pre-step of removing proteins present in a sample. According to one embodiment, the pre-step may include protein precipitation by acid treatment.
또 다른 양상은 상기 항체를 생산하기 위한 면역용 항원을 제조하는 방법을 제공하는 것이다. Another aspect is to provide a method of preparing an antigen for immunization to produce the antibody.
일 양상은 대칭적 디메틸아르기닌과 특이적으로 결합하는 항체 또는 이의 항원-결합 단편을 제공한다. One aspect provides an antibody or antigen-binding fragment thereof that specifically binds symmetric dimethylarginine.
본 명세서에서, 대칭적 디메틸아르기닌(SDMA: symmetric dimethylarginine)은 하기 화학식 1로 표시되는 화학 구조를 갖는 물질로서, IUPAC 명명법 상 (2S)-2-amino-5-[(N, N'-dimethylcarbamimidoyl)amino]pentanoic acid로 지칭된다. 상기 대칭적 디메틸아르기닌은 비대칭적 디메틸아르기닌(ADMA: asymmetric dimethylarginine)의 외인성 이성질체로서, 신부전, 사구체신염 등과 같은 신장 질환을 진단할 수 있는 바이오 마커 중 하나로 알려져 있으나, 이와 유사한 구조를 공유하는 이성질체 또는 물질과의 교차 반응성은 그 검출 효율을 저하시키는 요인으로 작용하고 있다.In the present specification, symmetric dimethylarginine (SDMA) is a substance having a chemical structure represented by the following Formula 1, and (2S)-2-amino-5-[(N, N'-dimethylcarbamimidoyl) in the IUPAC nomenclature It is referred to as amino]pentanoic acid. The symmetric dimethylarginine is an exogenous isomer of asymmetric dimethylarginine (ADMA), and is known as one of biomarkers capable of diagnosing kidney diseases such as renal failure and glomerulonephritis, but isomers or substances that share a similar structure. The cross-reactivity with and acts as a factor to lower the detection efficiency.
[화학식 1][Formula 1]
본 명세서에서 사용된 용어, "항체(antibody)"는 용어 "면역글로불린(immunoglobulin: Ig)"과 상호교환적으로 사용된다. 완전한 항체는 2개의 전장(full length) 경쇄 및 2개의 전장 중쇄를 가지는 구조이며 각각의 경쇄는 중쇄와 이황화 결합(disulfide bond: SS-bond)으로 결합한다. 항체는 예를 들면, IgA, IgD, IgE, IgG, 또는 IgM일 수 있다. 상기 항체는 동물 유래 항체, 마우스-인간 키메라 항체(chimeric antibody), 인간화 항체(humanized antibody), 또는 인간 항체일 수 있다. 상기 항체는 혈장 세포(plasma cell) 또는 하이브리도마 세포에 의해 생산될 수 있다.The term "antibody" as used herein is used interchangeably with the term "immunoglobulin (Ig)". A complete antibody is a structure having two full-length light chains and two full-length heavy chains, and each light chain is bound to a heavy chain by a disulfide bond (SS-bond). Antibodies can be, for example, IgA, IgD, IgE, IgG, or IgM. The antibody may be an animal-derived antibody, a mouse-human chimeric antibody, a humanized antibody, or a human antibody. The antibody may be produced by plasma cells or hybridoma cells.
본 명세서에서 사용된 용어, "항원 결합 단편(antigen-binding fragment)"은 면역글로불린 전체 구조에 대한 그의 단편으로, 항원이 결합할 수 있는 부분을 포함하는 폴리펩티드의 일부를 말한다. 예를 들어, 항원 결합 단편은 scFv, (scFv)2, Fv, Fab, Fab', Fv F(ab')2, 또는 이들의 조합일 수 있으며, 이는 유전자 재조합 기술을 통해 얻을 수 있다. 본 명세서에서는, 종종 편의를 위해, 전장 항체 및 항원-결합 항체 단편을 포괄적으로 지칭할 목적으로 용어 "항체"를 사용하기도 한다.As used herein, the term "antigen-binding fragment" refers to a fragment thereof for the entire structure of an immunoglobulin, and refers to a part of a polypeptide including a portion capable of binding an antigen. For example, the antigen-binding fragment may be scFv, (scFv)2, Fv, Fab, Fab', Fv F(ab')2, or a combination thereof, which can be obtained through genetic recombination techniques. In this specification, for convenience, the term “antibody” is often used for the purpose of generically referring to full-length antibodies and antigen-binding antibody fragments.
본 명세서에서 사용된 용어, "특이적으로 결합(specifically binding)" 은 당업자에게 통상적으로 알려진 의미와 동일한 것으로서, 항원 및 항체가 상호 작용하여 항원-항체 복합체를 형성하는 것을 의미할 수 있으며, 또한 면역학적 반응을 지칭하는 것일 수도 있다.The term "specifically binding" as used herein has the same meaning as commonly known to those skilled in the art, and may mean that an antigen and an antibody interact to form an antigen-antibody complex, and also It may also refer to a scientific reaction.
상기 중쇄(heavay chain)는 5가지(γ, δ, α, μ, ε) 종류가 있으며, 중쇄가 항체의 종류를 결정짓는다. 상기 중쇄에서 αa와 γ는 약 450개, μ 와 ε는 약 550개의 아미노산으로 구성되어 있으며, 두 영역, 즉 가변 영역과 불변 영역으로 이루어져 있다. 상기 경쇄(light chain)는 λ, κ 2가지 종류가 있으며 대략 211 내지 217개의 아미노산으로 구성되어 있으며, 경쇄는 불변 영역과 가변 영역이 연속적으로 이루어져 있다. 상기 가변 영역(variable region)은 항체에서 항원이 결합하는 영역을 말한다.There are five types of heavy chains (γ, δ, α, μ, ε), and the heavy chain determines the type of antibody. In the heavy chain, αa and γ are composed of about 450 amino acids, and μ and ε are composed of about 550 amino acids, and are composed of two regions, that is, a variable region and a constant region. The light chain has two types, λ and κ, and consists of approximately 211 to 217 amino acids, and the light chain has a constant region and a variable region consecutively. The variable region refers to a region to which an antigen binds in an antibody.
본 명세서에서 사용되는 용어, "단일클론항체(monoclonal antibody)"는 당해 분야에 공지된 용어로서 단일 항원성 부위에 대해서 지시되는 고도의 특이적인 항체를 의미한다. 상이한 에피토프(항원 결정기)들에 대해 지시되는 상이한 항체들을 포함하는 다클론항체와는 다르게, 단일클론항체는 항원상의 단일 결정기에 대해서 지시된다. 단일클론항체는 항원-항체 결합을 이용하는 진단 및 분석학적 분석법의 선택성과 특이성을 개선시키는 장점이 있으며, 또한 하이브리도마 배양에 의해 합성되기 때문에 다른 면역글로불린에 의해 오염되지 않는 또 다른 장점을 갖는다. 일 구체예에 따르면, 다클론항체와 단일클론항체 모두 사용될 수 있으며, 단일클론항체가 바람직하게 사용될 수 있다.As used herein, the term "monoclonal antibody" is a term known in the art and refers to a highly specific antibody directed against a single antigenic site. Unlike polyclonal antibodies, which contain different antibodies directed against different epitopes (antigen determinants), monoclonal antibodies are directed against a single determinant on the antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding, and also have another advantage of not being contaminated by other immunoglobulins because they are synthesized by hybridoma culture. According to one embodiment, both polyclonal antibodies and monoclonal antibodies may be used, and monoclonal antibodies may be preferably used.
일 구체예에 있어서, 상기 항체 (항-SDMA 항체) 또는 이의 항원-결합 단편은 서열번호 1의 아미노산 서열로 이루어지는 중쇄 상보성 결정 영역1(Heavy chain complementarity determining region1: HCDR1), 서열번호 2의 아미노산 서열로 이루어지는 중쇄 상보성 결정 영역2(HCDR2), 및 서열번호 3의 아미노산 서열로 이루어지는 중쇄 상보성 결정 영역3(HCDR3)을 포함하는 중쇄 가변 영역; 및 서열번호 4의 아미노산 서열로 이루어지는 LCDR1(Light chain complementarity determining region1: LCDR1), 서열번호 5의 아미노산 서열로 이루어지는 경쇄 상보성 결정 영역2(LCDR2), 및 서열번호 6의 아미노산 서열로 이루어지는 경쇄 상보성 결정 영역3(LCDR3)을 포함하는 경쇄 가변 영역을 포함할 수 있다. 상기 CDRs은 Kabat (See, Kabat, et al., 5th Ed., National Institutes of Health Publication 91-3242, Bethesda, Md., 1991)에 따라 정의된다. In one embodiment, the antibody (anti-SDMA antibody) or antigen-binding fragment thereof is a heavy chain complementarity determining region 1 (HCDR1) consisting of the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2 A heavy chain complementarity determining region 2 (HCDR2) consisting of, and a heavy chain complementarity determining region 3 (HCDR3) consisting of the amino acid sequence of SEQ ID NO: 3; And LCDR1 (Light chain complementarity determining region 1: LCDR1) consisting of the amino acid sequence of SEQ ID NO: 4, light chain complementarity determining region 2 (LCDR2) consisting of the amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region consisting of the amino acid sequence of SEQ ID NO: 6. It may include a light chain variable region including 3 (LCDR3). The CDRs are defined according to Kabat (See, Kabat, et al., 5th Ed., National Institutes of Health Publication 91-3242, Bethesda, Md., 1991).
서열번호 1-3의 HCDR1-3 및 서열번호 4-6의 LCDR4-6을 포함하는 일 구체예에 따른 항-ADMA 항체는 IgG, IgD, IgA로부터 유도된 것일 수 있다. 일 구체예에 따른 항-ADMA 항체는 IgG1, IgG2, IgG3, 또는 IgG4로부터 유도된 것 혹은 이들의 hybrid일 수 있다. 일 구현예에 따른 항-SDMA 항체는 서열번호 1-3의 HCDR1-3 및 서열번호 4-6의 LCDR4-6을 포함하는 IgG 항체일수 있다.The anti-ADMA antibody according to an embodiment comprising HCDR1-3 of SEQ ID NO: 1-3 and LCDR4-6 of SEQ ID NO: 4-6 may be derived from IgG, IgD, and IgA. The anti-ADMA antibody according to an embodiment may be derived from IgG1, IgG2, IgG3, or IgG4, or a hybrid thereof. The anti-SDMA antibody according to an embodiment may be an IgG antibody comprising HCDR1-3 of SEQ ID NO: 1-3 and LCDR4-6 of SEQ ID NO: 4-6.
일 구체예에 있어서, 상기 항체 또는 이의 결합 단편은 서열번호 7의 아미노산 서열로 이루어지는 중쇄 가변 영역; 및 서열번호 8의 아미노산 서열로 이루어지는 경쇄 가변 영역을 포함할 수 있다. In one embodiment, the antibody or binding fragment thereof is a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7; And it may include a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.
일 구현예에 따른 항-SDMA항체는, 서열번호 1의 HCDR1, 서열번호 2의 HCDR2, 및 서열번호 3의 HCDR3을 포함하며, SDMA에 대해 특이적으로 결합하고, 서열번호 7의 아미노산 서열에 대해 적어도 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 또는 99% 서열동일성을 갖는 항체를 포함한다. 또 다른 구현예에 따른 항-SDMA항체는, 서열번호 4의 LCDR1, 서열번호 5의 LCDR2, 및 서열번호 6의 LCDR3을 포함하며, SDMA에 대해 특이적으로 결합하고, 서열번호 8의 아미노산 서열에 대해 적어도 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 또는 99% 서열동일성을 갖는 항체를 포함한다. The anti-SDMA antibody according to an embodiment comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, specifically binds to SDMA, and against the amino acid sequence of SEQ ID NO: 7 Antibodies having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. The anti-SDMA antibody according to another embodiment comprises LCDR1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6, specifically binds to SDMA, and binds to the amino acid sequence of SEQ ID NO: 8 Antibodies having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to.
일 구현예에 따른 항-SDMA 항체는 서열번호 9의 중쇄 및 서열번호 10의 경쇄를 포함할 수 있다. The anti-SDMA antibody according to an embodiment may include a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10.
단백질에 대하여 기재할 때, "보존적 치환(conservative substitution)"은 단백질의 활성을 실질적으로 변화시키지 않는 단백질의 아미노산 조성 변화를 지칭할 수 있다. 따라서, 특정 아미노산 서열의 "보존적으로 변형된 변이"는 단백질 활성에 중대한 영향을 미치지 않는 아미노산으로의 치환, 또는 유사한 특성(산성, 염기성, 양성, 또는 음성으로 하전, 극성 또는 비-극성 등)을 갖고, 중요한 아미노산의 활성을 실질적으로 변경시키지 않는, 다른 아미노산으로의 치환을 지칭할 수 있다. 기능적으로 유사한 아미노산을 제공하는 보존적 치환에 대한 테이블은 해당 기술 분야에 잘 알려져있다. 이하 6개의 군은 각각 일반적으로 서로간 보존적 치환된 아미노산을 포함한다. When describing a protein, "conservative substitution" may refer to a change in the amino acid composition of a protein that does not substantially change the activity of the protein. Thus, a "conservatively modified mutation" of a particular amino acid sequence is a substitution with an amino acid that does not significantly affect protein activity, or a similar property (acid, basic, positive, or negatively charged, polar or non-polar, etc.) And does not substantially alter the activity of an important amino acid, and may refer to a substitution with another amino acid. Tables for conservative substitutions giving functionally similar amino acids are well known in the art. The following six groups each generally contain amino acids conservatively substituted with each other.
Alanine(A), Serine(S), Threonin(T);Alanine (A), Serine (S), Threonin (T);
Aspartic acid(D), Glutamic acid(E);Aspartic acid (D), Glutamic acid (E);
Asparagine(N), Glutamine(Q);Asparagine (N), Glutamine (Q);
Arginine(R), Lysine(K);Arginine (R), Lysine (K);
Isoleucine(I), Leucine(L), Methionine(M), Valine(V): 및Isoleucine (I), Leucine (L), Methionine (M), Valine (V): and
Phenylalanine(F), Tyrosine(Y), Tryptophan(W).Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
일 구현예에 따르면, 상기 항체는 유리(free) 대칭적 디메틸아르기닌을 약 112 μg/dl 또는 그 이하의 농도로 포함하는 생물학적 시료에서 유리 대칭적 디메틸아르기닌을 선별적으로 (selectively) 검출하는데 사용할 수 있다.According to one embodiment, the antibody can be used to selectively detect free symmetric dimethylarginine in a biological sample containing a free symmetric dimethylarginine at a concentration of about 112 μg/dl or less. have.
또 다른 일 구현예에 따르면, 상기 생물학적 시료는 유리 대칭적 디메틸아르기닌을 약 112 μg/dl 또는 그 이하, 약 62 μg/dl 또는 그 이하, 약 37 μg/dl 또는 그 이하, 약 24.5 μg/dl 또는 그 이하, 약 18.2 μg/dl 또는 그 이하, 혹은 약 11 μg/dl 또는 그 이하의 농도로 포함할 수 있다.According to another embodiment, the biological sample contains about 112 μg/dl or less, about 62 μg/dl or less, about 37 μg/dl or less, about 24.5 μg/dl of free symmetric dimethylarginine. Or less, about 18.2 μg/dl or less, or about 11 μg/dl or less.
본 명세서에 사용된 "유리 SDMA (free SDMA)"는 폴리펩티드 혹은 단백질 사슬의 일부를 구성하지 않고 유리된 상태로 (free) 있는 SDMA를 의미한다. As used herein, “free SDMA” refers to SDMA that does not form part of a polypeptide or protein chain and is free.
일 구현예에 따른 항체는, 동일한 조건에서 측정했을 때, 유리 대칭적 디메틸아르기닌에 대한 반응성을 100%로 간주했을 때, 비대칭적 디메틸아르기닌에 대해 25 % 또는 그 이하, 모노메틸아르기닌에 대해 25 % 또는 그 이하, 아르기닌에 대해 25 % 또는 이하의 반응성을 나타내는 것일 수 있다. 또 다른 일 구현예에 따른 항체는 동일한 조건에서 측정했을 때, 유리 대칭적 디메틸아르기닌에 대한 반응성을 100%로 간주했을 때, 비대칭적 디메틸아르기닌에 대해 15 % 또는 그 이하, 모노메틸아르기닌에 대해 15 % 또는 그 이하, 아르기닌에 대해 15 % 또는 이하의 반응성을 나타내는 것일 수 있다. 또 다른 일 구현예에 따른 항체는 동일한 조건에서 측정했을 때, 유리 대칭적 디메틸아르기닌에 대한 반응성을 100%로 간주했을 때, 비대칭적 디메틸아르기닌에 대해 10 % 또는 그 이하, 모노메틸아르기닌에 대해 10 % 또는 그 이하, 아르기닌에 대해 10 % 또는 이하의 반응성을 나타내는 것일 수 있다. 또 다른 일 구현예에 따른 항체는 동일한 조건에서 측정했을 때, 유리 대칭적 디메틸아르기닌에 대한 반응성을 100%로 간주했을 때, 비대칭적 디메틸아르기닌에 대해 5 % 또는 그 이하, 모노메틸아르기닌에 대해 5 % 또는 그 이하, 아르기닌에 대해 5 % 또는 이하의 반응성을 나타내는 것일 수 있다. 또 다른 일 구현예에 따른 항체는 동일한 조건에서 측정했을 때, 유리 대칭적 디메틸아르기닌에 대한 반응성을 100%로 간주했을 때, 비대칭적 디메틸아르기닌에 대해 1 % 또는 그 이하, 모노메틸아르기닌에 대해 1 % 또는 그 이하, 아르기닌에 대해 1 % 또는 이하의 반응성을 나타내는 것일 수 있다. The antibody according to an embodiment is 25% or less for asymmetric dimethylarginine, 25% for asymmetric dimethylarginine or less, when the reactivity to free symmetric dimethylarginine is considered as 100% when measured under the same conditions. Or less, may be one exhibiting a reactivity of 25% or less with respect to arginine. The antibody according to another embodiment is 15% or less for asymmetric dimethylarginine, 15% or less for asymmetric dimethylarginine, as measured under the same conditions as 100% of the reactivity to free symmetric dimethylarginine. % Or less, and may have a reactivity of 15% or less with respect to arginine. The antibody according to another embodiment is 10% or less for asymmetric dimethylarginine, 10% or less for asymmetric dimethylarginine, as measured under the same conditions, when the reactivity to free symmetric dimethylarginine is considered to be 100%. % Or less, and may exhibit reactivity of 10% or less with respect to arginine. The antibody according to another embodiment is 5% or less for asymmetric dimethylarginine, 5% or less for asymmetric dimethylarginine, as measured under the same conditions as 100% of the reactivity to free symmetric dimethylarginine. % Or less, and may exhibit a reactivity of 5% or less with respect to arginine. The antibody according to another embodiment is 1% or less for asymmetric dimethylarginine, 1% or less for asymmetric dimethylarginine, as measured under the same conditions, when the reactivity to free symmetric dimethylarginine is considered to be 100%. % Or less, and may exhibit 1% or less reactivity to arginine.
일 구체예에 있어서, 상기 항체는 대칭적 디메틸아르기닌의 2번 아민기를 변형시킨 유도체를 포함하는 면역용 항원, 즉, 하기 화학식 2의 대칭적 디메틸아르기닌의 유도체와 담체 단백질(carrier protein)이 컨쥬게이션된 면역용 항원을 사용하여 제조된 것일 수 있다. In one embodiment, the antibody is an antigen for immunization comprising a derivative having modified amine group 2 of symmetric dimethylarginine, that is, conjugation of a derivative of symmetric dimethylarginine represented by the following formula (2) and a carrier protein It may be prepared using an antigen for immunization.
[화학식 2][Formula 2]
상기 화학식 2에서, 상기 R1은 -XR2 또는 -R2이고; 상기 X는 O 또는 S이고, 상기 R2는 -(CH2)n-R3이며; 및 상기 n은 1 내지 5의 정수이고, R3는 히드록시(OH), 티올(SH), 또는 카르복시일 수 있다. 예를 들어, 상기 R1은 -XR2 또는 -R2이고; 상기 X는 O이고, 상기 R2는 -(CH2)n-R3이며; 및 상기 n은 1 내지 3의 정수이고, R3는 카르복시일 수 있다. 예를 들어, 상기 R1은 -XR2 이고; 상기 X는 O이고, 상기 R2는 -(CH2)n-R3이며; 및 상기 n은 1 내지 3의 정수이고, R3는 카르복시일 수 있다.In Formula 2, R 1 is -XR 2 or -R 2 ; X is O or S, and R 2 is -(CH 2 ) n -R 3 ; And n is an integer of 1 to 5, and R 3 may be hydroxy (OH), thiol (SH), or carboxy. For example, R 1 is -XR 2 or -R 2 ; X is O, and R 2 is -(CH 2 ) n -R 3 ; And n is an integer of 1 to 3, and R 3 may be carboxy. For example, R 1 is -XR 2 ; X is O, and R 2 is -(CH 2 ) n -R 3 ; And n is an integer of 1 to 3, and R 3 may be carboxy.
상기 항체는 마우스 등의 개체에 전술한 면역용 항원을 투여하여 면역화를 진행한 후, 상기 면역화된 마우스로부터 분리한 polyclonal 항체이거나, 상기 면역화된 마우스로부터 얻은 비장세포와 골수종 세포의 융합을 통해 얻은 하이브리도마로부터 얻은 모노클로날 항체일 수 있다. 폴리클로날 항체나 모노클로날항체를 수득하는 방법으로는, 당업계에 알려진 공지의 기술이 비제한적으로 적용될 수 있다. The antibody is a polyclonal antibody isolated from the immunized mouse after undergoing immunization by administering the aforementioned antigen for immunization to an individual such as a mouse, or a high-level antibody obtained through fusion of splenocytes and myeloma cells obtained from the immunized mouse. It may be a monoclonal antibody obtained from bridoma. As a method of obtaining a polyclonal antibody or a monoclonal antibody, known techniques known in the art may be applied without limitation.
상기 담체 단백질은 소혈청알부민, 인간혈청알부민, 개혈청알부민, 오브알부민, 코케인 증후군 그릅 A 단백질, 및 키홀 림펫 헤모시아닌으로 이루어진 군으로부터 선택되는 어느 하나일 수 있으나, 이에 제한되는 것은 아니다. The carrier protein may be any one selected from the group consisting of bovine serum albumin, human serum albumin, dog serum albumin, ovalbumin, cocaine syndrome group A protein, and keyhole limpet hemocyanin, but is not limited thereto.
화학식 2의 대칭적 디메틸아르기닌 유도체와 담체 단백질로 이루어진 conjugate는 아래 화학식 4로 표시할 수 있다.A conjugate consisting of a symmetric dimethylarginine derivative of Formula 2 and a carrier protein can be represented by Formula 4 below.
[화학식 4][Formula 4]
상기 화학식 4에서, 상기 R1 '은 -X-(CH2)n-R3' 또는 -(CH2)n-R3'이고; 상기 X는 O 또는 S이고, 상기 n은 1 내지 5의 정수이고, R3 '는 -O-, -S-, 또는 -C(O)O-이고, x 는 1-34 의 정수, 그리고 Protein은 담체 단백질이다. 예를 들어, 상기 R 1 ' 은 -X-(CH2)n-R3'이고, 상기 X는 O이고, 상기 n은 1 내지 3의 정수이고, R 3 ' 는 -C(O)O- 일 수 있고, Protein은 BSA이고, x는 1-34의 정수일 수 있다.In Formula 4, R 1 ′ is -X-(CH 2 ) n -R 3 ′ Or -(CH 2 ) n -R 3 '; Wherein X is O or S, n is an integer of 1 to 5, R 3 ′ is -O-, -S-, or -C(O)O-, x is an integer of 1-34, and Protein Is a carrier protein. For example, R 1 ′ is -X-(CH 2 ) n -R 3 ′, X is O, n is an integer of 1 to 3, R 3 ′ is -C(O)O- May be, Protein is BSA, and x may be an integer of 1-34.
일 실시예에 따르면, 상기 면역용 항원은 하기 화학식 3의 대칭적 디메틸아르기닌의 유도체와 소혈청알부민이 결합된 것일 수 있다. According to an embodiment, the antigen for immunization may be a combination of a symmetric dimethylarginine derivative represented by Formula 3 below and bovine serum albumin.
[화학식 3][Formula 3]
일 구체예에 있어서, 상기 항체는 수탁번호 KCLRF-BP-00463인 하이브리도마 세포에 의해 생산되는 대칭적 디메틸아르기닌에 특이적으로 결합하는 단일클론 항체일 수 있다. 또한, 상기 단일클론 항체는 IgG1-카파의 면역글로불린 아이소타입(isotype)일 수 있다.In one embodiment, the antibody may be a monoclonal antibody that specifically binds to symmetric dimethylarginine produced by hybridoma cells with accession number KCLRF-BP-00463. In addition, the monoclonal antibody may be an immunoglobulin isotype of IgG1-kappa.
일 실시예에 따르면, 상기 항체와 생물학적 시료 내 존재하는 대칭적 디메틸아르기닌간 특이적인 결합을 확인할 수 있었으며, 특히, 상기 항체는 16040ng/ml 이하의 대칭적 디메틸아르기닌 농도 조건 하에서, 모노메틸아르기닌, 비대칭적 디메틸아르기닌, 또는 L-아르기닌과의 교차 반응성은 관찰되지 않았다. 상기 생물학적 시료는 전혈, 혈청, 혈장, 척수액, 림프액, 복강액, 뇨, 혈액 세포, 기관, 조직, 타액, 눈물 및 시험 관 내 세포 배양물의 시료일 수 있다. According to an embodiment, a specific binding between the antibody and symmetric dimethylarginine present in a biological sample could be confirmed.In particular, the antibody was monomethylarginine, asymmetric under the condition of a symmetric dimethylarginine concentration of 16040ng/ml or less. No cross-reactivity with red dimethylarginine or L-arginine was observed. The biological sample may be whole blood, serum, plasma, spinal fluid, lymph fluid, peritoneal fluid, urine, blood cells, organs, tissues, saliva, tears, and samples of in vitro cell culture.
따라서, 일 실시예에 따른 항체 또는 이의 항원-결합 단편은 생물학적 시료 내 대칭적 디메틸아르기닌을 특이적으로 검출할 수 있고, 나아가, 대칭적 디메틸아르기닌의 수준과 관련된 질환, 예를 들어 신장 질환을 효과적으로 진단하는데 이용할 수 있다. Thus, the antibody or antigen-binding fragment thereof according to an embodiment can specifically detect symmetric dimethylarginine in a biological sample, and furthermore, effectively prevent diseases related to the level of symmetric dimethylarginine, for example, kidney disease. Can be used for diagnosis.
다른 양상은 대칭적 디메틸아르기닌과 특이적으로 결합하는 항체 또는 이의 항원-결합 단편을 생산하는, 수탁번호 KCLRF-BP-00463인 하이브리도마 세포를 제공한다. Another aspect provides a hybridoma cell with accession number KCLRF-BP-00463, which produces an antibody or antigen-binding fragment thereof that specifically binds symmetric dimethylarginine.
하기 하이브리도마 세포에서 언급된 용어 또는 요소 중 상기 항체에 대한 설명에서 언급된 것과 같은 것은, 상기한 바와 같다. Among the terms or elements mentioned in the hybridoma cells below, those mentioned in the description of the antibody are as described above.
일 실시예에서는 마우스의 비장세포를 확보하고, 이를 마우스 골수종 세포와 융합하여 배양한 후, 대칭적 디메틸아르기닌과 특이적으로 결합하는 항체가 확인된 하이브리도마 세포만을 선택하였다. 이후, 상기 하이브리도마 세포를 한국세포주연구재단(KCLRF, Korean Cell Line Research Foundation)에 기탁하여 2018년 11월 9일 수탁번호 KCLRF-BP-00463를 부여 받았다.In one embodiment, mouse splenocytes were obtained, fused with mouse myeloma cells, and cultured, and then only hybridoma cells in which antibodies specifically binding to symmetric dimethylarginine were confirmed were selected. Thereafter, the hybridoma cells were deposited with the Korean Cell Line Research Foundation (KCLRF) and received accession number KCLRF-BP-00463 on November 9, 2018.
상기 항체를 생산하는 하이브리도마 세포는 이를 시험관 내 또는 생체 내에서 다량으로 배양하는데 사용될 수 있다. 상기 하이브리도마 세포가 생산하는 항체는 정제하지 않고 사용할 수 있으나, 최선의 결과를 얻기 위하여 공지된 방법에 따라 고순도 예컨대, 95% 이상으로 정제하여 사용할 수 있다. 상기 항체는 예를 들어 투석, 염 친전, 크로마토그래피 등의 정제방법을 이용하여 배양 배지 또는 복수액(ascites fluid)으로부터 분리될 수 있다.Hybridoma cells that produce the antibodies can be used to cultivate them in large quantities in vitro or in vivo. The antibody produced by the hybridoma cells may be used without purification, but may be used after purification with high purity, such as 95% or more, according to a known method in order to obtain the best results. The antibody can be isolated from a culture medium or ascites fluid using a purification method such as dialysis, salt electrophilicity, chromatography, or the like.
또 다른 양상은 상기의 항체 또는 이의 항원-결합 단편을 포함하는 시료 내 대칭적 디메틸아르기닌 검출용 조성물을 제공한다. Another aspect provides a composition for detecting symmetric dimethylarginine in a sample comprising the antibody or antigen-binding fragment thereof.
하기 조성물에서 언급된 용어 또는 요소 중 상기 항체에 대한 설명에서 언급된 것과 같은 것은, 상기한 바와 같다. Among the terms or elements mentioned in the following compositions, those mentioned in the description of the antibody are as described above.
상기 조성물은 유효성분으로 대칭적 디메틸아르기닌과 특이적으로 결합하는 항체 또는 이의 항원-결합 단편을 포함하며, 상기 항체를 이용하여 시료 내 대칭적 디메틸아르기닌 항원에 대하여 형성된 항원-항체 복합체를 검출함으로써, 대칭적 디메틸아르기닌을 특이적으로 검출할 수 있다. The composition comprises an antibody or antigen-binding fragment thereof that specifically binds symmetric dimethylarginine as an active ingredient, and by detecting an antigen-antibody complex formed against symmetric dimethylarginine antigen in a sample using the antibody, Symmetrical dimethylarginine can be specifically detected.
본 명세서에서 사용된 용어, "항원-항체 복합체"는, 시료 중의 대칭적 디메틸아르기닌 항원과 이를 인지하는 상기 항체 또는 이의 항원-결합 단편을 의미하며, 이러한 항원-항체 복합체는 비색법(colormetric method), 전기화학법(electrochemical method), 형광법(fluorimetric method), 발광법(luminometry), 입자계수법(particle counting method), 육안측정법(visual assessment) 및 섬광계수법(scintillation counting method)으로 이루어진 군에서 선택되는 임의의 방법으로 검출할 수 있다. As used herein, the term "antigen-antibody complex" refers to a symmetric dimethylarginine antigen in a sample and the antibody or antigen-binding fragment thereof that recognizes it, and the antigen-antibody complex is a colormetric method, Any selected from the group consisting of electrochemical method, fluorescence method, luminometry, particle counting method, visual assessment, and scintillation counting method. It can be detected by the method.
상기 조성물은 항체를 액체 중에서 포함하는 액체 조성물 형태로 제공될 수 있다. 상기 액체 성분은 상기 항체를 용해시킬 수 있고 유지시킬 수 있는 것일 수 있다. 예를 들어, 물, 또는 PBS와 같은 버퍼 용액일 수 있으며, 상기 조성물은 상기 항체를 안정하게 유지하는 물질을 더 포함할 수 있다.The composition may be provided in the form of a liquid composition containing the antibody in a liquid. The liquid component may be one capable of dissolving and maintaining the antibody. For example, it may be water or a buffer solution such as PBS, and the composition may further include a material that stably maintains the antibody.
또 다른 양상은 상기의 항체 또는 이의 항원-결합 단편을 포함하는 시료 내 대칭적 디메틸아르기닌 검출용 키트를 제공한다. Another aspect provides a kit for detecting symmetric dimethylarginine in a sample comprising the antibody or antigen-binding fragment thereof.
하기 키트에서 언급된 용어 또는 요소 중 상기 항체 및 조성물에 대한 설명에서 언급된 것과 같은 것은, 상기한 바와 같다. Among the terms or elements mentioned in the following kits, those mentioned in the description of the antibody and composition are as described above.
또한, 상기 키트는 대칭적 디메틸아르기닌을 면역분석 방법에 따라 검출하는데 이용될 수 있다. 이러한 면역분석은 종래에 개발된 다양한 면역분석(immunoassay) 또는 면역염색(immunostaining) 프로토콜에 따라 실시될 수 있다. 상기 면역분석 또는 면역염색 포맷은 방사능면역분석, 방사능면역침전, 면역침전, ELISA(enzyme-linked immunosorbent assay), 캡처-ELISA, 억제 또는 경쟁 분석, 샌드위치 분석, 유세포 분석(flow cytometry), 면역형광염색 면역친화성 정제, 면역 크로마토그래피(immunochromatography), 및 이들의 조합으로부터 선택된 하나 이상을 포함할 수 있으며, 이 뿐만 아니라, 당업계에 알려진 공지의 기술이 비제한적으로 적용될 수 있다. In addition, the kit can be used to detect symmetric dimethylarginine according to an immunoassay method. These immunoassays can be performed according to various immunoassays or immunostaining protocols previously developed. The immunoassay or immunostaining format is radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA (enzyme-linked immunosorbent assay), capture-ELISA, inhibition or competition analysis, sandwich analysis, flow cytometry, immunofluorescence staining. It may include one or more selected from immunoaffinity purification, immunochromatography, and combinations thereof, and, in addition, known techniques known in the art may be applied without limitation.
예를 들어, 본 출원의 키트가 방사능면역분석 방법에 따라 실시되는 경우, 방사능동위원소(예를 들어, C14, I125, P32 및 S35)로 표지된 항체가 대칭적 디메틸아르기닌을 검출하는 데 이용될 수 있다.For example, when the kit of the present application is carried out according to a radioimmunoassay method, an antibody labeled with a radioactive isotope (e.g., C14, I125, P32 and S35) will be used to detect symmetric dimethylarginine. I can.
또한, 예를 들어, 본 출원의 키트가 ELISA 방식으로 실시되는 경우, 특정 구체예는 (i) 분석하고자 하는 미지의 시료를 고체 기질의 표면에 코팅하는 단계; (ⅱ) 대칭적 디메틸아르기닌에 대한 일차 항체와 상기 시료를 반응시키는 단계; (ⅲ) 상기 단계 (ⅱ)의 결과물을 효소가 결합된 이차항체와 반응시키는 단계; 및 (ⅳ) 상기 효소의 활성을 측정하는 단계를 포함할 수 있다. In addition, for example, when the kit of the present application is carried out by ELISA method, specific embodiments include the steps of: (i) coating an unknown sample to be analyzed on the surface of a solid substrate; (Ii) reacting the sample with a primary antibody against symmetric dimethylarginine; (Iii) reacting the result of step (ii) with a secondary antibody to which an enzyme is bound; And (iv) measuring the activity of the enzyme.
상기 고체 기질로 적합한 것은 탄화수소 폴리머(예를 들어, 폴리스티렌 및 폴리프로필렌), 유리, 금속 또는 젤이며, 마이크로타이터 플레이트일 수 있다.Suitable as the solid substrate are hydrocarbon polymers (eg, polystyrene and polypropylene), glass, metal or gel, and may be microtiter plates.
상기 이차항체에 결합된 효소는 발색반응, 형광반응, 발광반응 또는 적외선 반응을 촉매하는 효소를 포함할 수 있으며, 예를 들어, 알칼린 포스파타아제, β-갈락토시다아제, 호스 래디쉬 퍼옥시다아제, 루시퍼라아제 및 사이토크롬 P450을 포함한다. 상기 이차항체에 결합하는 효소로서 알칼린 포스파타아제가 이용되는 경우에는, 기질로서 브로모클로로인돌일 포스페이트(BCIP), 니트로 블루 테트라졸리움(NBT)나프톨-AS-B1-포스페이트(naphthol-AS-B1-phosphate) 및 ECF(enhanced chemifluorescence)와 같은 발색 반응 기질이 이용되고, 호스래디쉬퍼옥시다아제가 이용되는 경우에는 클로로나프톨, 아미노에틸카바졸, 디아미노벤지딘, D-루시페린, 루시게닌(비스-N-메틸아크리디늄 니트레이트), 레소루핀 벤질 에테르, 루미놀, 암플렉스 레드 시약(10-아세틸-3,7-디하이드록시페녹사진), HYR(p-phenylenediamine-HCl and pyrocatechol), TMB(tetramethylbenzidine), ABTS(2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o-페닐렌디아민(OPD) 및 나프톨/파이로닌, 글루코스 옥시다아제와 t-NBT(nitroblue tetrazolium) 및 m-PMS(phenzaine methosulfate)과 같은 기질이 이용될 수 있다.The enzyme bound to the secondary antibody may include an enzyme that catalyzes a color development reaction, a fluorescence reaction, a light emission reaction or an infrared reaction, for example, alkaline phosphatase, β-galactosidase, horse radish fur Oxidase, luciferase and cytochrome P450. When alkaline phosphatase is used as the enzyme that binds to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT) naphthol-AS-B1-phosphate (naphthol-AS-) is used as a substrate. B1-phosphate) and ECF (enhanced chemifluorescence) are used, and when horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N -Methylacridinium nitrate), resorupine benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine) ), ABTS (2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol/pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate) can be used.
본 출원의 키트가 캡처-ELISA 방식으로 실시되는 경우, 특정 구체예는 (i) 포획항체(capturing antibody)로서 대칭적 디메틸아르기닌에 대한 항체를 고체 기질의 표면에 코팅하는 단계; (ⅱ) 포획 항체와 시료를 반응시키는 단계; (ⅲ) 상기 단계 (ⅱ)의 결과물을 시그널을 발생시키는 레이블이 결합되어 있고, 대칭적 디메틸아르기닌에 대한 검출항체 (예, 본 출원의 항체 또는 항원 결합 단편)와 반응시키는 단계; 및 (ⅳ) 상기 레이블로부터 발생하는 시그널을 측정하는 단계를 포함할 수 있다.When the kit of the present application is carried out in a capture-ELISA method, a specific embodiment includes the steps of: (i) coating an antibody against symmetric dimethylarginine as a capturing antibody on the surface of a solid substrate; (Ii) reacting the capture antibody with the sample; (Iii) reacting the result of step (ii) with a detection antibody (eg, an antibody or antigen-binding fragment of the present application) to which a label for generating a signal is bound and a symmetric dimethylarginine; And (iv) measuring a signal generated from the label.
상기 검출 항체는 검출 가능한 시그널을 발생시키는 표지물질 (label) 가지고 있다. 상기 표지물질은 화학물질(예를 들어, 바이오틴), 효소(알칼린 포스파타아제, β-갈락토시다아제, 호스 래디쉬 퍼옥시다아제 및 사이토크롬 P450), 방사능물질(예를 들어, C14, I125, P32 및 S35), 형광물질(예를 들어, 플루오레신), 발광물질, 화학발광물질(chemiluminescent) 및 FRET(fluorescence resonance energy transfer)을 포함할 수 있다. The detection antibody has a label that generates a detectable signal. The labeling substances include chemical substances (e.g., biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase, and cytochrome P450), radioactive substances (e.g., C14, I125 , P32 and S35), a fluorescent material (eg, fluorescein), a luminescent material, a chemiluminescent material, and a fluorescence resonance energy transfer (FRET).
상기 ELISA 방법 및 캡처-ELISA 방법에서 최종적인 효소의 활성 측정 또는 시그널의 측정은 당업계에 공지된 다양한 방법에 따라 실시될 수 있다. 이러한 시그널의 검출은 대칭적 디메틸아르기닌의 정량적 분석을 가능하게 한다. 만일, 레이블로서 바이오틴이 이용된 경우에는 스트렙타비딘으로, 루시퍼라아제가 이용된 경우에는 루시페린으로 시그널을 용이하게 검출할 수 있다.In the ELISA method and the capture-ELISA method, the final enzyme activity measurement or signal measurement may be performed according to various methods known in the art. The detection of these signals enables quantitative analysis of symmetric dimethylarginine. If biotin is used as a label, streptavidin can be used, and when luciferase is used, a signal can be easily detected with luciferin.
또 다른 양상은 상기의 항체 또는 이의 항원 결합 단편과 시료를 접촉시키는 단계; 및 상기 항체 또는 항원-결합 단편과 대칭적 디메틸아르기닌의 복합체를 검출하는 단계를 포함하는, 시료 내 대칭적 디메틸아르기닌을 검출하는 방법을 제공한다.Another aspect is the step of contacting the antibody or antigen-binding fragment thereof and a sample; And it provides a method for detecting symmetric dimethyl arginine in a sample comprising the step of detecting a complex of the antibody or antigen-binding fragment and symmetric dimethyl arginine.
하기 방법에서 언급된 용어 또는 요소 중 상기 항체 및 조성물에 대한 설명에서 언급된 것과 같은 것은, 상기한 바와 같다. Among the terms or elements mentioned in the following methods, those mentioned in the description of the antibody and composition are as described above.
본 명세서에서 사용되는 용어, "개체"는 대칭적 디메틸아르기닌의 수준과 관련된 질병, 예를 들어 신장 질환의 발병 여부 또는 진행 가능성을 확인하거나 발병 위험도를 예측하고자 하는 개체를 의미한다. 상기 개체는 상기 질환이 발병할 수 있는 동물이라면 그 종류를 한정하지 않으나, 예를 들어, 포유동물일 수 있다.As used herein, the term "individual" refers to a disease related to the level of symmetric dimethylarginine, for example, an individual who wants to determine whether or not a kidney disease is onset or progression or to predict the risk of the onset. The type of the individual is not limited as long as it is an animal capable of developing the disease, but may be, for example, a mammal.
본 명세서에서 사용되는 용어, "시료"는 개체의 체외로 분리된 조직, 세포, 전혈, 혈청, 혈장, 타액, 객담, 뇌척수액, 뇨와 같은 시료 등을 포함할 수 있으며, 예를 들어, 혈청, 혈장, 또는 혈액 시료일 수 있다.As used herein, the term "sample" may include samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, urine, etc. separated from the body of an individual, for example, serum, It may be plasma or a blood sample.
상기 검출하는 단계는 효소면역분석법(ELISA), 웨스턴 블로팅(Western Blotting), 면역형광(Immunofluorescence), 면역크로마토그래피(immunochromatography), 면역조직화학염색(Immunohistochemistry staining), 유세포분석법(Flow cytometry), 면역세포화학법, 방사능면역분석법(RIA), 면역침전분석법(Immunoprecipitation Assay), 면역확산분석법(Immunodiffusion assay), 보체 고정 분석법(Complement Fixation Assay) 및 단백질 칩 Protein Chip)으로 이루어진 군에서 선택된 어느 하나를 수행하는 것을 포함할 수 있으며, 이 뿐만 아니라, 당업계에 알려진 공지의 기술이 비제한적으로 적용될 수 있다. The detecting step includes enzyme immunoassay (ELISA), western blotting, immunofluorescence, immunochromatography, immunochromatography, immunohistochemistry staining, flow cytometry, and immunity. Cell chemistry, radioimmunoassay (RIA), immunoprecipitation assay (Immunoprecipitation Assay), immunodiffusion assay (Immunodiffusion assay), Complement Fixation Assay (Complement Fixation Assay) and protein chip protein chip) It may include, and, as well as this, known techniques known in the art may be applied without limitation.
또한, 또 다른 양상은 시료 내 존재하는 단백질을 침전시키고, 이의 상층액을 수득하는 단계; 상기 상층액에 대칭적 디메틸아르기닌과 특이적으로 결합하는 항체 또는 이의 항원-결합 단편을 접촉시켜 복합체를 형성하는 단계; 및 상기 항체 또는 항원-결합 단편과 대칭적 디메틸아르기닌의 복합체를 검출하는 단계를 포함하는 시료 내 대칭적 디메틸아르기닌을 검출하는 방법을 제공한다.In addition, another aspect is the step of precipitating a protein present in a sample and obtaining a supernatant thereof; Forming a complex by contacting the supernatant with an antibody or antigen-binding fragment thereof that specifically binds symmetric dimethylarginine; And detecting a complex of the antibody or antigen-binding fragment and symmetric dimethylarginine. It provides a method of detecting symmetric dimethylarginine in a sample.
하기 방법에서 언급된 용어 또는 요소 중 상기 개체, 시료, 및 검출하는 단계에 대한 설명에서 언급된 것과 같은 것은, 상기한 바와 같다. Among the terms or elements mentioned in the following method, those mentioned in the description of the object, sample, and detecting step are as described above.
일 실시예에 따르면, 시료에 대한 전처리를 통하여 시료 내 단백질을 침전시킨 후, 상기 시료의 상층액을 대칭적 디메틸아르기닌과 특이적으로 결합하는 항체 또는 이의 항원-결합 단편과 접촉시킴으로써, 대칭적 디메틸아르기닌의 검출 효능을 향상시킬 수 있었다. According to an embodiment, after the protein in the sample is precipitated through pretreatment of the sample, the supernatant of the sample is contacted with an antibody or antigen-binding fragment thereof that specifically binds symmetric dimethylarginine, thereby symmetric dimethyl The detection efficiency of arginine could be improved.
일 구체예에 있어서, 상기 대칭적 디메틸아르기닌과 특이적으로 결합하는 항체 또는 이의 항원-결합 단편은 전술한 항체 또는 이의 항원 결합 단편일 수 있다. In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds symmetric dimethylarginine may be the aforementioned antibody or antigen-binding fragment thereof.
상기 단백질을 침전시키는 단계는예를 들어, 시료에 트리클로로아세트산(Trichloroacetic acid: TCA), 메탄올, 에탄올, 폴리에틸렌 글리콜(Polyethylene glycol: PEG), 덱스트란, 및 황산 암모늄으로 이루어지는 군으로부터 선택되는 어느 하나를 첨가하는 단계를 포함할 수 있다. Precipitating the protein is, for example, trichloroacetic acid (TCA), methanol, ethanol, polyethylene glycol (PEG), dextran, and any one selected from the group consisting of ammonium sulfate in the sample. It may include the step of adding.
일 구체예에 있어서, 상기 시료에 단백질 침전 물질을 첨가하는 단계는 염산, 트리클로로아세트산, 황산 암모늄, 또는 에탄올을 포함하는 단백질 침전 물질과 시료를 각각 1:10 내지 1:3, 1:8 내지 1:3, 1:6 내지 1:3, 또는 1:5 내지 1:3의 부피비로 혼합하는 것일 수 있으며, 이때, 상기 혼합물을 5 내지 30분, 5 내지 25분, 5 내지 20분, 또는 5 내지 15분 동안 추가로 인큐베이션하는 것일 수 있다. 상기 단계에서, 첨가 물질의 양, 시료와 첨가 물질간 반응시간, 시료를 인큐베이션하는 시간 등의 세부 조건은 적의 변경 가능하다. In one embodiment, the step of adding the protein precipitation material to the sample includes a protein precipitation material including hydrochloric acid, trichloroacetic acid, ammonium sulfate, or ethanol and a sample of 1:10 to 1:3, and 1:8 to, respectively. It may be mixed in a volume ratio of 1:3, 1:6 to 1:3, or 1:5 to 1:3, in which case, the mixture is 5 to 30 minutes, 5 to 25 minutes, 5 to 20 minutes, or It may be an additional incubation for 5 to 15 minutes. In the above step, detailed conditions such as the amount of the additive material, the reaction time between the sample and the additive material, and the time for incubating the sample can be appropriately changed.
또한, 상기 상층액을 수득하는 단계는, 시료에 전술한 물질을 첨가하고, 임의적으로 (optionally) 전술한 조건에서 인큐베이션한 후, 이를 원심분리하고, 이로부터 상층액을 수득하는 것일 수 있다. In addition, the step of obtaining the supernatant may include adding the above-described material to a sample, optionally incubating under the above-described conditions, and then centrifuging it, thereby obtaining a supernatant.
또한, 상기 상층액을 수득하는 단계와 항체 또는 항원-결합 단편과 대칭적 디메틸아르기닌의 복합체를 검출하는 단계 사이에서, 상기 상층액과 적절한 버퍼를 혼합하여 중화시키는 단계를 추가로 포함할 수도 있다. In addition, between the step of obtaining the supernatant and the step of detecting the complex of the antibody or antigen-binding fragment and the symmetric dimethylarginine, the step of neutralizing the supernatant by mixing an appropriate buffer may be further included.
또 다른 양상은 상기 화학식 2의 대칭적 디메틸아르기닌의 유도체와 담체 단백질을 컨쥬이션시키는 단계를 포함하는, 대칭적 디메틸아르기닌과 특이적으로 결합하는 항체를 생산하기 위한 면역용 항원을 제조하는 방법을 제공한다.Another aspect provides a method of preparing an antigen for immunization for producing an antibody that specifically binds symmetric dimethylarginine, comprising the step of conjugating a symmetric dimethylarginine derivative of Formula 2 with a carrier protein. do.
하기 방법에서 언급된 용어 또는 요소 중 항체, 및 시료 등에 대한 설명에서 언급된 것과 같은 것은, 상기한 바와 같다. Among the terms or elements mentioned in the following method, those mentioned in the description of the antibody, the sample, and the like are as described above.
상기 대칭적 디메틸아르기닌의 유도체는 2번 아민기에 작용기가 도입되는 방식으로 변형되는 것으로서, 그 작용기 말단에는 담체 단백질과의 컨쥬게이션을 위하여, 히드록시, 티올, 또는 카르복시가 도입되어 있을 수 있다. 상기 대칭적디메틸아르기닌의 유도체는 당업계에 알려진 공지의 기술을 적용하여 다양한 적의 변경 가능하다. 일 구현예에 따르면, 대칭적 디메틸아르기는의 유도체는 상기 화학식 (2)의 화합물일 수 있다. 일 구현예에 따르면, 대칭적 디메틸아르기닌의 유도체는 상기 화학식 (3)의 화합물일 수 있다.The symmetric dimethylarginine derivative is modified in such a way that a functional group is introduced into the second amine group, and hydroxy, thiol, or carboxy may be introduced at the end of the functional group for conjugation with a carrier protein. The symmetric dimethylarginine derivative can be modified in various ways by applying known techniques known in the art. According to an embodiment, the derivative of the symmetric dimethylar group may be a compound of Formula (2). According to one embodiment, the symmetric derivative of dimethylarginine may be a compound of Formula (3).
일 구쳬예에 있어서, 상기 컨쥬게이션시키는 단계는 상기 화학식 2로 표시되는 대칭적 디메틸아르기닌의 유도체 말단과 담체 단백질 표면의 작용기 사이에 결합을 형성하는 것일 수 있으며, 예를 들어, 대칭적 디메틸아르기닌 (화학식 2)의 유도체 내 R3의 히드록시, 티올, 또는 카르복시와 담체 단백질 표면의 작용기간 결합을 형성하는 것일 수 있고, 예를 들어, 대칭적 디메틸아르기닌의 유도체 내 R3의 카르복시와 담체 단백질 표면의 아민기간 결합을 형성하는 것 일 수 있으나, 이에 제한되는 것은 아니다. 일 실시예에 따르면, 대칭적 디메틸아르기닌의 2번 아민기를 변형시킨 유도체를 포함하는 면역용 항원을 사용하여 항체를 제조하였으며, 이렇게 해서 얻은 상기 항체는 혈청 시료 내 존재하는 대칭적 디메틸아르기닌과 선택적으로 특이적인 결합을 보였을 뿐만 아니라, 모노메틸아르기닌, 비대칭적 디메틸아르기닌, 또는 L-아르기닌과의 교차 반응성은 관찰되지 않았다. 따라서, 일 실시예에 따른 면역용 항원은 대칭적 디메틸아르기닌과 특이적으로 결합하는 항체를 생산하는데 사용될 수 있다. In one embodiment, the conjugating step may be to form a bond between the terminal of the derivative of the symmetric dimethylarginine represented by Formula 2 and the functional group on the surface of the carrier protein, for example, symmetric dimethylarginine ( It may be to form a functional bond between the hydroxy, thiol, or carboxy of R 3 in the derivative of formula 2) and the surface of the carrier protein, for example, the carboxy of R 3 in the derivative of symmetric dimethylarginine and the carrier protein surface It may be to form a bond between the amine group, but is not limited thereto. According to an embodiment, an antibody was prepared using an antigen for immunization comprising a derivative of amine group 2 modified with symmetric dimethylarginine, and the antibody obtained in this way was selectively used with symmetric dimethylarginine present in a serum sample. In addition to showing specific binding, no cross-reactivity with monomethylarginine, asymmetric dimethylarginine, or L-arginine was observed. Therefore, the antigen for immunization according to an embodiment can be used to produce an antibody that specifically binds to symmetric dimethylarginine.
한편, 상기 대칭적 디메틸아르기닌은 신부전, 사구체신염 등과 같은 신장 질환을 진단할 수 있는 바이오 마커 중 하나로 알려져 있다. 따라서, 또 다른 양상은 상기의 항체 또는 이의 항원-결합 단편을 포함하는 개체의 신장 질환을 진단하기 위한 조성물 및 키트; 및 개체로부터 분리된 시료에 상기 항체 또는 이의 항원 결합 단편을 접촉시키는 단계를 포함하는 신장 질환의 진단 및 치료에필요한 정보를 제공하는 방법을 제공한다. Meanwhile, the symmetric dimethylarginine is known as one of biomarkers capable of diagnosing kidney diseases such as renal failure and glomerulonephritis. Accordingly, another aspect is a composition and kit for diagnosing kidney disease in an individual comprising the antibody or antigen-binding fragment thereof; And contacting the antibody or antigen-binding fragment thereof with a sample isolated from an individual. It provides a method of providing information necessary for diagnosis and treatment of kidney disease.
상기 시료에서 대칭적 디메틸아르기닌의 수준이 정상 대조군 시료의 수준보다 높으면, 상기 개체는 신장 질환이 발병하거나 진행될 가능성이 높은 것으로 판단할 수 있다. 상기 수준의 변화는 개체의 활성 수준이 정상 대조군 또는 음성 대조군에 비하여 유사한 수준 또는 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% 또는 1000% 이상 증가하는 것을 포함할 수 있다. If the level of symmetrical dimethylarginine in the sample is higher than that of the normal control sample, it may be determined that the individual has a high probability of developing or progressing kidney disease. The change in the level is similar to the level of the individual's activity compared to the normal or negative control, or 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, It may include an increase of 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% or more.
일 양상에 따른 대칭적 디메틸아르기닌 검출용 항체 또는 이의 항원-결합 단편 등에 따르면, 시료 내 유리 대칭적 디메틸아르기닌을 대칭적 디메틸아르기닌과 구조적으로 유사한 물질과의 교차 반응성 없이 특이적으로 검출할 수 있고, 이를 통하여, 대칭적 디메틸아르기닌의 수준과 관련된 질환을 효과적으로 진단할 수 있다. 대칭적 디메틸아르기닌과 구조적으로 유사한 물질은 비대칭적 디메틸아르기닌 (ADMA), N-모노메틸아르기닌 (NMMA), 혹은 아르기닌일 수 있다.According to the symmetric dimethylarginine detection antibody or antigen-binding fragment thereof according to an aspect, free symmetric dimethylarginine in a sample can be specifically detected without cross-reactivity with a substance structurally similar to symmetric dimethylarginine, Through this, it is possible to effectively diagnose diseases related to the level of symmetric dimethylarginine. A substance structurally similar to symmetric dimethylarginine may be asymmetric dimethylarginine (ADMA), N-monomethylarginine (NMMA), or arginine.
도 1은 실시예 4에서 일 구현예에 따른 항체를 이용하여 측정한 IC50 (SDMA)이다. 1 is an IC 50 (SDMA) measured using an antibody according to an embodiment in Example 4.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
실시예 1. 대칭적 디메틸아르기닌의 검출을 위한 면역용 항원 제조 Example 1. Preparation of antigen for immunity for detection of symmetric dimethylarginine
본 실시예에서는 대칭적 디메틸아르기닌(이하, SDMA로 명명함)에 특이적으로 결합하는 항체를 제조하기 위한 면역용 항원을 제조하였다. 구체적으로, SDMA의 SMCC(succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate)를 이용하여 2번 탄소에 결합된 아민기(-NH2)와의 공유결합, 즉 아마이드 결합을 형성시켜 SDMA 유도체를 제조한 뒤, 여기에 담체 단백질과 말레아마이드 결합을 진행하여 면역용 항원을 제조 및 사용하였다. 일례로서, 본 실시예에서는 하기 화학식 3으로 표시되는 유도체(이하, 제1 SDMA 유도체로 명명함.)를 포함하는 면역용 항원을 제조 및 사용하였다.In this example, an antigen for immunization was prepared to prepare an antibody that specifically binds to symmetric dimethylarginine (hereinafter referred to as SDMA). Specifically, the SDMA derivative is formed by forming a covalent bond, that is, an amide bond, with an amine group (-NH 2 ) bonded to carbon 2 using SDMA's SMCC (succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate). After preparation, the carrier protein and maleamide were bound thereto to prepare and use an antigen for immunity. As an example, in this example, an antigen for immunization was prepared and used, including a derivative represented by the following formula (3) (hereinafter referred to as the first SDMA derivative).
[화학식 3] [Formula 3]
[반응식 1][Scheme 1]
이후, 제 1 SDMA 유도체와 소혈청 알부민(BSA, 20mg, Sigma사) 을 결합하기 위하여 소혈청 알부민을 녹인 용액(100mM PBS pH7.2)을 첨가하여 이를 30분동안 교반 하였다. 이후, 상기 혼합 용액을 PD-10 컬럼에 투과시킴으로써, 소혈청 알부민과 결합된 제2의 SDMA 유도체를 포함하는 용액을 수득하였다. 상기 소혈청 알부민에 결합된 제2의 SDMA 유도체의 합성 과정은 하기 반응식 2에 나타낸 바와 같다.Then, in order to combine the first SDMA derivative with bovine serum albumin (BSA, 20mg, Sigma), a solution (100mM PBS pH7.2) in which bovine serum albumin was dissolved was added and stirred for 30 minutes. Thereafter, the mixed solution was permeated through a PD-10 column to obtain a solution containing a second SDMA derivative bound with bovine serum albumin. The synthesis process of the second SDMA derivative bound to bovine serum albumin is as shown in Scheme 2 below.
[반응식 2][Scheme 2]
이하의 실시예에서는 상기 상기 소혈청 알부민에 결합된 SDMA 유도체를 면역용 항원으로 사용하였다.In the following examples, the SDMA derivative bound to the bovine serum albumin was used as an antigen for immunization.
실시예 2. SDMA의 검출을 위한 항체의 제조 Example 2. Preparation of antibodies for detection of SDMA
본 실시예에서는 상기 실시예 1에서 제조된 면역용 항원을 이용하여 SDMA 검출을 위한 항체, 및 상기 항체를 생산하기 위한 하이브리도마 세포주 등을 제조하였다. In this example, an antibody for SDMA detection and a hybridoma cell line for producing the antibody were prepared using the immunization antigen prepared in Example 1 above.
2-1. 면역원의 제조2-1. Preparation of immunogen
상기 실시예 1에서 제조된 면역용 항원을 사용하여 다음과 같이 면역원을 제작하였다. 1차 면역시에 사용하는 면역원은 완전 프로인트 보조제(Complete Freund's Ajuvant: Sigma 사)를 1:1로 유액화(emulsion)하여 제조하였고, 추가적인 면역시에 사용하는 면역원은 불완전 프로인트 보조제 (Incomplete Freund's Ajuvant: Sigma 사)를 1:1로 유액화하여 제조하였다.Using the antigen for immunization prepared in Example 1, an immunogen was prepared as follows. The immunogen used for the first immunization was prepared by emulsifying a complete Freund's Ajuvant (Sigma) 1:1, and the immunogen used for the additional immunization was an incomplete Freund's adjuvant (Incomplete Freund's). Ajuvant: Sigma) was prepared by emulsifying 1:1.
2-2. 면역2-2. immune
상기의 방법으로 제조된 면역원을 BALB/C 마우스, 6주령, 암컷에 다음과 같이 면역하였다. 200 ㎕/마리의 양으로 1주 간격으로 4회 복강 주사하고 일주일 후에 항원을 인산염 완충 용액(Phosphate buffered saline: PBS)에 20% 농도(v/v)로 희석하여 1일 간격으로 3회 꼬리 정맥에 50㎕/회 주사하였다. 3차 면역 후에는 꼬리 정맥에서 얻어낸 혈청으로 중간 역가 검사를 실시하였다.The immunogen prepared by the above method was immunized to BALB/C mice, 6 weeks old, and females as follows. Intraperitoneal injection at 200 µl/dog at intervals of 1 week 4 times, and after a week, dilute antigen at 20% concentration (v/v) in phosphate buffered saline (PBS) and tail vein 3 times at 1 day intervals Was injected into 50 μl/time. After the third immunization, an intermediate titer test was performed with serum obtained from the tail vein.
2-3. 하이브리도마 세포주 제작2-3. Hybridoma cell line production
면역 후에 마우스의 비장 세포를 떼어내어 70μm 세포 strainer(BD falcon사)를 통해 걸러내었다. 걸러진 비장세포를 원심분리하여 침전물로 획득하고 적혈구 용해 버퍼(Red blood cell lysis buffer, Sigma사)를 5ml 섞어준 뒤 4분간 방치하여 적혈구를 제거하였다. 적혈구가 제거된 비장세포는 둘베코이글배지 (Dulbecco's Modified Eagle's Medium: DMEM, Gibco사)를 넣어 3회 세척하고 세포 카운팅을 실시하였다. 비장세포와 융합시킬 세포는 마우스 유래 골수종 세포인 SP2/0-Ag 14(ATCC CRL-1581) 세포주이며, 혼합비율은 5:1(비장세포: SP2/0)가 되게 섞어주었다. 혼합된 세포액은 DMEM으로 2회 세척 후 PEG1500 (Roche사)를 사용하여 융합하였다. 융합은 PEG1500을 1분간 1.7ml 점적하고, 30초간 정치 후 DMEM을 1분간 1ml 점적하고, 30초간 정치 후 DEME을 1분간 2ml 점적하고 30초간 6ml 점적하고, 30초간 정치 후 DMEM을 30초간 10ml 점적하였다. 융합이 완료된 세포액은 원심분리하여 침전물로 획득하고 HAT (PAA사)와 항생제 (Gibco사), 소태아혈청 (Fetal bovine serum, Hyclone 사)이 10% 첨가된 DMEM (이하 - HAT 배지)과 잘 섞어준 뒤 200 ㎕/웰의 용량으로 96 웰 플레이트에 분주한 뒤 3일간 배양기에서 성장시켰다.After immunization, the spleen cells of the mice were removed and filtered through a 70 μm cell strainer (BD Falcon). The filtered splenocytes were centrifuged to obtain a precipitate, mixed with 5 ml of red blood cell lysis buffer (Sigma), and left to stand for 4 minutes to remove red blood cells. The splenocytes from which red blood cells were removed were washed 3 times with Dulbecco's Modified Eagle's Medium (DMEM, Gibco), and cell counting was performed. The cells to be fused with splenocytes were mouse-derived myeloma cells, SP2/0-Ag 14 (ATCC CRL-1581) cell line, and the mixing ratio was 5:1 (splenocytes: SP2/0). The mixed cell solution was washed twice with DMEM and then fused with PEG1500 (Roche). For fusion, 1.7 ml of PEG1500 was added for 1 minute, and 1 ml of DMEM was added for 1 minute after standing for 30 seconds. After standing for 30 seconds, 2 ml of DEME was added for 1 minute, 6 ml for 30 seconds, and 10 ml of DMEM for 30 seconds after standing for 30 seconds. I did. After the fusion is completed, the cell fluid is centrifuged to obtain a precipitate, and mixed well with DMEM (hereinafter-HAT medium) containing 10% HAT (PAA), antibiotics (Gibco), and fetal bovine serum (Hiclone). After giving, it was dispensed into a 96-well plate at a volume of 200 μl/well and grown in an incubator for 3 days.
2-4. 세포 배양2-4. Cell culture
융합이 완료된 세포주는 37℃, 5%의 CO2, 가습 조건이 유지되는 세포배양기에서 성장시켰으며, 2일 간격으로 일주일 간 HAT 배지를 교체하였다. The fusion-completed cell line was grown in a cell incubator maintained at 37°C, 5% CO 2 , and humidified conditions, and the HAT medium was replaced for one week at intervals of 2 days.
2-5. 양성 클론 세포주 검색2-5. Search for positive clonal cell lines
HAT 배지로 선별이 완료된 세포주는 효소연결면역흡광도분석법(Enzyme linked immunosorbent assay, ELISA)으로 양성 클론을 검색하였다. 구체적으로, 세포주가 자라고 있는 각각의 배양액을 요막강액(음성 대조군)과 1:1로 섞어주어 37℃에서 한 시간 동안 반응시켜 비특이적인 항체를 중화시킨 뒤, 96 웰 흡착 플레이트에(Costar 사) SDMA-KLH (Keyhole limpet hemocyanin) 접합체가 2.5μg/ml의 농도로 코팅된 플레이트에 100㎕/웰의 용량으로 분주하고 37℃에서 1 시간 동안 반응시켰다. 1 시간 반응 후 5회 세척하고 항 마우스 IgG-HRP (Horseradish Peroxidase) 접합체와 항 마우스 IgM-HRP 접합체를 100㎕/웰의 용량으로 분주하고 37℃에서 30분간 반응시켰다. 30분 반응 후 5회 세척하고 기질액 (TMB-one)을 100㎕/웰의 용량으로 분주하여 15분간 발색시켰다. 발색이 완료 후 반응정지액을 100㎕/웰의 용량을 분주하고, 450nm 파장에서 흡광도를 측정하여 높은 수치를 나타내는 클론을 선별하였다. 선별된 양성클론은 24-웰 플레이트로 옮긴 뒤 3일간 배양하고 SDMA-KLH, NMMA-KLH, ADMA-KLH, L-Arginine-KLH 접합체가 각각 2.5μg/ml 농도로 코팅된 플레이트를 이용하여 위와 동일한 방법으로 2차 검색하여 최종 SDMA 특이적 반응을 나타내는 양성 클론 후보군을 선별해내었다. 최종 선별된 양성 클론은 Anti-SDMA 8F #14-8로 명명하고, 부다페스트 조약에 따른 국제기탁기관인 한국세포주 연구재단 (Cancer Research Institute at Seoul National University College of Medicine, at 28 Yongon-dong,. Chongno-gu, Seoul, 110-744, Korea)에 수탁번호 KCLRF-BP-00463로 2018년 11월 9일에 기탁되었다. Cell lines that were screened with HAT medium were screened for positive clones by enzyme linked immunosorbent assay (ELISA). Specifically, each culture medium in which the cell line is growing is mixed 1:1 with the allantoic fluid (negative control) and reacted at 37°C for an hour to neutralize non-specific antibodies, and then to a 96-well adsorption plate (Costar). A plate coated with SDMA-KLH (Keyhole limpet hemocyanin) conjugate at a concentration of 2.5 μg/ml was dispensed at a volume of 100 μl/well and reacted at 37° C. for 1 hour. After the reaction for 1 hour, washing was performed 5 times, and the anti-mouse IgG-HRP (Horseradish Peroxidase) conjugate and the anti-mouse IgM-HRP conjugate were dispensed at a dose of 100 μl/well and reacted at 37° C. for 30 minutes. After the reaction for 30 minutes, washing was performed 5 times, and a substrate solution (TMB-one) was dispensed at a volume of 100 μl/well, followed by color development for 15 minutes. After the color development was completed, 100 µl/well of the reaction stop solution was dispensed, and the absorbance was measured at a wavelength of 450 nm to select clones showing high values. The selected positive clones were transferred to a 24-well plate, cultured for 3 days, and the same as above using a plate coated with SDMA-KLH, NMMA-KLH, ADMA-KLH, and L-Arginine-KLH conjugates at a concentration of 2.5 μg/ml, respectively. A secondary search was performed by the method to select a candidate group of positive clones showing a final SDMA-specific reaction. The final selected positive clone was named Anti-SDMA 8F #14-8, and the Korea Cell Line Research Foundation, an international depository institution under the Budapest Treaty (Cancer Research Institute at Seoul National University College of Medicine, at 28 Yongon-dong,. Chongno- gu, Seoul, 110-744, Korea) under the accession number KCLRF-BP-00463 on November 9, 2018.
2-6. 항체의 정제 및 시퀀스 분석2-6. Antibody purification and sequence analysis
최종 선별된 양성클론은 T75(NUNC사) 배양 접시로 옮긴 뒤 3일간 배양하여 1x106 cell/ml 세포 밀도로 0.5ml씩 마우스의 복강에 주사한 뒤, 이로부터 일주일 후 마우스 복수액을 채취하였다. 상기 마우스 복수액을 원심분리(3000rpm, 10분, 4℃)한 후, 이를 결합버퍼(Binding buffer, Thermo scientific 사)와 1:1 비율로 섞어준 뒤 상온에서 1시간 동안 반응시켰다. 이로부터 1시간 후 이를 다시 원심분리(3000rpm, 10분, 4℃)한 후, 1.12μm 구멍크기를 가지는 필터를 사용하여 가라앉지 않은 물질을 재차 걸러내었다. 이후 사익 복수액은 단백질 G 레진 (Pierce사)이 들어있는 컬럼에 넣어주어, 항체와 단백질 G 간의 결합을 유도하였다. 복수액이 컬럼을 모두 통과한 후 인산염완충용액으로 3회 세척한 뒤 용출버퍼(elution buffer, Thermo scientific 사)를 사용하여 용출시켰다. 용출된 항체는 다시 PD-10 컬럼(GE heathcare사)에 적용하고 인산염완충용액으로 용출시켜, 항체를 최종 정제하였다. The final selected positive clone was transferred to a T75 (NUNC) culture dish, cultured for 3 days, and injected into the peritoneal cavity of the mouse at a cell density of 0.5 ml at 1×10 6 cells/ml, and a week later, mouse ascites fluid was collected. After centrifuging the mouse ascites solution (3000rpm, 10 minutes, 4°C), it was mixed with a binding buffer (Thermo Scientific) in a 1:1 ratio and reacted at room temperature for 1 hour. After 1 hour, it was centrifuged again (3000 rpm, 10 minutes, 4° C.), and the unsettled material was again filtered using a filter having a pore size of 1.12 μm. Afterwards, the ascites solution was put into a column containing protein G resin (Pierce) to induce binding between the antibody and protein G. After the ascites solution passed through the column, it was washed three times with a phosphate buffer solution, and then elution was performed using an elution buffer (Thermo Scientific). The eluted antibody was again applied to a PD-10 column (GE heathcare) and eluted with a phosphate buffer solution, and the antibody was finally purified.
또한, 상기 정제된 항체(IgG1, kappa)의 시퀀스 분석 결과, 상기 항체의 중쇄 가변 영역 및 경쇄 가변 영역의 서열 정보는 표 1에 나타낸 바와 같다. 전장 중쇄 및 전장 경쇄는 각각 서열번호 9와 10으로 표시된다.In addition, as a result of sequence analysis of the purified antibodies (IgG1, kappa), sequence information of the heavy chain variable region and light chain variable region of the antibody are as shown in Table 1. Full-length heavy chain and full-length light chain are represented by SEQ ID NOs: 9 and 10, respectively.
실시예 3. 시료 내 유리 SDMA의 검출능 평가 Example 3. Evaluation of detection ability of free SDMA in samples
본 실시예에서는 상기 실시예 2에서 제조된 항체를 사용하여, 항원-항체 복합체 형성에 따른 시료 내 유리(free) SDMA의 검출능을 평가하였다. 이와 함께, 시료의 전처리 과정이 유리 SDMA의 검출능에 미치는 영향을 확인하고자 하였다. In this example, the antibody prepared in Example 2 was used to evaluate the detection ability of free SDMA in a sample according to the formation of an antigen-antibody complex. In addition, it was attempted to confirm the effect of sample pretreatment on the detection ability of free SDMA.
3-1. 실험 과정3-1. Experimental process
정상 개체(Normal canine)의 혈청에 유리 SDMA를 첨가하여, 각각 112 μg/dl, 62 μg/dl, 37 μg/dl, 24 μg/dl, 18 μg/dl, 또는 11 μg /dl 농도의 유리 SDMA를 포함하는 시료를 준비하였다. 이후, 상기 시료에 아래와 같은 실험조건별 방법으로 전처리 조건이 위 실시예 2-6에서 얻은 항체의 SDMA 검출능을 확인하였다.Free SDMA at a concentration of 112 μg/dl, 62 μg/dl, 37 μg/dl, 24 μg/dl, 18 μg/dl, or 11 μg/dl, respectively, by adding free SDMA to the serum of normal canine A sample containing was prepared. Thereafter, the SDMA detection ability of the antibody obtained in Example 2-6 above was confirmed in the pretreatment conditions of the sample by the following experimental conditions.
(a) 전처리 없음(a) No pretreatment
상기 시료에 50ul 검체 희석액 버퍼(0.1M tris ph 8.0) 50ul 를 혼합하고, 이후, 시료에 Europium 과 Conjugation 된 항체 접합체액을 첨가하여, 항원-항체 복합체의 형성을 유도하였다. 이후, 형성된 항원-항체 접합체 복합체를 형광리더기를 사용하여 검출하였으며, 각각의 시료 내 반응 물질의 농도를 측정하였다.50ul of a 50ul sample dilution buffer (0.1M tris ph 8.0) was mixed with the sample, and then, Europium and conjugated antibody conjugates were added to the sample to induce formation of an antigen-antibody complex. Thereafter, the formed antigen-antibody conjugate complex was detected using a fluorescent reader, and the concentration of the reaction substance in each sample was measured.
(b) HCL 처리(b) HCL treatment
상기와 같이 제조된 각 시료 검체 100ul에 5N HCl 25ul 를 혼합하여 단백질 석출을 유도한 뒤 이를 10,000rpm으로 5분 동안 원심분리하여 상층액 50ul를 수득하였다. 상기 수득된 상층액과 버퍼(1M Tris, pH 11) 50ul를 혼합하여, pH 중화를 유도하였다 이후, 상기 전처리된 시료에 Europium 과 Conjugation 된 항체 접합체액을 첨가하여, 항원-항체 복합체의 형성을 유도하였다. 이후, 형성된 항원-항체 접합체 복합체를 형광리더기를 사용하여 검출하였으며, 각각의 시료 내 반응 물질의 농도를 측정하였다.After inducing protein precipitation by mixing 25 ul of 5N HCl in 100 ul of each sample sample prepared as described above, 50 ul of the supernatant was obtained by centrifuging it at 10,000 rpm for 5 minutes. 50ul of the obtained supernatant and buffer (1M Tris, pH 11) were mixed to induce pH neutralization. Then, Europium and conjugated antibody conjugate solution was added to the pretreated sample to induce formation of an antigen-antibody complex. I did. Thereafter, the formed antigen-antibody conjugate complex was detected using a fluorescent reader, and the concentration of the reaction substance in each sample was measured.
(c) TCA 처리(c) TCA treatment
상기와 같이 제조된 각 시료 100ul에 50% TCA (Trichloroacetic acid) 25ul를 혼합하여 pH Shock을 이용한 단백질 석출을 유도한 뒤 이를 10,000rpm으로 5분 동안 원심분리하고, 이로부터 상층액 50ul를 수득하였다. 상기 수득된 상층액과 버퍼(1M Tris ph 10.7) 50ul와 혼합하여, pH 중화를 유도하였다 이후, 상기 전처리된 시료에 Europium과 Conjugation 된 항체 접합체액을 첨가하여, 항원-항체 복합체의 형성을 유도하였다. 이후, 형성된 항원-항체 접합체 복합체를 형광리더기를사용하여 검출 하였으며, 각각의 시료 내 반응 물질의 농도를 측정하였다.100ul of each sample prepared as described above was mixed with 25ul of 50% Trichloroacetic acid (TCA) to induce protein precipitation using pH Shock, followed by centrifugation at 10,000rpm for 5 minutes, and 50ul of a supernatant was obtained therefrom. The obtained supernatant was mixed with 50 ul of buffer (1M Tris ph 10.7) to induce pH neutralization. Then, Europium and conjugated antibody conjugate solution was added to the pretreated sample to induce the formation of an antigen-antibody complex. . Thereafter, the formed antigen-antibody conjugate complex was detected using a fluorescent reader, and the concentration of the reaction substance in each sample was measured.
(d) Ammonium Sulfate 처리(d) Ammonium Sulfate treatment
상기와 같이 제조된 각 시료 100ul에 70% Ammonium sulfate 25ul를 혼합하여 단백질 석출을 유도한 뒤 이를 10,000rpm으로 5분 동안 원심분리하고, 수득된 상층액 50ul와 버퍼(0.1M Tris, pH 8) 50ul와 혼합한 후 Europium 과 Conjugation 된 항체 접합체액을 첨가하여, 항원-항체 복합체의 형성을 유도하였다. 이후, 형성된 항원-항체 접합체 복합체를 형광리더기를 사용하여 검출하였으며, 각각의 시료 내 반응 물질의 농도를 측정하였다.100ul of each sample prepared as described above was mixed with 25ul of 70% ammonium sulfate to induce protein precipitation, and then centrifuged for 5 minutes at 10,000rpm, and 50ul of the obtained supernatant and 50ul of buffer (0.1M Tris, pH 8) After mixing with Europium, the conjugated antibody conjugated with Europium was added to induce the formation of an antigen-antibody complex. Thereafter, the formed antigen-antibody conjugate complex was detected using a fluorescent reader, and the concentration of the reaction substance in each sample was measured.
3-2. 실험 결과3-2. Experiment result
위 실시예 3-1에서 얻어진 결과를 하기 표 2에 나타내었다. The results obtained in Example 3-1 above are shown in Table 2 below.
전처리HCl
Pretreatment
전처리TCA
Pretreatment
Sulfate 전처리Amonium
Sulfate pretreatment
상기 표 2에 나타낸 바와 같이, 일 실시예에 따른 항체는 시료를 전처리한 후, 시료 내 유리 SDMA를 검출한 경우, 상기 항체의 검출능이 더욱 향상됨을 확인하였다. As shown in Table 2, when the antibody according to an embodiment detects free SDMA in the sample after pretreating the sample, it was confirmed that the detection ability of the antibody was further improved.
실시예 4. SDMA 항체 특성(Characterization of Anti-SDMA Antibodies) - 교차 반응성 평가Example 4. Characterization of Anti-SDMA Antibodies-Evaluation of Cross Reactivity
본 실시예에서는 상기 실시예 2-6에서 제조된 항체의 시료 내 유리(free) SDMA에 대한 특이적인 반응을 확인하고자 하였고, 이와 함께, 모노메틸아르기닌(NMMA), 비대칭적 디메틸아르기닌(ADMA), 및 L-아르기닌(L-Arginine)에 대한 상기 항체의 교차 반응성을 평가하였다.In this example, it was attempted to confirm the specific reaction of the antibody prepared in Example 2-6 to free SDMA in the sample, and with this, monomethylarginine (NMMA), asymmetric dimethylarginine (ADMA), And L-arginine (L-Arginine) to evaluate the cross-reactivity of the antibody.
시료내 SDMA 농도가 (예를 들어 4ug/dL인 경우)인 형광신호 값을 1,000 FIS(Fluorescence intensity signal)를 reactivity 100%로 정하고, SDMA를 이 시료에 첨가하여 형광신호값이 50%로 줄어드는 농도 (즉, 500 FSI로 되는 농도)를 half maximal inhibitory concentration (IC50) SDMA라고 정의한다. 그 결과, SDMA IC50에 해당하는 농도 값을 50.7ug/dl로 확인하였다.The concentration at which the fluorescence signal value with the SDMA concentration in the sample (for example, 4 ug/dL) is set to 1,000 FIS (Fluorescence intensity signal) as 100% reactivity, and the fluorescence signal value is reduced to 50% by adding SDMA to this sample. (I.e., 500 FSI) is defined as the half maximal inhibitory concentration (IC50) SDMA. As a result, the concentration value corresponding to the SDMA IC50 was confirmed to be 50.7ug/dl.
한편, 각 NMMA, ADMA 및 L-Arginine을 첨가하였을 때 500FIS로 줄어드는 각 물질의 농도를 IC50 (Cross reacting compound)로 정의한 후, % Cross reactivity는 아래의 수식을 적용하여 구하였다: Meanwhile, the concentration of each substance reduced to 500 FIS when each NMMA, ADMA and L-Arginine was added was defined as IC 50 (Cross reacting compound), and the% Cross reactivity was calculated by applying the following formula:
% 교차반응성= IC50(SDMA)/IC50(Cross reacting compound).% Cross-reactive = IC 50 (SDMA)/IC 50 (Cross reacting compound).
4-1. 실험 방법 4-1. Experimental method
(a) IC(a) IC 5050 (SDMA) (SDMA)
1X PBS에 SDMA 0 / 25 / 50 / 100 / 250 / 500 / 1,000 / 2,500 / 5,000 ug/dl 농도가 되도록 제조하였다. 상기와 같이 제조된 각 시료 100ul에 50% TCA (Trichloroacetic acid) 25ul를 혼합하여 이를 10000rpm으로 5분 동안 원심분리하고, 이로부터 상층액 50ul를 수득하였다. 상기 수득된 상층액을 버퍼(1M Tris ph 10.7) 와 혼합하여, pH 중화를 유도하였다. 이후, 상기 전처리된 시료에 Europium 과 Conjugation 된 항체 접합체 7ul를 첨가하여, SDMA-항체 복합체의 형성을 유도하였다. 이후, 형성된 SDMA-항체 복합체를 면역형광법을 사용하여 검출하였으며, 각각의 시료 내 SDMA의 농도를 측정하였다.It was prepared in 1X PBS to a concentration of
결과를 아래 표 3과 얻어진 IC50 (SDMA)를 도 1에 나타내었다:The results are shown in Table 3 below and the obtained IC 50 (SDMA) in FIG. 1:
(b) IC(b) IC 5050 (Cross reacting compound)(Cross reacting compound)
각각의 ADMA, NMMA, L-Arginine을 100 / 250 / 500 / 1,000 / 2,500 / 5,000 / 10,000 / 25,000 / 50,000 ug/dl로 1X PBS에 넣어 제조하였다.Each of ADMA, NMMA, and L-Arginine was prepared by putting it in 1X PBS at 100/250/500 / 1,000 / 2,500 / 5,000 / 10,000 / 25,000 / 50,000 ug/dl.
상기와 같이 제조된 각 시료 100ul에 50%TCA(Trichloroacetic acid) 25ul를 혼합하여 이를 10,000rpm으로 5분 동안 원심분리하고, 이로부터 상층액 50ul를 수득하였다. 상기 수득된 상층액을 버퍼(1M Tris ph 10.7)와 혼합하여, pH 중화를 유도하였다 이후, 상기 전처리된 시료에 Europium과 Conjugation된 항체 접합체 7ul를 첨가하여, 항원-항체 복합체의 형성을 유도하였다. 이후, 형성된 항원-항체 복합체를 면역형광법을 사용하여 검출 하였으며, 각각의 시료 내 반응 물질의 농도를 측정하였다. 그 결과를 아래 표 4에 나타내었다.100ul of each sample prepared as described above was mixed with 25ul of 50% Trichloroacetic acid (TCA) and centrifuged for 5 minutes at 10,000rpm, and 50ul of a supernatant was obtained therefrom. The obtained supernatant was mixed with a buffer (1M Tris ph 10.7) to induce pH neutralization. After that, 7ul of the antibody conjugate conjugated with Europium was added to the pretreated sample to induce the formation of an antigen-antibody complex. Thereafter, the formed antigen-antibody complex was detected using immunofluorescence, and the concentration of the reaction substance in each sample was measured. The results are shown in Table 4 below.
위 결과로부터 IC50(NMMA) 6,324ug/dl, IC50(ADMA) 50,000ug/dl, IC50(L-Arginine) > 50,000ug/dl를 얻을 수 있었다.From the above results, IC 50 (NMMA) 6,324 ug/dl, IC 50 (ADMA) 50,000 ug/dl, and IC 50 (L-Arginine)> 50,000 ug/dl were obtained.
위에서 얻은 IC50 (SDMA), IC50 (NMMA), IC50(ADMA), 및 IC50 (L-Arginine)을 아래 식에 대응하여 교차반응성 (Cross reactivity)을 구하고, 그 결과를 아래 표 5에 나타내었다: IC 50 (SDMA), IC 50 (NMMA), IC 50 (ADMA), and IC 50 (L-Arginine) obtained above were calculated for cross reactivity according to the equation below, and the results are shown in Table 5 below. Indicated:
% 교차반응성 = (IC50 (SDMA) / IC50 (Cross reacting compound)% Cross-reactive = (IC 50 (SDMA) / IC 50 (Cross reacting compound)
(ug/dl)IC50
(ug/dl)
위 결과에서 확인되는 바와 같이, 본 발명의 한 구현예에 따른 항-SDMA 항체는 SDMA에 대해 우수한 선택성과 민감성을 나타내고, 구조적 유사물질인 NMMA, ADMA 및 L-Arginine에 대해서는 1% 미만의 교차 반응성을 나타내었다.As can be seen from the above results, the anti-SDMA antibody according to an embodiment of the present invention exhibits excellent selectivity and sensitivity to SDMA, and less than 1% cross-reactivity to structural analogs such as NMMA, ADMA and L-Arginine. Shown.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects.
<110> BIONOTE, INC. <120> An antibody specifically binding symmetric dimethylarginine and uses thereof <130> PN124844 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 1 Gly Phe Ala Phe Ser Ser Tyr Asp Met Ser 1 5 10 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 2 Tyr Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val Lys 1 5 10 15 Gly <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 3 Leu Gly Tyr Tyr Trp Tyr Phe Asp Val 1 5 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 4 Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr Trp 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 5 Asp Thr Ser Asn Leu Ala Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 6 Gln Gln Trp Ser Ser Tyr Pro Leu Thr 1 5 <210> 7 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> heavy chain variable region <400> 7 Glu Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Tyr Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 8 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region <400> 8 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 9 <211> 442 <212> PRT <213> Artificial Sequence <220> <223> Full length heavy chain <400> 9 Glu Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Tyr Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Pro 180 185 190 Arg Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro 210 215 220 Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys 245 250 255 Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp 260 265 270 Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser 305 310 315 320 Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly 325 330 335 Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln 340 345 350 Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe 355 360 365 Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu 370 375 380 Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn Thr Asn Gly Ser Tyr Phe 385 390 395 400 Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn 405 410 415 Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr 420 425 430 Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 10 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Full length light chain <400> 10 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala Pro 100 105 110 Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly 115 120 125 Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn 130 135 140 Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn 145 150 155 160 Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser 165 170 175 Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr 180 185 190 Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe 195 200 205 Asn Arg Asn Glu Cys 210 <110> BIONOTE, INC. <120> An antibody specifically binding symmetric dimethylarginine and uses thereof <130> PN124844 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 1 Gly Phe Ala Phe Ser Ser Tyr Asp Met Ser 1 5 10 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 2 Tyr Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val Lys 1 5 10 15 Gly <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 3 Leu Gly Tyr Tyr Trp Tyr Phe Asp Val 1 5 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 4 Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr Trp 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 5 Asp Thr Ser Asn Leu Ala Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 6 Gln Gln Trp Ser Ser Tyr Pro Leu Thr 1 5 <210> 7 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> heavy chain variable region <400> 7 Glu Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Tyr Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 8 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region <400> 8 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 9 <211> 442 <212> PRT <213> Artificial Sequence <220> <223> Full length heavy chain <400> 9 Glu Val Lys Leu Gln Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Tyr Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Ser Val Thr Val Pro Ser Ser Pro 180 185 190 Arg Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro 210 215 220 Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys 245 250 255 Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp 260 265 270 Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser 305 310 315 320 Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly 325 330 335 Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln 340 345 350 Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe 355 360 365 Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu 370 375 380 Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn Thr Asn Gly Ser Tyr Phe 385 390 395 400 Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn 405 410 415 Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr 420 425 430 Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 <210> 10 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Full length light chain <400> 10 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala Pro 100 105 110 Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly 115 120 125 Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn 130 135 140 Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn 145 150 155 160 Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser 165 170 175 Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr 180 185 190 Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe 195 200 205 Asn Arg Asn Glu Cys 210
Claims (21)
서열번호 4의 아미노산 서열로 이루어지는 LCDR1, 서열번호 5의 아미노산 서열로 이루어지는 LCDR2, 및 서열번호 6의 아미노산 서열로 이루어지는 LCDR3을 포함하는 경쇄 가변 영역을 포함하고, 대칭적 디메틸아르기닌 (Symmetric dimethylarginine: SDMA)과 특이적으로 결합하는 항체 또는 이의 항원-결합 단편.A heavy chain variable region comprising HCDR1 consisting of the amino acid sequence of SEQ ID NO: 1, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 3; And
A light chain variable region including LCDR1 consisting of the amino acid sequence of SEQ ID NO: 4, LCDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and LCDR3 consisting of the amino acid sequence of SEQ ID NO: 6, and symmetric dimethylarginine (SDMA) An antibody or antigen-binding fragment thereof that specifically binds to.
서열번호 8의 아미노산 서열로 이루어지는 경쇄 가변 영역을 포함하는, 항체 또는 이의 항원-결합 단편.The method according to claim 1, wherein the antibody comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7; And
An antibody or antigen-binding fragment thereof, comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.
서열번호 8의 아미노산 서열에 대해 95% 또는 그 이상의 서열 일치성을 갖는 아미노산 서열로 이루어지는 경쇄 가변 영역을 포함하는, 항체 또는 이의 항원-결합 단편.The method according to claim 2, wherein the antibody comprises a heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity to the amino acid sequence of SEQ ID NO: 7; And
An antibody or antigen-binding fragment thereof comprising a light chain variable region consisting of an amino acid sequence having 95% or more sequence identity to the amino acid sequence of SEQ ID NO: 8.
서열번호 1의 아미노산 서열로 이루어지는 HCDR1, 서열번호 2의 아미노산 서열로 이루어지는 HCDR2, 및 서열번호 3의 아미노산 서열로 이루어지는 HCDR3을 포함하는 중쇄 가변 영역을 포함하고,
하기 화학식 2의 대칭적 디메틸아르기닌의 유도체와 담체 단백질(carrier protein)이 컨쥬게이션된 면역용 항원을 사용하여 제조된 것인, 항체 또는 이의 항원-결합 단편:
[화학식 2]
상기 화학식 2에서,
상기 R1은 -XR2 또는 -R2이고;
상기 X는 O 또는 S이고, 상기 R2는 -(CH2)n-R3이며; 및
상기 n은 1 내지 5의 정수이고, R3는 히드록시(OH), 티올(SH), 또는 카르복시이다. Specifically binds to free symmetrical dimethylarginine (free SDMA),
A heavy chain variable region including HCDR1 consisting of the amino acid sequence of SEQ ID NO: 1, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 3,
An antibody or antigen-binding fragment thereof, prepared using an antigen for immunization in which a derivative of symmetric dimethylarginine of the following formula (2) and a carrier protein are conjugated:
[Formula 2]
In Chemical Formula 2,
R 1 is -XR 2 or -R 2 ;
X is O or S, and R 2 is -(CH 2 ) n -R 3 ; And
Wherein n is an integer of 1 to 5, R 3 is hydroxy (OH), thiol (SH), or carboxy.
[화학식 3]
. The method of claim 8, wherein the symmetric derivative of Formula 2 is represented by the following Formula (3), an antibody or antigen-binding fragment thereof:
[Formula 3]
.
[화학식 4]
상기 화학식 4에서, 상기 R1 '은 -X-(CH2)n-R3' 또는 -(CH2)n-R3'이고; 상기 X는 O 또는 S이고, 상기 n은 1 내지 5의 정수이고, R3 '는 -O-, -S-, 또는 -C(O)O-이고, x 는 1-34 의 정수, 그리고 Protein은 담체 단백질이다.A method for producing the antibody or antigen-binding antibody of claim 1, comprising the step of inducing an immune response in a host organism by using a conjugate of a symmetric dimethylarginine derivative and a carrier protein represented by the following Formula 4 as an immunogen. :
[Formula 4]
In Formula 4, R 1 ′ is -X-(CH 2 ) n -R 3 ′ Or -(CH 2 ) n -R 3 '; Wherein X is O or S, n is an integer of 1 to 5, R 3 ′ is -O-, -S-, or -C(O)O-, x is an integer of 1-34, and Protein Is a carrier protein.
상기 상층액에 청구항 1항의 항체 또는 이의 항원-결합 단편을 접촉시켜 유리 SDMA-항체 혹은 항원-결합 단편 복합체를 형성하는 단계; 및
상기유리 SDMA-항체 또는 항원-결합 단편 복합체를 검출하는 단계를 포함하는 것인, 시료내 유리 SDMA를 검출하는 방법.Precipitating the protein in the sample by adding trichloroacetic acid (TCA) to a sample containing free symmetrical dimethylarginine (SDMA) and protein, and obtaining a supernatant thereof;
Forming a free SDMA-antibody or antigen-binding fragment complex by contacting the supernatant with the antibody of claim 1 or an antigen-binding fragment thereof; And
A method for detecting free SDMA in a sample comprising the step of detecting the free SDMA-antibody or antigen-binding fragment complex.
상기 상층액에 청구항 2항의 항체 또는 이의 항원-결합 단편을 접촉시켜 유리 SDMA-항체 혹은 항원-결합 단편 복합체를 형성하는 단계; 및
상기유리 SDMA-항체 또는 항원-결합 단편 복합체를 검출하는 단계를 포함하는 것인, 시료 내 유리 SDMA를 검출하는 방법.Precipitating the protein in the sample by adding trichloroacetic acid (TCA) to a sample containing free symmetrical dimethylarginine (SDMA) and protein, and obtaining a supernatant thereof;
Forming a free SDMA-antibody or antigen-binding fragment complex by contacting the supernatant with the antibody or antigen-binding fragment thereof of claim 2; And
A method for detecting free SDMA in a sample comprising the step of detecting the free SDMA-antibody or antigen-binding fragment complex.
상기 상층액에 청구항 8항의 항체 또는 이의 항원-결합 단편을 접촉시켜 유리 SDMA-항체 혹은 항원-결합 단편 복합체를 형성하는 단계; 및
상기유리 SDMA-항체 또는 항원-결합 단편 복합체를 검출하는 단계를 포함하는 것인, 시료 내 유리 SDMA를 검출하는 방법.Precipitating the protein in the sample by adding trichloroacetic acid (TCA) to a sample containing free symmetrical dimethylarginine (SDMA) and protein, and obtaining a supernatant thereof;
Forming a free SDMA-antibody or antigen-binding fragment complex by contacting the supernatant with the antibody of claim 8 or an antigen-binding fragment thereof; And
A method for detecting free SDMA in a sample comprising the step of detecting the free SDMA-antibody or antigen-binding fragment complex.
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EP3948280A4 (en) * | 2019-04-03 | 2022-12-28 | ARK Diagnostics, Inc. | Antibodies to symmetrically dimethylated arginine analytes and use thereof |
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