KR20120045822A - Methods for analyzing quantitatively and qualitatively active forms of enzymes - Google Patents
Methods for analyzing quantitatively and qualitatively active forms of enzymes Download PDFInfo
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- KR20120045822A KR20120045822A KR1020100107640A KR20100107640A KR20120045822A KR 20120045822 A KR20120045822 A KR 20120045822A KR 1020100107640 A KR1020100107640 A KR 1020100107640A KR 20100107640 A KR20100107640 A KR 20100107640A KR 20120045822 A KR20120045822 A KR 20120045822A
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Abstract
본 발명은 효소의 활성형의 정성 및 정량적 분석방법에 관한 것이다. 본 발명은 효소 활성형 억제제 및 결합제를 이용하여 시료내 효소 활성형을 분석함으로써 단순 결합제(예컨대, 항체)를 이용하여 효소 활성형을 분석하는 방법보다 빠르고 정확하게 분석할 수 있는 장점을 가진다. 본 발명은 단순 결합제를 이용하여 효소 활성형을 분석하는 방법보다 작은 장비를 이용하여 복잡하지 않고 간단하게 시료내에 존재하는 효소 활성형을 분석할 수 있다. 본 발명은 타겟 효소 또는 효소의 활성형에 억제제 및 결합제를 동시에 이용함으로써 신속하고 특이적으로 분석할 수 있으며, 이러한 본 발명의 특성을 이용하여 약품(drug)을 고속으로 그리고 대량(high-throughput)으로 스크리닝을 할 수 있다. 또한, 본 발명은 약품 투여 대상체(예컨대, 동물 또는 인간)에서의 약품의 효능 및 투여량에 대한 임상 모니터링을 위한 POCT(Point of care testing)에 적용될 수 있다.The present invention relates to methods for the qualitative and quantitative analysis of active forms of enzymes. The present invention has the advantage that it can be analyzed faster and more accurately than the method of analyzing the enzyme active form using a simple binder (eg, antibody) by analyzing the enzyme active form in the sample using the enzyme active inhibitor and the binder. The present invention can analyze the enzyme active form present in a sample without being complicated by using a smaller device than a method for analyzing the enzyme active form using a simple binder. The present invention can be analyzed quickly and specifically by simultaneously using an inhibitor and a binder in a target enzyme or an active form of an enzyme, and utilizing the characteristics of the present invention, a drug is rapidly and high-throughput. Screening can be done. The invention may also be applied to point of care testing (POCT) for clinical monitoring of the efficacy and dosage of a drug in a drug administration subject (eg, animal or human).
Description
본 발명은 효소의 활성형의 정성 및 정량적 분석방법에 관한 것이다.
The present invention relates to methods for the qualitative and quantitative analysis of active forms of enzymes.
단백질들은 여러 가지 기능을 하기 위하여 한 가지 혹은 그 이상의 다양한 조절 경로를 이용하고 있다. 이러한 여러 가지 조절 경로에는 단백질의 특정 위치에서의 절단, 결실, 추가, 사이드 체인의 콘버트나 모디피케이션 등 다양한 방법들이 있다.Proteins use one or more different regulatory pathways to function. These different regulatory pathways include a variety of methods, such as cleavage, deletion, addition, side chain conversion, or modulation of proteins at specific locations.
단백질, 특히 효소는 다양한 메커니즘을 통하여 효소의 활성이 조절된다. 첫째, 최종 산물(product)에 의하여 효소의 활성이 조절되는 피드백 억제 조절, 둘째, 헤모글로빈에서의 O2, H+ 및 CO2의 상호 작용 또는 대장균(E. coli)에서의 ATCase(aspartate transcarbamoylase)의 활성 조절 기작과 같은 알로스테릭 조절(allosteric control), 셋째, 칼모둘린(calmodulin)이 세포 내의 Ca+2을 감지하여 다른 효소들을 활성화시키는 조절, 안티헤모필릭 인자(antihemophilic factor)가 세린 프로테아제의 활성을 증가시키 혈액 응고의 속도를 증가시키는 조절 또는 사이클릭 AMP이 프로틴 키나아제 A의 억제 서브유닛을 제거시킴으로서 키나아제의 활성시키는 조절과 같은 조절 단백질에 의한 자극 및 억제, 넷째, 단백질 키나아제가 ATP에서 포스포릴 그룹을 떼어 효소에 붙임으로써 효소를 활성화시키는 조절기작 및 단백질 포스파타아제가 활성화된 효소로부터 포스포릴기를 가수분해하여 불활성화시키는 가역적 코발런트 모디피케이션, 그리고 다섯째, 모젠(zymogens) 또는 프로효소(proenzymes)의 펩티드 결합을 가수 분해를 통한 비가역적인 효소 활성 조절 메커니즘이 있다.Proteins, especially enzymes, regulate enzyme activity through a variety of mechanisms. Firstly, feedback inhibition regulation in which enzyme activity is regulated by end product, second, interaction of O 2 , H + and CO 2 in hemoglobin or aspartate transcarbamoylase in E. coli Allosteric control, such as activity regulation mechanisms, and third, calmodulin senses Ca +2 in cells to activate other enzymes, and antihemophilic factor serine protease Stimulation and inhibition by regulatory proteins, such as regulation of kinase by activating regulation or cyclic AMP by removing the inhibitory subunit of protein kinase A, which increases the rate of blood coagulation and fourth, protein kinases in ATP Regulatory mechanisms that activate enzymes by detaching phosphoryl groups and attaching them to enzymes and protein phosphatase-activated enzymes By hydrolyzing an poril have deactivate reversible cobalt parent parent difficile indications, and fifth, mojen (zymogens), or enzyme pro (proenzymes) irreversible enzyme activity regulatory mechanism a peptide bond by the hydrolysis of the.
특히, 효소적 활성을 위한 메커니즘 중에서 단백질 분해(proteolytic cleavage)에 의한 프로효소(proenzymes)으로부터 효소 활성형으로의 전환은 지모젠(zymogens) 또는 프로효소에서의 펩티드 결합을 가수 분해를 통하여 지모젠 또는 프로효소를 효소 활성형으로 전환시키는 비가역적인 효소 활성 조절 메커니즘이다. 예컨대, 단백질 분해에 의한 효소의 활성형은 리포프로틴-관련 포스포리파아제 A2(Lipoprotein-associated phospholipase: Lp-PLA2), 트롬빈, 유로키나아제, 트립신, 키모트립신, 엘라타아제 및 서브틸리신등과 같은 효모 활성형이 알려져 있다.In particular, in the mechanism for enzymatic activity, the conversion from proenzymes to enzymatically active form by proteolytic cleavage is carried out by hydrolysis of peptide bonds in zymogens or proenzymes through hydrolysis. It is an irreversible enzyme activity control mechanism that converts a proenzyme into an enzyme active form. For example, the active forms of enzymes by proteolysis are lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ), thrombin, urokinase, trypsin, chymotrypsin, elatases and subtilisin, etc. Yeast active forms such as are known.
효소가 활성형으로 전환된 후 기질과의 결합하는 경우 효소 활성형의 구조적인 형태 변화에 있어서 다음과 같은 2가지의 이론이 있다: 첫째, 락 앤 키(lock and key) 이론은 효소 활성형 억제제가 효소 활성형과 결합하는 경우 효소 활성형의 형태 변화에 영향을 주지 않는다는 이론이며, 인듀스드 핏(induced fit) 이론은 억제제가 효소 활성형과 결합하는 경우 효소 활성형의 형태가 변화된다는 이론이다.There are two theories about the structural change of the enzyme active form when it is bound to the substrate after the enzyme is converted to the active form: First, the lock and key theory is an enzyme active inhibitor. Is a theory that does not affect the morphological change of the enzyme active form when it is bound to the enzyme active form, and the induced fit theory is that the form of the enzyme active form changes when the inhibitor is bound to the enzyme active form. to be.
Lp-PLA2는 인간의 경우 PLA2G7 유전자에 의하여 암호화된 프로포스포리파아제 A2 가 단백질 분해에 의하여 활성화된 효소이며, Lp-PLA2는 441개의 아미노산으로 이루어진 45-kDa 단백질이다. 또한, 프로포스포리파아제 A2의 인간 mRNA 전장서열 및 단백질 서열은 접근번호 NM_005084 및 NP_005075에 각각 공지되어 있다. 또한, Lp-PLA2는 PAF(platelet-activating factor) 아세틸하이드로라아제이며, 아세틸 그룹의 가수분해를 통하여 PAF를 생물학적으로 불활성화 생산물인 LYSO-PAF 및 아세테이트로의 분해 과정을 촉매한다고 알려져 있다.Lp-PLA 2 is an enzyme in which prophospholipase A2 encoded by the PLA2G7 gene is activated by proteolysis, and Lp-PLA 2 is a 45-kDa protein composed of 441 amino acids. In addition, human mRNA full-length sequences and protein sequences of prophospholipase A 2 are known, respectively, under access numbers NM_005084 and NP_005075. In addition, Lp-PLA 2 is a platelet-activating factor (PAF) acetylhydrolase and is known to catalyze the decomposition of PAF into biologically inactive products LYSO-PAF and acetate through hydrolysis of acetyl groups.
Lp-PLA2는 혈액에서 저밀도 리포프로틴(low-density lipoprotein: LDL)과 함께 주로 이동하며, 약 20%는 고밀도 리포트로틴과 관련되어 있다고 알여져 있다. Lp-PLA2는 염증세포에 의하여 생산되며, LDL에서 산화된 포스포리피드(phospholipid)를 가수분해시킨다. 보다 구체적으로, Lp-PLA2는 LDL에 있는 리포리피드를 lysoPC(lysophosphatidylcholine)과 oxNEFA(xidized nonesterified fatty acids)으로 가수분해시키고, 산화된 LDL이 동맥내피를 투과한 후 마크로파지에 의하여 흡수되어 폼세포(foam cell)을 형성된다. 형성된 폰세포는 림프구에 의하여 용혈되어 잔여물이 점차 증가되어 혈관을 좁게 만들고 결국 동맥혈관 안으로 유출되어 혈관을 막거나 염증을 유발시킨다.Lp-PLA 2 migrates primarily with low-density lipoprotein (LDL) in the blood, and about 20% is known to be related to high-density reportrotin. Lp-PLA 2 is produced by inflammatory cells and hydrolyzes oxidized phospholipids in LDL. More specifically, Lp-PLA 2 hydrolyzes lipolipids in LDL into lysoPC (lysophosphatidylcholine) and oxNEFA (xidized nonesterified fatty acids), and the oxidized LDL penetrates the arterial endothelium and is absorbed by macrophages to form foam cells ( foam cells). The formed ponocytes are hemolyzed by lymphocytes and the residue gradually increases to narrow the blood vessels and eventually flows into the arterial vessels to block or cause inflammation.
종래 개발된 Lp-PLA2 활성 분석은 발과 또는 방사활성 Lp-PLA2 기질 및 카운터를 필요로 한다. 보다 구체적으로, 발광 또는 형광 기질인 Lp-PLA2-기질을 이용하여 Lp-PLA2 활성을 분석 경우 2단계의 세척과정을 필요하며 기질이 Lp-PLA2에 특이적이지 않기 때문에 특이성을 나타내기 위하여 특정 농도의 Lp-PLA2가 필요하다. 또한, 방사활성 Lp-PLA2 기질을 이용하여 Lp-PLA2 활성을 분석 경우 비절단 또는 절단된 방사활성 Lp-PLA2 기질의 침전을 위한 BSA(ovine serum albumin)을 첨가한 후 원심분리하고, 기질의 침전을 위하여 금속이온으로 케미컬 및 버퍼를 최적화하는 단계를 필요로 할 뿐 만 아니라, 활성 측정을 위하여 신틸레이션(scintillation) 카운터를 필요로 하며 POCT(Point of Care Testing)하기에 복잡하다는 문제점이 있다.Previously developed Lp-PLA 2 activity assays require ectopic or radioactive Lp-PLA 2 substrates and counters. More specifically, when analyzing Lp-PLA 2 activity using the luminescent or fluorescent substrate Lp-PLA 2 -substrate, two steps of washing process are required and specificity is shown because the substrate is not specific to Lp-PLA 2 . A specific concentration of Lp-PLA 2 is required. Further, after adding the radioactivity Lp-PLA 2 substrate BSA (ovine serum albumin) for the precipitation of the non-cutting or cutting radioactivity Lp-PLA 2 substrate, if analysis of the Lp-PLA 2 activity by using a centrifugation, In addition to the step of optimizing chemicals and buffers with metal ions for precipitation of substrates, scintillation counters are required for activity measurements and are complex to point of care testing (POCT). .
현재, 효소 활성형의 정량적 또는 정성적으로 분석하는 방법은 일반적으로 항원-항체 반응과 같은 면역학적 방법이 널리 이용되고 있다.At present, methods for quantitatively or qualitatively analyzing the enzyme active form are generally widely used immunological methods such as antigen-antibody reactions.
그러나, 이러한 면역학적 방법은 대부분 시그널 표지가 결합된 항체를 타깃 효소 또는 효소의 활성혈과 직접인 결합만으로 검출하는 방법이며, 검출 결과를 분석하는데 복잡한 단계를 이용함으로써 상당한 시간이 소요되고, 결과의 정확도면에서도 신뢰도가 매우 떨어진다는 문제점이 있다.However, most of these immunological methods detect only a signal-labeled antibody by binding directly to the target enzyme or active blood of the enzyme, and it takes considerable time by using complicated steps to analyze the detection result. In terms of accuracy, there is a problem that the reliability is very low.
따라서, 활성을 가지는 효소의 활성형을 정성적 및 정량적으로 검출 또는 분석할 수 있는 신규한 방법의 필요성이 대두되고 있다.
Therefore, there is a need for a new method capable of detecting or analyzing the active form of an enzyme having activity qualitatively and quantitatively.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.
본 발명자들은 활성을 가지는 효소의 활성형을 간단하면서 빠르게 정성적 또는 정량적으로 검출할 수 있는 방법을 발굴하고자 예의 연구 노력하였다. 그 결과, 효소의 활성형에 특이적으로 결합하는 효소의 활성형 억제제 및 결합제를 이용하여 시료내에 효소의 활성형의 존재 유무 또는 효소의 활성형이 얼만큼 존재하는지를 빠르고 정확하게 확인할 수 있음을 규명함으로써, 본 발명을 완성하게 되었다.The present inventors have made diligent research efforts to find a method for simple and rapid qualitative or quantitative detection of an active form of an enzyme having activity. As a result, by using the active type inhibitor and the binding agent of the enzyme that specifically binds to the active form of the enzyme, it is found that it is possible to quickly and accurately confirm whether the active form of the enzyme or the active form of the enzyme exists in the sample. The present invention has been completed.
따라서, 본 발명의 목적은 효소의 활성형의 정성적 및 정량적인 분석방법을 제공하는 데 있다.
Accordingly, it is an object of the present invention to provide a method for qualitative and quantitative analysis of an active form of an enzyme.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 다음 단계를 포함하는 효소의 활성형의 정성적 또는 정량적 분석방법을 제공한다:According to one aspect of the present invention, the present invention provides a method for qualitative or quantitative analysis of an active form of an enzyme comprising the following steps:
(a) 포획제(capturing agent)로서 상기 효소 활성성의 억제제에 분석하고자 하는 시료 내의 상기 효소 활성형을 접촉(contacting)시키는 단계;(a) contacting the enzyme active form in the sample to be analyzed with the inhibitor of enzyme activity as a capturing agent;
(b) 검출 가능한 시그널을 발생시키는 표지가 결합된 검출자(detector)를 상기 단계 (a)의 결과물에 접촉(contacting)시키는 단계로서, 상기 검출자는 상기 효소 또는 효소 활성형, 그리고 이들의 모두에 결합하는 결합제이며; 및(b) contacting a detector with a label that generates a detectable signal to the product of step (a), wherein the detector is capable of contacting the enzyme or enzyme active form, and all of Binding agent; And
(c) 상기 효소 또는 효소 활성형에 결합된 검출자의 표지로부터 발생되는 시그널을 측정하는 단계.
(c) measuring the signal generated from the label of the detector bound to the enzyme or enzyme active form.
본 발명의 다른 양태에 따르면, 본 발명은 다음 단계를 포함하는 효소의 활성형의 정성적 또는 정량적 분석방법을 제공한다:According to another aspect of the invention, the invention provides a method for qualitative or quantitative analysis of an active form of an enzyme comprising the following steps:
(a) 포획제로서 상기 효소 또는 효소 활성형, 그리고 이들 모두의 결합제에 분석하고자 하는 시료 내의 상기 효소 활성형을 접촉시키는 단계;(a) contacting the enzyme or enzyme active form, and both binders, as a capture agent with the enzyme active form in the sample to be analyzed;
(b) 검출 가능한 시그널을 발생시키는 표지가 결합된 검출자를 상기 단계 (a)의 결과물에 접촉시키는 단계로서, 상기 검출자는 상기 효소 활성형에 결합하는 억제제이며; 및(b) contacting a detector with a label that generates a detectable signal to the product of step (a), wherein the detector is an inhibitor that binds to the enzyme active form; And
(c) 상기 효소 활성형에 결합된 검출자의 표지로부터 발생되는 시그널을 측정하는 단계.
(c) measuring the signal generated from the label of the detector bound to the enzyme active form.
본 발명자들은 활성을 가지는 효소의 활성형을 간단하면서 빠르게 정성적 또는 정량적으로 검출할 수 있는 방법을 발굴하고자 예의 연구 노력하였으며, 그 결과, 효소의 활성형에 특이적으로 결합하는 효소의 활성형 억제제 및 결합제를 이용하여 시료내에 효소의 활성형의 존재 유무 또는 효소의 활성형이 얼만큼 존재하는지를 빠르고 정확하게 확인할 수 있음을 규명하였다.The present inventors have made diligent research to find a method that can detect the active form of an active enzyme simply and quickly and qualitatively or quantitatively. As a result, the active type inhibitor of the enzyme that specifically binds to the active form of the enzyme And using a binder, it was found that the presence or absence of an active form of the enzyme in the sample or how quickly the active form of the enzyme can be confirmed quickly and accurately.
본 발명은 효소의 활성형을 매우 특이적으로 정성 또는 정량적으로 분석할 수 있는 방법이며, 바람직하게는 본 발명은 효소의 활성형을 매우 특이적으로 정성 및 정량적으로 분석할 수 있는 방법이다.The present invention is a method capable of very specific qualitatively or quantitatively analyzing an active form of an enzyme, and preferably the present invention is a method capable of very specific qualitatively and quantitatively analyzing an active form of an enzyme.
본 발명의 명세서에서, 표현 “정성적 분석”은 시료내 타겟 효소 활성형의 존재 유무를 분석하는 것을 의미하고, 표현 “정량적 분석”은 시료내 타겟 효소 활성형의 농도를 정확히 분석하는 것을 의미한다.In the specification of the present invention, the expression "quantitative analysis" means analyzing the presence or absence of the target enzyme active form in the sample, and the expression "quantitative analysis" means accurately analyzing the concentration of the target enzyme active form in the sample. .
본 발명에 의하여 정성 또는 정량적으로 분석할 수 있는 효소의 활성형은 자연계에 존재하는 유기체에서 발견될 수 있는 모든 효소의 활성형이다.Active forms of enzymes that can be analyzed qualitatively or quantitatively by the present invention are active forms of all enzymes that can be found in organisms that exist in nature.
본 발명의 명세서에서, 표현 “효소의 활성형(active form) 또는 효소 활성형”은 알로스테릭 조절, 조절단백질에 의한 활성, 가역적 코발런트 모디피케이션 또는 단백질분해에 의하여 효소가 본래의 기능인 효소활성을 나타낼 수 있는 효소 형태 또는 구조를 의미한다.In the context of the present invention, the expression “active form or enzyme active form” refers to an enzyme in which the enzyme is inherently functioning by allosteric regulation, activity by a regulatory protein, reversible cobalt-based modulation or proteolysis. It refers to an enzyme form or structure that can exhibit activity.
본 발명의 바람직한 구현예에 따르면, 본 발명에 의하여 정성 및 정량적으로 분석할 수 있는 효소의 활성형은 예컨대, 알로스테릭 조절, 조절단백질에 의한 활성, 가역적 코발런트 모디피케이션 또는 단백질분해에 의한 형성된 효소의 활성형이며, 보다 바람직하게는 가역적 코발런트 모디피케이션 또는 단백질분해에 의한 효소의 활성형이고, 가장 바람직하게는 단백질 분해에 의한 효소의 활성형이다.According to a preferred embodiment of the present invention, the active forms of enzymes that can be quantitatively and quantitatively analyzed by the present invention are, for example, allosteric regulation, activity by regulatory proteins, reversible cobalt modification or proteolysis It is the active form of the enzyme formed, more preferably the active form of the enzyme by reversible cobalt modification or proteolysis, and most preferably the active form of the enzyme by proteolysis.
본 발명이 단백질 분해에 의한 효소의 활성형을 정성 또는 정량적으로 분석하는데 이용하는 방법인 경우, 바람직한 효소의 활성형은 Lp-PLA2(lipoprotein-associated phospholipase A2), TAFIa(activated thrombin-activatable fibrinolysis inhibitor), 혈액응고 인자 Ⅴ, 혈액응고 인자 Ⅶ, 혈액응고 인자 Ⅸ, 혈액응고 인자 Ⅹ, 혈액응고 인자 , 혈액응고 인자 , 트롬빈(thrombin), 트립신(trypsin), 키모트립신(chymotrypsin), 플라즈민(plasmin), u-PA(urokinase-type plasminogen activator), tPA(tissue plasminogen activator), 엘라스타아제(elastase), 서브틸리신(subtilisin), 칼리크레인(kallikrein), 카텝신 G(cathepsin G), 콜라겐, 콘카나발린 A, TPA(12-O-tetradecanoylphorbol-13 acetate) 또는 TGF-β(transforming growth factor-β)이며, 보다 바람직하게는 Lp-PLA2, TAFIa, 혈액응고 인자 Ⅴa, 혈액응고 인자 Ⅶ, 혈액응고 인자 Ⅸ, 혈액응고 인자 Ⅹ, 혈액응고 인자 , 혈액응고 인자 또는 트롬빈이며, 보다 더 바람직하게는 Lp-PLA2 또는 TAFIa이고, 가장 바람직하게는 Lp-PLA2이다.When the present invention is a method used for qualitatively or quantitatively analyzing the active form of an enzyme by proteolysis, the preferred active form of the enzyme is Lp-PLA 2 (lipoprotein-associated phospholipase A 2 ) or TAFIa (activated thrombin-activatable fibrinolysis inhibitor). ), Blood coagulation factor V, blood coagulation factor Ⅶ, blood coagulation factor Ⅸ, blood coagulation factor Ⅹ, blood coagulation factor, blood coagulation factor, thrombin (thrombin), trypsin, chymotrypsin, plasmin (plasmin) ), urokinase-type plasminogen activator (u-PA), tissue plasminogen activator (tPA), elastase, subtilisin, kallikrein, cathepsin G, collagen, Concanavalin A, TPA (12- O- tetradecanoylphorbol-13 acetate) or TGF-β (transforming growth factor-β), more preferably Lp-PLA 2 , TAFIa, blood coagulation factor Va, blood coagulation factor VII, Coagulation factor Ⅸ, blood coagulation factor Ⅹ, blood A clotting factor, a blood coagulation factor or thrombin, and more preferably, Lp-PLA 2 or TAFIa, and most preferably, Lp-PLA 2.
본 발명의 명세서에서 표현 “효소 억제제(enzyme inhibitor)”는 효소와 결합하여 효소의 활성을 감소시키는 분자를 의미한다. 상기 효소 억제제는 직접 또는 간접적으로 효소 활성부위에서 기질이 효소와 상호작용을 하는 것을 차단시키며, 가역 또는 비가역적으로 효과 결합을 할 수 있다. 약물(drug) 분자들은 생화학, 분자생물학, 유전체학, 프로테오믹스 및 약리학과 같은 다양한 분야에서 효소 억제제를 의미한다. 억제제는 유효성 및 특이적 결합 상호작용을 위하여 보다 높은 특이성이 요구되는 경우 다양한 특이성들을 가질 수 있다. As used herein, the expression “enzyme inhibitor” refers to a molecule that binds to an enzyme and reduces the activity of the enzyme. The enzyme inhibitor may directly or indirectly block the substrate from interacting with the enzyme at the enzyme active site, and may bind effectally or reversibly. Drug molecules refer to enzyme inhibitors in various fields such as biochemistry, molecular biology, genomics, proteomics and pharmacology. Inhibitors can have a variety of specificities where higher specificity is required for effectiveness and specific binding interactions.
바람직하게는, 본 발명에서 이용되는 억제제는 단백질(단편, 항체, 도메인등), 펩타이드(L- 및 D- 아미노산, 올리고펩타이드), PNA(Peptoids Nucleic acids; DNA aptamer 및 RNA aptamer), 유기 화합물, 나노입자, 무기염 및 이의 금속 유도체를 포함한다.Preferably, the inhibitors used in the present invention are proteins (fragments, antibodies, domains, etc.), peptides (L- and D-amino acids, oligopeptides), PNAs (Peptoids Nucleic acids; DNA aptamers and RNA aptamers), organic compounds, Nanoparticles, inorganic salts and metal derivatives thereof.
본 발명에서 이용하는 억제제는 효소의 활성형과 결합하여 효소의 활성형의 구조적인 형태를 변형 또는 변형시키지 않으면서 효소 활성형의 기능만을 억제시키는 물질을 의미한다. 바람직하게는, 본 발명에서 이용하는 억제제는 효소의 활성형과 결합하여 효소의 활성형의 구조적인 형태를 변형시키지 않으면서 효소의 활성형의 기능만을 억제시킬 수 있는 억제제이다.The inhibitor used in the present invention means a substance which binds to the active form of the enzyme and inhibits only the function of the enzyme active form without modifying or modifying the structural form of the active form of the enzyme. Preferably, the inhibitor used in the present invention is an inhibitor that can bind only to the active form of the enzyme and inhibit the function of the active form of the enzyme without modifying the structural form of the active form of the enzyme.
본 발명의 명세서에서, 효소의 활성형의 억제제를 설명하면서 사용하는 표현 “구조적인 형태를 변형”란 분자량, 아미노산 위치, 소수성, 친수성, 전하, 이차구조, 이종(heterogeneity), 길이, 극성, 용해도, 양친매성(amphipathic) 성질, 아미노산 서열 또는 면역원성이 변형되는 것을 의미한다.In the context of the present invention, the expression “modifying structural form” used to describe an inhibitor of the active form of an enzyme refers to molecular weight, amino acid position, hydrophobicity, hydrophilicity, charge, secondary structure, heterogeneity, length, polarity, solubility. Amphipathic nature, amino acid sequence or immunogenicity is meant to be altered.
또한, 본 발명에서 이용하는 억제제는 효소 활성형의 기질 결합 부위에 대하여 기질과 경쟁적 또는 비경쟁적 결합을 통하여 결합할 수 있는 억제제를 포함한다.In addition, the inhibitor used in the present invention includes an inhibitor capable of binding to a substrate binding site of an enzyme active type through competitive or non-competitive binding to the substrate.
본 발명의 바람직한 구현예에 따르면, 본 발명에서 이용하는 억제제는 효소 활성형의 기질 결합 부위에 대하여 기질과 경쟁적 결합을 하여 기질이 결합하는 것을 억제하는 억제제이다.According to a preferred embodiment of the present invention, the inhibitor used in the present invention is an inhibitor which inhibits the binding of the substrate by competitive binding with the substrate with respect to the substrate binding site of the enzyme active type.
본 발명에서 단계 (a) 또는 (b)에서의 효소 활성형의 억제제는 효소 활성형, 또는 프로효소 및 효소 활성형 모두에 결합할 수 있는 억제제를 포함한다.In the present invention, the inhibitor of the enzyme active form in step (a) or (b) includes an enzyme active form or an inhibitor capable of binding to both a proenzyme and an enzyme active form.
본 발명의 바람직한 구현예에 따르면, 본 발명에서 단계 (a) 또는 (b)에서의 효소 활성형의 억제제는 효소 활성형에만 특이적으로 결합하는 억제제이다.According to a preferred embodiment of the invention, the inhibitor of the enzyme active form in step (a) or (b) in the present invention is an inhibitor that specifically binds only to the enzyme active form.
본 발명에서 단계 (a)에서의 포획제는 직접적으로 효소 활성형과 결합할 수 있으며, 고체 기질(substrate) 표면에 결합된 상태에서 효소 활성형과 결합될 수 있다.In the present invention, the capture agent in step (a) may be directly coupled with the enzyme active form, and may be combined with the enzyme active form while bound to the solid substrate surface.
본 발명의 바람직한 구현예에 따르면, 본 발명에서 단계 (a)에서의 포획제는 는 고체 기질 표면상에 결합된 상태로 효소 활성형과 특이적으로 결합한다. According to a preferred embodiment of the present invention, in the present invention, the capture agent in step (a) specifically binds to the enzyme active form in a bound state on the solid substrate surface.
또한, 본 발명의 바람직한 구현예에 따르면, 본 발명에서 단계 (a)에서의 포획제가 고체 기질 표면상에 결합된 상태로 효소 활성형과 특이적으로 결합하는 경우에 이용될 수 있는 고체 기질은 기판, 나노입자, 나노 막대, 나노 와이어, 나노 큐브 또는 마이크로입자이며, 보다 바람직하게는 기판이다.Furthermore, according to a preferred embodiment of the present invention, in the present invention, the solid substrate which can be used when the capture agent in step (a) specifically binds to the enzyme active form in the state of being bound on the surface of the solid substrate is , Nanoparticles, nanorods, nanowires, nanocube, or microparticles, more preferably a substrate.
또한, 본 발명의 바람직한 구현예에 따르면, 본 발명에서 단계 (a)에서의 포획제가 기판 표면상에 결합된 상태로 효소 활성형과 특이적으로 결합하는 경우에 이용될 수 있는 기판은 유리, 금, 은, 알루미늄, 크롬, 실리콘, 게르마늄, 갈륨비소, 갈륨인, 규산질화물, 변형된 실리콘 니트로셀룰로오스, 폴리비닐리덴 플루오리드, 폴리스틸렌, 폴리테트라플루오로에틸렌, 폴리카보네이트, 나일론 또는 섬유이며, 보다 바람직하게는 유리, 금, 은 또는 알루미늄이고, 가장 바람직하게는 유리이다.In addition, according to a preferred embodiment of the present invention, in the present invention, the substrate which can be used when the capture agent in step (a) specifically binds to the enzyme active form in the bound state on the substrate surface is glass, gold , Silver, aluminum, chromium, silicon, germanium, gallium arsenide, gallium phosphorus, silicide nitride, modified silicon nitrocellulose, polyvinylidene fluoride, polystyrene, polytetrafluoroethylene, polycarbonate, nylon or fiber, more preferred Preferably glass, gold, silver or aluminum, most preferably glass.
본 발명에서 이용하는 기판에는 효소 활성형의 고정화를 위하여 당업계에 공지된 링커 분자가 부착될 수 있다.The substrate used in the present invention may be attached with a linker molecule known in the art for immobilization of an enzyme active form.
본 발명에서 단계 (b)에서의 효소의 활성형 억제제 또는 결합제는 직접적으로 효소 활성형과 결합할 수 있으며, 고체 기질 표면에 결합된 상태에서 효소 활성형과 결합될 수 있다.In the present invention, the active type inhibitor or binder of the enzyme in step (b) may be directly coupled with the enzyme active form, and may be combined with the enzyme active form while bound to the solid substrate surface.
본 발명의 바람직한 구현예에 따르면, 본 발명에서 단계 (b)에서의 활성형 억제제 또는 결합제가 고체 기질 표면상에 결합된 상태로 효소 활성형과 특이적으로 결합하는 경우에 이용될 수 있는 고체 기질은 기판, 나노입자, 나노 막대, 나노 와이어, 나노 큐브 또는 마이크로비드이며, 보다 바람직하게는 나노입자 또는 마이크로비드이고, 가장 바람직하게는 마이크로비드이다.According to a preferred embodiment of the present invention, in the present invention, the solid substrate which can be used when the active inhibitor or the binder in step (b) specifically binds to the enzyme active form while bound on the solid substrate surface Silver substrates, nanoparticles, nanorods, nanowires, nanocube, or microbeads, more preferably nanoparticles or microbeads, and most preferably microbeads.
본 발명에서 단계 (b)에서의 활성형 억제제 또는 결합제가 고체 기질 표면상에 결합된 상태로 효소 활성형과 특이적으로 결합하는 경우에 이용될 수 있는 고체 기질이 나노입자인 경우 금속나노입자가 바람직하며, 보다 바람직하게는 금 나노입자이다.In the present invention, when the active substrate inhibitor or binder in step (b) is specifically bound to the enzyme active form in the bound state on the solid substrate surface, the metal nanoparticles are used when the solid substrate is nanoparticles. Preferred is more preferably gold nanoparticles.
본 발명에서 이용하는 결합제는 효소 또는 효소의 활성형에 결합할 수 있는 결합제이다. 바람직하게는, 본 발명에서의 결합제는 효소의 활성형 또는 효소 및 효소의 활성형 모두에 결합할 수 있는 결합제이며, 보다 바람직하게는 효소의 활성형에 특이적으로 결합 할 수 있는 결합제이다.The binder used in the present invention is a binder capable of binding to an enzyme or an active form of the enzyme. Preferably, the binder in the present invention is a binder capable of binding the active form of the enzyme or both the enzyme and the active form of the enzyme, and more preferably a binder capable of specifically binding to the active form of the enzyme.
본 발명에서 효소의 활성형에 결합하는 앱타머는 올리고핵산 또는 펩타이드 분자이며, 앱타머의 일반적인 내용은 Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R . "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24):142727(1998)에 상세하게 개시되어 있다.The aptamer binding to the active form of the enzyme in the present invention is an oligonucleic acid or peptide molecule, the general content of aptamers are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95 (24): 142727 (1998).
본 발명의 바람직한 구현예에 따르면, 본 발명에서 이용하는 결합제는 올리고펩타이드, 모노클로날 항체, 폴리클로날 항체, 쉬메릭(chimeric) 항체, 리간드, PNA(Peptide nucleic acid) 또는 앱타머(aptamer)이며, 보다 바람직하게는 올리고펩타이드, 모노클로날 항체, 폴리클로날 항체 또는 쉬메릭 항체이고, 보다 더 바람직하게는 모노클로날 항체 또는 폴리클로날 항체이며, 가장 바람직하게는 모노클로날 항체이다.According to a preferred embodiment of the present invention, the binder used in the present invention is an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a PNA (Peptide nucleic acid) or an aptamer, More preferably, they are oligopeptides, monoclonal antibodies, polyclonal antibodies or chimeric antibodies, even more preferably monoclonal antibodies or polyclonal antibodies, and most preferably monoclonal antibodies.
본 발명은 효소의 활성형에 포획제와 먼저 결합시킨 후 포획제와 결합된 효소의 활성형에 결합제를 결합시킨 후 시그날 표지를 검출하는 단계를 통하여 실시되나, 효소 활성형에 포획제 및 결합제를 동시에 접촉시키는 단계를 통하여 실시될 수 있다.The present invention is carried out through the step of first binding the capture agent to the active form of the enzyme and then binding the binding agent to the active form of the enzyme combined with the capture agent and detecting a signal label. It may be carried out through the step of contacting at the same time.
본 발명의 바람직한 구형예에 따르면, 본 발명은 효소 활성형에 포획제 및 결합제를 동시에 접촉시키는 단계를 통하여 실시된다.According to a preferred embodiment of the invention, the invention is carried out through the step of simultaneously contacting the capture agent and the binder with the enzyme active form.
종래 타겟 프로효소 또는 효소의 활성형을 검출하기 위해서 이용되는 방법은 타겟 프로효소 또는 효소의 활성형에 직접 시그날 표지를 결합시키거나 시그날 표지가 결합된 항체를 결합시킨 후 시그날을 검출하는 방법을 이용하여 왔다. 그러나, 이러한 방법은 타겟 프로효소 또는 효소의 활성형을 검출하는데 복잡한 단계(다양한 시약 및 세척단계)로 인하여 많은 시간 및 장비가 요구되었다.Conventionally, a method used to detect an active form of a target proenzyme or enzyme uses a method of detecting a signal after binding a signal label directly to an active form of the target proenzyme or enzyme or binding an antibody having a signal label bound thereto. Has come. However, this method requires a lot of time and equipment due to the complex steps (various reagents and washing steps) for detecting the active form of the target proenzyme or enzyme.
본 발명은 포획자로서 효소 활성형의 억제제와 시스날 표지된 결합제를 함께 이용하여 타겟 효소의 활성형을 특이적으로 검출함으로써 간단하고 빠르게 결과를 확인할 수 있을 뿐 만 아니라 타겟 효소의 활성형에 대한 검출 정확도를 획기적으로 향상시킨 방법이다.The present invention provides a simple and fast result as well as a simple detection of a target enzyme by using a combination of an enzyme-activated inhibitor and a cisnal labeled binder as a capturer. It is a method of dramatically improving the detection accuracy.
본 발명에서 단계 (c)는 검출가능한 시그날 표지를 효소의 활성형 억제제 또는 결합제에 결합시킴으로써 실시된다.Step (c) in the present invention is carried out by binding a detectable signal label to an active inhibitor or binder of the enzyme.
본 발명의 바람직한 구현예에 따르면, 본 발명에서의 효소 활성형의 억제제 또는 결합제에 결합되는 검출가능한 시그날 표지(detectable signal label)는, 화학적 표지(예컨대, 바이오틴), 효소 표지(예컨대, 알칼린 포스파타아제, 퍼옥시다아제, β-갈락토시다아제 및 β-글루코시다아제), 방사능 표지(예컨대, I125 및 C14), 형광 표지(예컨대, 플루오레세인, TAMRA, Cy5, Cy3, HEX, TET, Dabsyl 및 FAM), 발광 표지, 화학발광(chemiluminescent) 표지, FRET(fluorescence resonance energy transfer) 표지 또는 금속 표지(예컨대, 금 및 은)이며, 보다 바람직하게는 효소 표지, 형광 표지 또는 발광 표지이고, 가장 바람직하게는 형광표지이다.According to a preferred embodiment of the present invention, a detectable signal label bound to an enzyme active inhibitor or binder in the present invention is a chemical label (e.g. biotin), an enzyme label (e.g. alkaline phosphate). Patases, peroxidases, β-galactosidase and β-glucosidase), radiolabels (eg I 125 and C 14 ), fluorescent labels (eg fluorescein, TAMRA, Cy5, Cy3, HEX, TET , Dabsyl and FAM), luminescent labels, chemiluminescent labels, fluorescence resonance energy transfer (FRET) labels or metal labels (e.g., gold and silver), more preferably enzyme labels, fluorescent labels or luminescent labels, Most preferably it is a fluorescent label.
본 발명에서의 단계 (c)를 통한 최종적인 결과 분석은 당업계에서 “리더” 또는 “스캔너”로 공지되어 있는 자동화된 장치를 이용하여 실시할 수 있다.The final result analysis through step (c) in the present invention can be carried out using an automated device known in the art as a "leader" or "scanner".
본 발명의 바람직한 구현예에 따르면, 본 발명에서는 단계 (c)는 광학 센서, 광원 및 광원검출기를 포함하는 광학 검출기, 흡광 또는 형광을 측정하는 광학 검출기, UV 검출기, 방사 검출기, 공초점현미경 검출기, CCD(charge-coupled device) 카메라 또는 마이크로플레이트 리더기를 이용하여 실시되며, 보다 바람직하게는 광학 센서, 광원 및 광원검출기를 포함하는 광학 검출기, 흡광 또는 형광을 측정하는 광학 검출기 또는 마이크로플레이트 리더기를 이용하여 실시되고, 가장 바람직하게는 흡광 또는 형광을 측정하는 광학 검출기를 이용하여 실시된다.According to a preferred embodiment of the present invention, step (c) in the present invention comprises an optical sensor, an optical detector including a light source and a light source detector, an optical detector for measuring absorption or fluorescence, a UV detector, a radiation detector, a confocal microscope detector, Implemented using a CCD (charge-coupled device) camera or a microplate reader, more preferably using an optical detector including an optical sensor, a light source and a light source detector, an optical detector or a microplate reader measuring absorbance or fluorescence. Most preferably, using an optical detector for measuring absorption or fluorescence.
본 발명은 시료내에 존재하는 효소의 활성형을 정성 및/또는 정량적으로 분석 할 수 있는 방법이다.The present invention is a method capable of qualitatively and / or quantitatively analyzing the active form of an enzyme present in a sample.
본 발명의 바람직한 구현예에 따르면, 본 발명에서 효소의 활성형을 포함하는 시료는 포유류, 조류, 파충류 또는 양서류로부터 수득된 시료이며, 보다 바람직하게는 포유류로부터 수득된 시료이고, 보다 더 바람직하게는 포유류의 혈액, 혈장, 혈청, 타액, 소변, 모유, 땀, 종양 적출액, 관절액, 척수액, 기관의 균질액, 정액 또는 질 분비물이며, 보다 더욱 바람직하게는 포유류의 혈액, 혈장, 혈청 또는 종양 적출액이고, 가장 바람직하게는 혈액이다.According to a preferred embodiment of the present invention, the sample comprising the active form of the enzyme in the present invention is a sample obtained from a mammal, a bird, a reptile or an amphibian, more preferably a sample obtained from a mammal, even more preferably Mammalian blood, plasma, serum, saliva, urine, breast milk, sweat, tumor extract, joint fluid, spinal fluid, organ homogenate, semen or vaginal secretion, and more preferably mammalian blood, plasma, serum or tumor extract Liquid, most preferably blood.
본 발명은 질환 또는 질병을 일으키는 관여하는 효소 활성형을 검출 및 분석하는 이용될 수 있다. 프로효소로부터 효소 활성형으로 전환되어 질환 또는 질병을 야기하는 효소 활성형이라면 제한없이 적용이 가능하다.The present invention can be used to detect and analyze the enzyme active forms involved in causing a disease or condition. Any enzyme active type that converts from a proenzyme to an enzyme active type to cause a disease or a disease can be applied without limitation.
본 발명의 바람직한 구현예에 따르면, 본 발명에서 분석하고자하는 효소의 활성형은 동맥경화증에 관여하는 효소 활성형이며, 보다 바람직하게는 죽상동맥경화증, 세동맥경화증 또는 중막경화증에 관여하는 효소 활성이고, 보다 더 바람직하게는 협심증, 심근경색증, 뇌혈관성 치매, 일과성 뇌허헐발작, 뇌경색, 뇌출혈, 뇌졸중, 뇌혈전증, 뇌색전증 또는 말초혈관폐쇄증에 관여하는 효소 활성형이며, 보다 더욱 바람직하게는 협심증, 심근경색증 또는 뇌혈관성 치매에 관여하는 효소 활성형고, 가장 바람직하게는 심근경색증에 관여하는 효소 활성형이다.
According to a preferred embodiment of the present invention, the active form of the enzyme to be analyzed in the present invention is an enzyme active type involved in atherosclerosis, more preferably is an enzyme activity involved in atherosclerosis, arteriosclerosis or mesclerosis, Even more preferred is an enzyme-activated type involved in angina, myocardial infarction, cerebrovascular dementia, transient cerebral dementia, cerebral infarction, cerebral hemorrhage, stroke, thrombosis, cerebral embolism or peripheral vascular obstruction, and even more preferably angina, myocardial infarction or It is an enzyme active type involved in cerebrovascular dementia, and most preferably, an enzyme active type involved in myocardial infarction.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(ⅰ) 본 발명은 효소의 활성형의 정성적 및 정량적인 분석방법을 제공한다.(Iii) The present invention provides a method for qualitative and quantitative analysis of an active form of an enzyme.
(ⅱ) 본 발명은 효소 활성형 억제제 및 결합제를 이용하여 시료내 효소 활성형을 분석함으로써 단순 결합제(예컨대, 항체)를 이용하여 효소 활성형을 분석하는 방법보다 빠르고 정확하게 분석할 수 있는 장점을 가진다.(Ii) The present invention has the advantage that it can be analyzed faster and more accurately than the method of analyzing the enzyme active form using a simple binder (eg, an antibody) by analyzing the enzyme active form in a sample using an enzyme active inhibitor and a binder. .
(ⅲ) 본 발명은 단순 결합제를 이용하여 효소 활성형을 분석하는 방법보다 작은 장비를 이용하여 복잡하지 않고 간단하게 시료내에 존재하는 효소 활성형을 분석할 수 있다.(Iii) The present invention can analyze the enzyme active form present in the sample simply and not by using a smaller device than the method of analyzing the enzyme active form using a simple binder.
(ⅳ) 본 발명은 타겟 효소 또는 효소의 활성형에 억제제 및 결합제를 동시에 이용함으로써 신속하고 특이적으로 분석할 수 있으며, 이러한 본 발명의 특성을 이용하여 약품(drug)을 고속으로 그리고 대량(high-throughput)으로 스크리닝을 할 수 있다.(Iii) The present invention can be analyzed quickly and specifically by simultaneously using an inhibitor and a binder in a target enzyme or an active form of an enzyme, and using this characteristic of the present invention, a drug is rapidly and high -throughput).
(ⅴ) 또한, 본 발명은 약품 투여 대상체(예컨대, 동물 또는 인간)에서의 약품의 효능 및 투여량에 대한 임상 모니터링을 위한 POCT(Point of care testing)에 적용될 수 있다.
(Iii) The invention may also be applied to point of care testing (POCT) for clinical monitoring of the efficacy and dosage of a drug in a drug administration subject (eg, animal or human).
도 1은 본 발명을 이용하여 시료(sample)내에 존재하는 Lp-PLA2에 대한 정성 및 정량적으로 분석 과정을 나타낸 개략도이다. 도 1a는 평면 고상기질(solid substrate)의 표면에 고정 결합된 Lp-PLA2 억제제와 시그날 표지가 결합된 Lp-PLA2 결합제(항체)를 이용하여 시료내에 존재하는 Lp-PLA2를 정성 및 정량적으로 분석하는 방법을 나타낸 개략도이다. 도 1b는 평면 고상기질의 표면에 고정 결합된 Lp-PLA2 억제제와 시그날 표지가 결합된 Lp-PLA2 결합제(항체)를 이용하여 시료내에 존재하는 Lp-PLA2를 정성 및 정량적으로 분석하는 방법을 나타낸 개략도이며, 시료내 Lp-PLA2 억제제가 존재하는 경우 Lp-PLA2는 Lp-PLA2 억제제와의 결합에 의하여 평면 고상기질 표면에 결합된 Lp-PLA2 억제제와 결합하지 않고 분리되어 시그날를 나타내지 않는다. 본 발명을 이용하는 경우, 시료내 Lp-PLA2의 포함유무 및 포함된 Lp-PLA2의 양을 분석할 수 있다.1 is a schematic diagram showing the qualitative and quantitative analysis of Lp-PLA 2 present in a sample using the present invention. Figure 1a qualitatively and quantitatively the Lp-PLA 2 present in the sample using a planar solid phase substrate (solid substrate) fixedly coupled to the Lp-PLA 2 inhibitors and signal Lp-PLA 2 binding agent (antibody) labeled is bonded to the surface of the Is a schematic diagram showing the method of analysis. Figure 1b is a method of analyzing qualitatively and quantitatively the Lp-PLA 2 present in the sample by using the Lp-PLA 2 inhibitors and signal Lp-PLA 2 binding agent (antibody), a labeled binding fixed to the surface of the planar solid substrate a is a schematic view showing, when the I Lp-PLA 2 inhibitor sample exists Lp-PLA 2 is separated rather than combined with the Lp-PLA 2 inhibitor binding to planar solid substrate surface by a combination of the Lp-PLA 2 inhibitors sigeunalreul Not shown. When using the present invention, it is possible to analyze the presence or absence of Lp-PLA 2 in the sample and the amount of Lp-PLA 2 contained.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .
실시예Example
다른 별도의 언급이 없는 경우, 고체/고체는 (중량/중량) 부 또는 %, 고체/액체는 (중량/부피) 부 또는 %, 그리고 액체/액체는 (부피/부피) 부 또는 %이다.
Unless stated otherwise, solids / solids are (weight / weight) parts or%, solids / liquids are (weight / volume) parts or%, and liquids / liquids are (volume / volume) parts or%.
실시예 1: Apo C-Ⅲ(Apolipoprotein C-Ⅲ)가 코팅된 칩을 이용한 LPL(lipoprotein lipase) 활성 측정Example 1 Measurement of Lipoprotein Lipase (LPL) Activity Using Apo C-III (Apolipoprotein C-III) Coated Chip
실험 재료 및 장비Experimental Materials and Equipment
본 실험에 이용된 실험 재료 및 장비는 다음과 같다:The experimental materials and equipment used in this experiment are as follows:
Apo C-Ⅲ(A3106, Sigma, 미국), LPL 항체(mouse anti-lipoprotein lipase monoclonal antibody, Unconjugated; 클론: LPL.A4, Cat# : ab21356, Abcam, 미국), 아비딘(A9275-25MG, Sigma, 미국), EDC(-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; 22980, Thermo, 미국), NHS(N-hydroxysulfosuccinimide sodium salt; 56485, Fluka, 미국), 술포-NHS-바이오틴(EZ-Link Sulfo-NHS-LC-LC-Biotin; 21338, PIERCE, 미국), 형광 파티클(Fluorescent particle; FluorSpheres carboxylate-modified microspheres, 0.2 ㎛, dark red fluorescent (660/680) *2% solids*, F8807, Molecular Probes), BSA(Bovine serum albumin; Bovostar, BSAS 0.5, BOVOGEN, 오수트레일리아), 덱스트란(Dextran; Dextran from Leuconostoc sp., 31389, Fluka, 미국), Tween 20(P1379, Sigma, 미국), 투석 막(Dialysis membrane; Spectra/pro dialysis membrane 12,000-14,000, 132 697, Spectrum Lab, 미국), 원심분리기(MICRO 17RT, Hanil Science, 대한민국), FREND 리더기(나오엔텍, 대한민국), UV 스펙트로포토미터(spectrophotometer; UV-1800, SHIMADZU, 일본), 인큐베이터(HB-101M, HANBAEK, 대한믹국)
Apo C-III (A3106, Sigma, USA), LPL antibody (mouse anti-lipoprotein lipase monoclonal antibody, Unconjugated; clone: LPL.A4, Cat #: ab21356, Abcam, USA), avidin (A9275-25MG, Sigma, USA ), EDC (-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride; 22980, Thermo, USA), NHS (N-hydroxysulfosuccinimide sodium salt; 56485, Fluka, USA), sulfo-NHS-biotin (EZ-Link Sulfo- NHS-LC-LC-Biotin; 21338, PIERCE, USA), Fluorescent particles; FluorSpheres carboxylate-modified microspheres, 0.2 μm, dark red fluorescent (660/680) * 2% solids *, F8807, Molecular Probes), BSA (Bovine serum albumin; Bovostar, BSAS 0.5, BOVOGEN, Australia), Dextran (Dextran from Leuconostoc sp., 31389, Fluka, USA), Tween 20 (P1379, Sigma, USA), Dialysis membrane (Dialysis membrane; Spectra / pro dialysis membrane 12,000-14,000, 132 697, Spectrum Lab, USA), Centrifuge (MICRO 17RT, Hanil Science, Korea), FREND Reader (Nao Entec, Korea), UV Specification Spectrophotometer (UV-1800, SHIMADZU, Japan), incubator (HB-101M, HANBAEK, Korea Mix)
Apo C-Ⅲ 바이오티닐레이션(biotinylation)Apo C-III biotinylation
PBS(phosphate buffered saline)을 이용하여 1 ㎎/㎖로 희석된 Apo C-Ⅲ를 제조하고, 1차 증류수를 이용하여 10 mM 바이오틴을 제조하였다. 그 다음, 1 ㎎/㎖ 농도의 1 ㎖ Apo C-Ⅲ에 10 mM 바이오틴 27 ㎕를 혼합한 후 상온에서 1시간 30분 교반 반응시켰다. 상온에서 반응시킨 반응물을 투석 막에 넣고 PBS에 담가 투석하였으며, 1 ℓ PBS로 3시간 마다 총 3회 교체하였다. 투석된 반응물을 280 ㎚에서 흡광도를 측정하여 농도를 확인한 후 냉장 보관 하였다.
Apo C-III diluted to 1 mg / ml was prepared using PBS (phosphate buffered saline), and 10 mM biotin was prepared using primary distilled water. Then, 27 μl of 10 mM biotin was mixed in 1 ml Apo C-III at a concentration of 1 mg / ml, followed by stirring for 1 hour and 30 minutes at room temperature. The reaction reacted at room temperature was placed in a dialysis membrane, soaked in PBS, and dialyzed, and replaced with 1 L PBS three times every three hours. The dialysis reactant was measured by measuring absorbance at 280 nm, and then refrigerated.
안티-LPL 형광 콘쥬게이트Anti-LPL Fluorescent Conjugate
pH 6.2, 50 mM MES 버퍼를 이용하여 1 ㎎/㎖ 안티-LPL를 제조하였다. 제조된 1 ㎎/㎖ 안티-LPL 용액에 50 mM과 100 mM의 농도가 되도록 EDC와 NHS를 각각 첨가하였다. 혼합된 용액에 0.2 중량/부피% 형광 파티클을 즉시 혼합한 후 상온에서 3시간 동안 교반 반응시켰다. 교반된 반응물을 12,000 g로 15분간 원심분리한 후 상층액을 제거하였다. 상층액이 제거된 반응물의 침전물을 PBS로 재 부유시킨 다음 12,000 g로 15분간 원심분리한 후 상층액을 제거시켰다. 그 다음, 상층액이 제거된 침전물을 1% BSA가 첨가된 PBS로 재부유시키고, 형광 파티클의 중량/부피%가 0.2가 되도록하였다. 상기와 같은 방법으로 제조된 안티-LPL 형광 콘쥬게이트를 냉장 보관하였다.
1 mg / ml anti-LPL was prepared using pH 6.2, 50 mM MES buffer. EDC and NHS were added to the prepared 1 mg / ml anti-LPL solution so as to have a concentration of 50 mM and 100 mM. 0.2 wt / vol% fluorescent particles were immediately mixed with the mixed solution, followed by stirring at room temperature for 3 hours. The reaction was centrifuged at 12,000 g for 15 minutes and then the supernatant was removed. The precipitate of the reactant from which the supernatant was removed was resuspended in PBS and then centrifuged at 12,000 g for 15 minutes to remove the supernatant. The supernatant was then resuspended in PBS with 1% BSA and the weight / volume of fluorescent particles was 0.2. The anti-LPL fluorescent conjugate prepared by the above method was refrigerated.
칩 제조Chip manufacturing
PBS 용액에 아비딘을 100 ㎍/㎖이 되도록 희석한 후 50 mM과 100 mM의 농도가 EDC와 NHS를 각각 첨가하여 아비딘 용액을 제조하였다. 제조된 아비딘 용액을 카르복실기가 유도된 PMMA(poly(methyl methacrylate) 슬라이드 위에 1.5 ㎕ 돗팅(dotting) 하였다. 아비딘 용액이 돗팅된 슬라이드를 37℃ 인큐베이터에서 1시간 30분 동안 인큐베이션시켰다. 인큐베이션된 슬라이드를 pH 7.4, 0.1M Tris 용액으로 세척한 후 슬라이드상에서 아비딘이 돗팅 되었던 위치에 10 ㎍/㎖로 희석된 바이오틴화된 APO C-Ⅲ를 1.5 ㎕로 돗팅하였다. APO C-Ⅲ의 희석은 1% BSA 포함된 PBS 용액으로 하였다. 바이오틴화된 APO C-Ⅲ로 돗팅된 슬라이드를 37℃ 인큐베이터에서 1시간 30분 동안 인큐베이션시켰다. 인큐베이션시킨 슬라이드를 1% BSA, 0.05% Tween 20 및 0.5% 덱스트란이 첨가된 PBS 용액으로 세척한 후 상온에서 건조시켰다. 그 다음, 1% BSA, 0.01% Tween20 및 3% 덱스트란이 첨가된 pH 7.4, 0.1 M Tris 용액을 이용하여 0.2 중량/부피%로 희석시킨 안티-LPL 형광 콘쥬게이트를 1.2 ㎕의 양으로 돗팅시킨 후 건조시켰다. 건조된 슬라이드의 유동을 유도 및 제어할 수 있는 또 다른 슬라이드와 접합하여 시험 할 수 있는 칩을 제조하였다.
The avidin solution was prepared by diluting avidin to 100 μg / ml in PBS solution and adding EDC and NHS at 50 mM and 100 mM, respectively. The prepared avidin solution was doted by 1.5 μl onto a carboxyl group-induced poly (methyl methacrylate) PMMA slide, and the incubated slides were incubated in a 37 ° C. incubator for 1 hour and 30 minutes. After washing with 7.4, 0.1 M Tris solution, 1.5 μl of the biotinylated APO C-III diluted to 10 μg / ml was placed at the position where avidin was doted on the slide.The dilution of APO C-III included 1% BSA. The PBS solution was then incubated with biotinylated APO C-III for 1 hour and 30 minutes in an incubator at 37 ° C. The incubated slides were added with 1% BSA, 0.05% Tween 20 and 0.5% dextran. Washed with PBS solution and dried at room temperature, then diluted to 0.2 wt / vol% with pH 7.4, 0.1 M Tris solution added with 1% BSA, 0.01% Tween20 and 3% dextran -LPL fluorescent conjugate was doted in an amount of 1.2 [mu] l and dried, A chip was prepared that could be tested by bonding with another slide capable of inducing and controlling the flow of the dried slide.
시험 방법Test Methods
제조된 칩의 샘플 주입(inlet) 부위에 샘풀(lipoprotein lipase 가 존재하거나 또는 존재하지 않는 환자 혈청 또는 혈장)을 30 ㎕를 떨어뜨린다. 샘플을 떨어뜨린지 5분이 경과 하면 시험되는 칩을 FREND 리더기에 삽입시킨다. 약 40초 후에 FREND 리더기의 디스플레이 화면에 샘플내 LPL의 존재 정도가 정량적으로 표시되며, 이러한 정량적인 표시는 LPL의 활성화 장도를 나타낸다.
30 [mu] l of the sample (lipoprotein lipase with or without patient serum) is dropped at the sample inlet of the prepared chip. Five minutes after dropping the sample, insert the chip under test into the FREND reader. After about 40 seconds, the presence of LPL in the sample is quantitatively displayed on the display screen of the FREND reader, and this quantitative indication indicates the degree of activation of the LPL.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
Claims (40)
(a) 포획제(capturing agent)로서 상기 효소 활성형의 억제제에 분석하고자 하는 시료 내의 상기 효소 활성형을 접촉(contacting)시키는 단계;
(b) 검출 가능한 시그널을 발생시키는 표지가 결합된 검출자(detector)를 상기 단계 (a)의 결과물에 접촉(contacting)시키는 단계로서, 상기 검출자는 상기 효소 또는 효소 활성형, 그리고 이들의 모두에 결합하는 결합제이며; 및
(c) 상기 효소 또는 효소 활성형에 결합된 검출자의 표지로부터 발생되는 시그널을 측정하는 단계.
Qualitative or quantitative analysis of the active form of the enzyme comprising the following steps:
(a) contacting said enzyme active form in a sample to be analyzed with an inhibitor of said enzyme active form as a capturing agent;
(b) contacting a detector with a label that generates a detectable signal to the product of step (a), wherein the detector is capable of contacting the enzyme or enzyme active form, and all of Binding agent; And
(c) measuring the signal generated from the label of the detector bound to the enzyme or enzyme active form.
(a) 포획제로서 상기 효소 또는 효소 활성형, 그리고 이들 모두의 결합제에 분석하고자 하는 시료 내의 상기 효소 활성형을 접촉시키는 단계;
(b) 검출 가능한 시그널을 발생시키는 표지가 결합된 검출자를 상기 단계 (a)의 결과물에 접촉시키는 단계로서, 상기 검출자는 상기 효소 활성형에 결합하는 억제제이며; 및
(c) 상기 효소 활성형에 결합된 검출자의 표지로부터 발생되는 시그널을 측정하는 단계.
Qualitative or quantitative analysis of the active form of the enzyme comprising the following steps:
(a) contacting the enzyme or enzyme active form, and both binders, as a capture agent with the enzyme active form in the sample to be analyzed;
(b) contacting a detector with a label that generates a detectable signal to the product of step (a), wherein the detector is an inhibitor that binds to the enzyme active form; And
(c) measuring the signal generated from the label of the detector bound to the enzyme active form.
3. The method according to claim 1 or 2, wherein the active form of the enzyme is an active form of an enzyme activated by allosteric regulation, activity by a regulatory protein, reversible cobalt modification or proteolysis.
4. The method of claim 3, wherein the active form of the enzyme is an active form of the enzyme activated by reversible cobalt modification or proteolysis.
5. The method according to claim 4, wherein the active form of the enzyme is an active form of an enzyme activated by proteolysis.
The method according to claim 5, wherein the active form of the enzyme activated by proteolysis is Lp-PLA 2 (lipoprotein-associated phospholipase A 2 ), TAFIa (activated thrombin-activatable fibrinolysis inhibitor), blood coagulation factor V, blood coagulation factor Ⅶ , Blood coagulation factor Ⅸ, blood coagulation factor Ⅹ, blood coagulation factor, blood coagulation factor, thrombin, trypsin, chymotrypsin, plasmin, u-PA (urokinase-type plasminogen activator ), tissue plasminogen activator (tPA), elastase, subtilisin, kallikrein, kallikrein, cathepsin G, collagen, concanavalin A, TPA (12- O- ) tetradecanoylphorbol-13 acetate) or TGF-β (transforming growth factor-β).
The method according to claim 6, wherein the active form of the enzyme is Lp-PLA 2 , TAFIa, blood coagulation factor Va, blood coagulation factor Ⅶ, blood coagulation factor Ⅸ, blood coagulation factor Ⅹ, blood coagulation factor, blood coagulation factor or thrombin How to feature.
8. The method of claim 7, wherein the active form of the enzyme is Lp-PLA 2 or TAFIa.
The method of claim 8, wherein the active form of the enzyme is Lp-PLA 2 .
The method according to claim 1 or 2, wherein the active type inhibitor of the enzyme inhibits the binding of the substrate by competitively binding the substrate to the substrate binding site of the enzyme active type.
The method according to claim 1 or 2, wherein the active type inhibitor of the enzyme specifically binds only to the enzyme active type.
The method of claim 1 or 2, wherein the capture agent is bound to a solid substrate.
The method of claim 12, wherein the solid substrate is a substrate, nanoparticles, nanorods, nanowires, nanocube, or microparticles.
The method of claim 13, wherein the solid substrate is a substrate.
15. The silicide nitride, modified silicon nitrocellulose, polyvinylidene fluoride, polystyrene, polytetrafluoro, wherein the substrate is glass, gold, silver, aluminum, chromium, silicon, germanium, gallium arsenide, gallium. Ethylene, polycarbonate, nylon or fiber.
The method of claim 15, wherein the substrate is glass, gold, silver or aluminum.
The method of claim 16, wherein the substrate is glass.
The method according to claim 1 or 2, wherein the active inhibitor or binder of the enzyme in step (b) is bound to a solid substrate.
The method of claim 18, wherein the solid substrate is a substrate, nanoparticles, nanorods, nanowires, nanocube, or microbeads.
20. The method of claim 19, wherein the solid substrate is nanoparticles or microbeads.
The method of claim 20, wherein the solid substrate is microbeads.
The method of claim 1 or 2, wherein the binding agent is an oligopeptide, monoclonal antibody, polyclonal antibody, chimeric antibody, ligand, PNA (Peptide nucleic acid) or aptamer (aptamer), characterized in that How to.
The method of claim 22, wherein said binding agent is an oligopeptide, a monoclonal antibody, a polyclonal antibody, or a chimeric antibody.
The method of claim 23, wherein said binding agent is a monoclonal antibody.
The method according to claim 1 or 2, wherein the method is carried out through the step of simultaneously contacting a capture agent and a binder with an enzyme active form.
The label according to claim 1 or 2, wherein the label bound to the binder is a compound label, an enzyme label, a radiolabel, a fluorescent label, a luminescent label, a chemiluminescent label, a FRET (fluorescence resonance energy transfer) label or a metal label. How to.
27. The method of claim 26, wherein the label bound to the binder is an enzyme label, a fluorescent label, or a luminescent label.
28. The method of claim 27, wherein the label bound to the binder is a fluorescent label.
The method according to claim 1 or 2, wherein step (c) comprises an optical sensor, an optical detector including a light source and a light source detector, an optical detector for measuring absorption or fluorescence, a UV detector, a radiation detector, a confocal microscope detector, a CCD. (charge-coupled device) A method comprising using a camera or a microplate reader.
30. The method of claim 29, wherein step (c) is carried out using an optical sensor, an optical detector comprising a light source and a light source detector, an optical detector or a microplate reader measuring absorbance or fluorescence.
31. The method of claim 30, wherein step (c) is performed using an optical detector that measures absorbance or fluorescence.
The method according to claim 1 or 2, wherein the sample is a sample obtained from a mammal, a bird, a reptile or an amphibian.
33. The method of claim 32, wherein said sample is a sample obtained from a mammal.
34. The method of claim 33, wherein the sample is blood, plasma, serum, saliva, urine, breast milk, sweat, tumor extract, joint fluid, spinal fluid, organ homogenate, semen or vaginal secretion.
35. The method of claim 34, wherein said sample is blood, plasma, serum or tumor extract.
36. The method of claim 35, wherein said sample is blood.
3. The method according to claim 1 or 2, wherein the enzyme active form is an enzyme active form involved in atherosclerosis.
38. The method according to claim 37, wherein the enzyme active type is an enzyme active type involved in atherosclerosis, arteriosclerosis, or mesclerosis.
39. The method of claim 38, wherein the enzyme active type is an enzyme active type involved in angina pectoris, myocardial infarction, cerebrovascular dementia, transient cerebral dementia, cerebral infarction, cerebral hemorrhage, stroke, cerebrothrombosis, cerebral embolism or peripheral vascular obstruction.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020100107640A KR20120045822A (en) | 2010-11-01 | 2010-11-01 | Methods for analyzing quantitatively and qualitatively active forms of enzymes |
| EP11786904.0A EP2579040A4 (en) | 2010-05-25 | 2011-05-25 | QUALITATIVE AND QUANTITATIVE ANALYTICAL METHOD FOR ANALYSIS OF THE TYPE OF ACTIVITY OF AN ENZYME THAT IS ACTIVATED BY PROTEOLYSIS |
| KR1020127020306A KR101255828B1 (en) | 2010-05-25 | 2011-05-25 | Qualitative and quantitative analytical method for analyzing the activity type of an enzyme that is activated by proteolysis |
| PCT/KR2011/003843 WO2011149272A2 (en) | 2010-05-25 | 2011-05-25 | Qualitative and quantitative analytical method for analyzing the activity type of an enzyme that is activated by proteolysis |
| US13/699,868 US20130071835A1 (en) | 2010-05-25 | 2011-05-25 | Qualitative and Quantitative Analytical Method for Analyzing the Activity Type of an Enzyme that is Activated by Proteolysis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020100107640A KR20120045822A (en) | 2010-11-01 | 2010-11-01 | Methods for analyzing quantitatively and qualitatively active forms of enzymes |
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| KR20120045822A true KR20120045822A (en) | 2012-05-09 |
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| KR1020100107640A Pending KR20120045822A (en) | 2010-05-25 | 2010-11-01 | Methods for analyzing quantitatively and qualitatively active forms of enzymes |
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