JP2008131908A - Method for analyzing von willebrand factor-decomposing enzyme - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for analyzing a Von Willebrand factor-decomposing enzyme using a substrate capable of being cut off by the Von Willebrand factor-decomposing enzyme, capable of accurately determining the active amount of ADAMTS13 (a pseudonym of the Von Willebrand factor-decomposing enzyme) in an extremely short time, and a kit for the analysis. <P>SOLUTION: This method of analysis is provided by performing the contact of a test specimen with the substrate in liquid in the presence of a cationic water-soluble polysaccharide interacting with the substrate and also capable of forming a complex. The kit for the analysis contains (1) the substrate capable of being cut off by the Von Willebrand factor-decomposing enzyme and (2) the cationic water-soluble polysaccharide capable of interacting with the substrate and forming the complex. <P>COPYRIGHT: (C)2008,JPO&amp;INPIT

Description

The present invention relates to a method for analyzing a von Willebrand factor (hereinafter referred to as vWF) degrading enzyme and a kit for analysis contained in a test sample.
In recent years, the clinical value of the activity measurement of vWF-degrading enzyme [hereinafter referred to as ADAMTS13 (also referred to as vWF-degrading enzyme)] has been recognized in the detection of the degree of platelet thrombus formation or the detection of thrombosis. According to the method of the present invention, it is possible to realize an ADAMTS13 analysis method that is excellent in simplicity, rapidity, and sensitivity, and further, the amount of enzyme activity is quantified finely by a fully automatic analyzer by an extremely short time measurement. be able to. Note that the term “analysis” in this specification includes “detection” for determining the presence or absence of an analyte, and “measurement” for quantitatively or semi-quantitatively determining the amount or activity of the analyte. included.

  ADAMTS13 was originally suggested to be involved in the development of thrombotic thrombocytopenic purpura (hereinafter referred to as “TTP”), which is very serious and has a high fatality rate, and is purified from plasma ( Non-patent document 1), the gene was determined by cDNA cloning. In fact, it has been clarified that gene mutation of ADAMTS13 significantly reduces vWF degradation activity (Non-patent Document 2). In recent years, an enzyme immunoassay method using a monoclonal antibody or a polyclonal antibody against ADAMTS13 has been developed (Patent Document 1), and a method for detecting the cause of thrombosis associated with platelet aggregation and the tendency of thrombosis in thrombosis has been established. Using this, it has been found that the concentration of ADAMTS13 in the plasma of various thrombotic patients is significantly lower than that in healthy individuals.

  As a method for measuring the activity of ADAMTS13, a vWF giant multimer detection method combining SDS-agarose electrophoresis and autoradiography or Western blotting has been used (Non-patent Document 3). In recent years, FRETS-vWF73, in which the A2 domain 73 residue, which is a specific degradation site of vWF by ADAMTS13, is introduced, and a fluorescent group and a quenching group are introduced into the peripheral site, has been developed so that ADAMTS13 activity can be easily measured. (Non-Patent Document 4).

  However, these methods for measuring activity are actually very complicated, require a special fluorescence detector for the measurement, and take a long time from the measurement of the clinical specimen to the output of the result. It is unbearable for use in the field of clinical diagnostic tests.

International Publication No. 2004/029242 Pamphlet K. Fujikawa et al., “Blood”, (USA), 2001, Vol. 98, p. 1662-6 G. G. Levy et al., “Nature” (UK), 2001, Vol. 413, p. 488-494 M. Furlan et al., “Blood”, (USA), 1996, Vol. 87, p. 4223-4234 Kokame K et al. “British Journal of Haematology” (UK), 2005, 129, 93-100.

  As described above, the activity measurement of ADAMTS13 is expected to be very useful clinically and become the center of elucidation of various disease states in the future. In the diagnosis of such pathological conditions, it is expected that simple and extremely rapid quantitative measurement of ADAMTS13 activity is required. In order to realize these required elements, unprecedented quantitative measurement of ADAMTS13 activity is indispensable. For example, short-term quantitative measurement of ADAMTS13 activity by a fully automated device that places minimal burden on the measurer is an example. It can be considered.

  As a result of extensive research, the present inventor has quantitatively measured ADAMTS13 activity using a fully automated apparatus using a magnetic particle capable of capturing a fragment serving as a substrate for von Willebrand factor and an enzyme labeling partner capable of recognizing the fragment. Furthermore, by adding a cationic water-soluble polysaccharide capable of interacting with the substrate and forming a complex in a solution in which ADAMTS13 and the substrate are in contact with each other, the substrate is cleaved by ADAMTS13. An easy situation occurs, and as a result, enzyme activity is remarkably enhanced, and quantitative measurement of ADAMTS13 activity in an extremely short time which has not been possible in the past can be realized, and the present invention has been completed here.

  Therefore, an object of the present invention is to provide an analysis method and an analysis kit capable of accurately quantifying the amount of ADAMTS13 activity in an extremely short time which has not been conventionally achieved.

Therefore, the present invention
[1] In a method for analyzing von Willebrand factor-degrading enzyme using a substrate cleavable by von Willebrand factor-degrading enzyme, the contact between the test sample and the substrate in the liquid interacts with the substrate, and the complex A method for analyzing von Willebrand factor-degrading enzyme, which is carried out in the presence of a cationic water-soluble polysaccharide capable of forming
[2] The analysis method according to [1], wherein the cationic water-soluble polysaccharide is a polysaccharide having glucose as a structural unit;
[3] The cationic water-soluble polysaccharide is a polysaccharide having glucose as a structural unit and having a tertiary amino group and / or a quaternary ammonium group as a functional group exhibiting a cationic property. Analysis method;
[4] The analysis method according to [1], wherein the cationic water-soluble polysaccharide is DEAE-dextran;
[5] The analysis method according to [1] to [4], wherein the analysis is performed using a fully automatic analyzer;
[6] The analysis according to [1] to [5], wherein an insoluble carrier capable of capturing a substrate fragment generated by cleaving the substrate with a von Willebrand factor-degrading enzyme and / or an uncut substrate is used. Method;
[7] The analysis method according to [6], wherein the insoluble carrier is a particle;
[8] The analysis method according to [7], wherein the particles are latex particles or magnetic particles;
[9] The substrate is labeled with a labeling substance on the substrate fragment side that is cleaved and released by von Willebrand factor-degrading enzyme, or has an amino acid sequence that can be specifically recognized. Analysis method of [8];
[10] The analysis method according to [9], wherein the labeling substance is a fluorescent substance, a luminescent substance, a coloring substance, or an enzyme;
[11] The analysis method according to [6] to [8], wherein a partner that specifically binds to neoantigen generated by cleavage of the substrate by von Willebrand factorase is used;
[12] A von, comprising (1) a substrate cleavable by von Willebrand factor-degrading enzyme, and (2) a cationic water-soluble polysaccharide capable of interacting with the substrate to form a complex. The present invention relates to a kit for analysis of Wilbrand factorase.

  In the present invention, by adding a cationic water-soluble polysaccharide that interacts with a substrate that can be cleaved by ADAMTS13 and forms a complex, a situation in which the substrate is easily cleaved by ADAMTS13 occurs. It was found that the enzymatic activity to cleave is greatly improved compared to the case where it does not coexist. By applying this discovery, the time required for the conventional method for measuring ADAMTS13 activity (particularly the contact time between ADAMTS13 and the substrate) could be significantly shortened. As a result, simple and extremely rapid quantitative measurement, as typified by measurement by a fully automated apparatus, is achieved.

In the analysis method of the present invention, at least a substrate that can be cleaved by ADAMTS13, that is, a full length of von Willebrand factor or a fragment of a portion that becomes the substrate is used. The analysis method of the present invention is, for example,
(1) A test sample that may contain ADAMTS13 and a substrate that can be cleaved by ADAMTS13 interact with the substrate to form a complex in the presence of a cationic water-soluble polysaccharide that can form a complex. The step of contacting in,
(2) a step of separating the substrate fragment generated by cleaving the substrate with ADAMTS13 and / or the uncut substrate, and (3) a step of analyzing the separated substrate fragment and / or the substrate. be able to.

  The analysis method of the present invention can be carried out without using a partner (preferably an antibody) that can specifically react with a substrate cleavable by ADAMTS13 or a substrate fragment after cleavage, or the partner. Can also be implemented. As an embodiment to be carried out without using the partner, for example, a method of confirming a substrate fragment after cleavage by electrophoresis, as a substrate, for example, a fluorescent group and a quenching group are introduced around the cleavage site of the substrate, Examples thereof include a method using a substrate [for example, FRETS-vWF73 (Non-patent Document 4)] capable of detecting fluorescence by the cleavage.

  In the analysis method of the present invention, measurement using an insoluble carrier is preferable in that simple and rapid quantitative measurement can be performed. In an embodiment using an insoluble carrier, for example, a substrate that can be cleaved by ADAMTS13 can be used in a state of being bound in advance to the insoluble carrier, or a substrate after contacting with a test sample (cleaved by ADAMTS13). Resulting substrate fragments and / or uncut substrate) can also be contacted with an insoluble carrier.

In the analysis method of the present invention, in an embodiment using an insoluble carrier, for example,
(1) A test sample that may contain ADAMTS13 and a substrate that can be cleaved by ADAMTS13 interact with the substrate to form a complex in the presence of a cationic water-soluble polysaccharide that can form a complex. The step of contacting in,
(2) After or simultaneously with the step (1), a step of contacting an insoluble carrier (if desired, a labeling partner or the like) capable of capturing the substrate or the cleaved substrate fragment;
(3) separating the liquid from the insoluble carrier; and (4) analyzing the substrate or substrate fragment remaining on the insoluble carrier and / or the substrate or substrate fragment in the liquid released from the insoluble carrier. be able to.

Furthermore, in the analysis method of the present invention, for example, based on the mode of labeling of the labeling substance used, for example,
(A) an embodiment using a labeling partner that has a sequence or structure that can be recognized by another substance on the substrate fragment side that is released by cleavage of ADAMTS13, and which can recognize this;
(B) an embodiment in which the substrate is directly labeled with a labeling substance on the side of the substrate fragment that is liberated by cleavage of ADAMTS13, or (c) a substrate that specifically binds to neoantigen generated by cleavage with ADAMTS13 and labeled Embodiments using a labeling partner that is labeled with a substance are included.

  The test sample that can be analyzed by the analysis method of the present invention is not particularly limited as long as it is a sample that may contain the analysis target vWF-degrading enzyme (ADAMTS13). For example, a biological liquid sample, In particular, blood, serum, plasma, urine, cerebrospinal fluid and the like can be mentioned.

  The target enzyme that can be analyzed by the analysis method of the present invention is typically ADAMTS13, which is currently considered as a vWF-degrading enzyme. The enzyme is not particularly limited as long as it is an enzyme that can be cleaved between [methionine (1606)]. This means that, for example, when a vWF-degrading enzyme other than ADAMTS13 is discovered in the future, or when a control factor that forms a complex with ADAMTS13 and adjusts the cleavage activity is discovered, etc. And

  The enzyme substrate used in the analysis method of the present invention is not particularly limited as long as it is a substance that can be specifically cleaved by a protease between tyrosine (1605) and methionine (1606) of the A2 domain of vWF. The full length of vWF or a fragment of the portion serving as its substrate can be used, and those containing the full length of vWF or the shortest region of the portion serving as its specific substrate are preferred. The substrate can be prepared by a well-known method, for example, a method of collecting a crude parasite extract or purified product from human or animal body fluids, or artificially cultivating a biologically derived extract or purified product. Examples thereof include a method of obtaining from a living organism, a method of producing nucleic acid or protein by gene recombination technology or cell culture technology, or a method of synthesizing a polypeptide or hapten by chemical synthesis technology.

The cationic water-soluble polysaccharide used in the present invention is not particularly limited as long as it can form a complex with a substrate and can form a form that can be easily cleaved by ADAMTS13.
For example, Glc (glucose, Gal (galactose), Man (mannose), GlcUA (glucuronic acid), IdoA (iduronic acid), Fuc (fucose) ), GlcN (D-glucosamine: glucosamine, GlcNH ₂ ), GlcNAc (N-acetylglucosamine), GalNAc (N-acetylgalactosamine), NeuAc (N-acetylneuraminic acid), NeuGc (N-glycolylneuraminic acid) Glucose is preferable, and the water-soluble polysaccharide part can be composed of only one kind of these structural units, or can be composed of two or more kinds of structural units.

  Examples of the water-soluble polysaccharide include cellulose (eg, carboxymethylcellulose) ether, sialic acid, dextran, pullulan, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan, diethylaminoethyl (DEAE) -dextran, heparin. Hyaluronic acid and the like can be mentioned, and examples of the cationic water-soluble polysaccharide used in the present invention include those obtained by chemically modifying the functional groups of these water-soluble polysaccharides to impart cationic properties. Of the water-soluble polysaccharides, chitosan or DEAE-dextran that originally has a cationic property can be subjected to the chemical modification, but without being subjected to the chemical modification, the cationic water-soluble polysaccharide is used as it is. Can be used. As the water-soluble polysaccharide, chitosan or DEAE-dextran is preferable in that it has a cationic property.

  Cationic water-soluble polysaccharides are, for example, primary amino groups, secondary amino groups, tertiary amino groups, quaternary ammonium groups, amidino as basic functional groups for exerting their cationic properties. Group, a guanidino group, an imidazole group, etc. are mentioned, For example, it is preferable that they are a tertiary amino group and a quaternary ammonium group. Further, the most suitable basic functional group used in the present invention is a DEAE group which is a tertiary amino group, or a DEAE-DEAE group which is a tandem structure in which a quaternary ammonium group and a tertiary amino group are bonded. Is preferred.

  As the cationic water-soluble polysaccharide used in the present invention, DEAE-dextran is most preferable. In general, DEAE-dextran that is commercially available includes both a DEAE group and a DEAE-DEAE group. In this specification, “DEAE-dextran” is a DEAE group and / or DEAE-DEAE as a basic functional group. Means dextran containing groups. The molecular weight of DEAE-dextran is determined by the number of constituent glucose units, and is usually 10,000 to 2,000,000, of which 100,000 to 1,000,000 are preferred, 300 1,000 to 700,000 are particularly preferred. The N content is usually 0.1 to 20%, preferably 0.5 to 10%, and particularly preferably 1 to 5%.

  As an insoluble carrier that can be used in the method of the present invention, any form of an insoluble carrier that can capture an enzyme substrate or a cleaved substrate fragment can be used. A material capable of physically adsorbing a partner specific to a substrate fragment of the above, or a material having an appropriate functional group capable of supporting by a chemical bond [for example, various plastics (for example, polypropylene, polystyrene, polycarbonate, polyamide, or polytetrafluoro (Ethylene) plates, tubes, beads, chips, glass, magnetic carriers, latex particles, or films can be used. In view of easy operation during B / F separation and ease of application to measurement by a fully automated apparatus, insoluble magnetic particles are preferably used here.

  The particle size of the insoluble magnetic particles is usually 0.05 μm to 5 μm, preferably insoluble magnetic particles having a particle size in the range of 0.3 μm to 3 μm. Examples of the physical adsorption method include a method of directly immobilizing proteins on insoluble magnetic particles. As a method of chemically supporting, for example, amino groups, carboxyl groups, mercapto groups, hydroxyl groups, aldehyde groups, epoxy groups and the like existing on the surface of magnetic particles are chemically modified to bind to proteins. Using a functional group that can be directly immobilized on the particle, a method of immobilizing a spacer molecule by introducing a chemical bond between the particle and the substrate molecule, and binding other proteins such as albumin by the above method Then, a method of further binding an antibody or the like from above, or a method of chemically binding an antibody or the like to another protein such as albumin and then chemically binding the composite protein to the particle can be mentioned. In general, the amount of protein to be supported can be appropriately determined depending on the surface area of the insoluble carrier particles used, the amount of functional groups, and the like.

  As a labeling partner that can be used in the method of the present invention, if the substrate has a sequence or structure that can be recognized by other substances on the substrate fragment side that is released by cleavage of ADAMTS13, this is recognized. For example, an antibody or a fragment thereof [for example, Fab ′, scFv, V-domain proteins, etc.] or Affibody And aptamers. In the case of using an antibody, the antibody can be appropriately selected according to one having a sequence or structure that can be recognized by other substances, and is previously a substrate or a human or mouse, rabbit, rat, goat, sheep or the like. An antiserum obtained by immunizing the fragment, an affinity-purified polyclonal antibody, a monoclonal antibody, or the like can be used.

  A labeling partner that can be used in the method of the present invention and is a substance that can specifically bind to neoantigen produced by cleaving the substrate with ADAMTS13 has a property of recognizing it. If it is, it will not specifically limit, For example, an antibody or its fragment | piece (for example, Fab ', scFv, V-domain protein etc.) or an affibody, an aptamer etc. are mentioned. When using an antibody as a labeling partner, the antibody can be appropriately selected depending on the substrate used, and is obtained in advance by immunizing a substrate, or a fragment thereof, to a human, mouse, rabbit, rat, goat, sheep or the like. Antisera, affinity-purified polyclonal antibodies, monoclonal antibodies, and the like can be used.

  In the present invention, when labeling a partner, a labeling substance is bound to a site different from the site cleaved by ADAMTS13. A method for preparing a labeled substrate (preferably an enzyme-labeled substrate) or a labeling partner (preferably an enzyme-labeled partner substance, particularly an enzyme-labeled antibody) is well known, and any method can be selected.

As the labeling substance used in the present invention, various known labeling substances can be used. For example, enzymes (for example, alkaline phosphatase, horseradish peroxidase, β-galactosidase, urease, glucose oxidase, luciferase, etc.), fluorescent substances (for example, , Fluorescein derivatives, rhodamine derivatives, etc.), rare earth or rare earth complexes capable of time-resolved fluorescence measurement (eg, europium, europium complexes, etc.), chemiluminescent materials (eg, acridinium esters, etc.), radioisotopes (eg, ¹²⁵ I, ³ H, ¹⁴ C, ³² P, etc.), and a coloring substance [eg, paranitroaniline (pNA)] can be used. That is, methods for measuring color development, fluorescence, time-resolved fluorescence, chemiluminescence, electrochemiluminescence, radioactivity, and the like can be used.

The means for detecting the change in the signal intensity derived from the labeling substance is appropriately selected depending on the type of the labeling substance and the characteristics of the measuring instrument, and uses a known method such as a colorimetric method, a fluorescence method, or a luminescence method. Can do. For example, when peroxidase is used as a labeling substance, it can also be detected by chemiluminescence using luminol as a substrate, and is particularly suitable for micro-measurement (for example, Koichiro Tanaka, Junji Ishikawa: an enzyme labeled with horseradish peroxidase) Comparison of fluorescence method and luminescence method in immunoassay, clinical test equipment / reagents, 11: 567-569, 1988). In addition, when alkaline phosphatase is used as the labeling substance, AMPPD [3- (2′-spiroadamantane) -4-methoxy-4- (3 ”phosphoroxy) phenyl-1,2-dioxetane], a CDP— It is preferable to use a known substrate such as Star ^™ [1,2-dioxetane (Tropics)] because it can be detected with higher sensitivity.

  Hereinafter, the analysis method of the present invention will be described in more detail by showing specific measurement procedures for each embodiment.

A labeling partner capable of recognizing a sequence or structure that can be recognized by another substance on the substrate fragment side that is released by cleavage of ADAMTS13, which is an embodiment of the analysis method of the present invention Is not limited to the following steps, for example,
(A1) A cation capable of interacting with a substrate by a test sample possibly containing ADAMTS13 and a substrate having a terminal sequence or structure that can be recognized by other substances to form a complex A step of contacting in a liquid in the presence of a water-soluble polysaccharide,
(A2) mixing the liquid, the insoluble carrier and the labeling partner, further separating the insoluble carrier and the other liquid, and (A3) a substrate that is captured by the insoluble carrier without being cleaved, and / or The step of analyzing the substrate fragments in the liquid that has been cleaved and released without being captured by the insoluble carrier can be included.
The insoluble carrier used in the step (A2) carries a partner that specifically recognizes the end of the substrate. Further, the labeling partner used in the step (A2) is obtained by labeling a partner capable of specifically recognizing the other end of the substrate.

  The substrate used in the step (A1) is a substrate having a sequence or structure that can be recognized by other substances at the terminal. This substrate has a specific cleavage site of ADAMTS13, and has a sequence or structure that can be recognized by other substances on both ends of the substrate, which is clearly different from this cleavage site. In the step (A1), when the cationic water-soluble polysaccharide capable of interacting with the substrate to form a complex, the substrate, and the test sample are contacted at the same time, ADAMTS13 is usually released as a single substrate. The substrate can be cleaved at the specific cleavage site more efficiently than when it is present. In addition, the cationic water-soluble polysaccharide capable of interacting with the substrate and forming a complex and the substrate are mixed in advance, and a complex of the cationic water-soluble polysaccharide and the substrate is formed, When a test sample containing ADAMTS13 is brought into contact with this, the substrate can be cleaved more efficiently at the specific cleavage site. The contact time between ADAMTS13 and the substrate throughout this step is not particularly limited, but in the method of the present invention, it is desirable to add a cationic water-soluble polysaccharide that can interact with the substrate and form a complex. In addition to having reactivity (amount corresponding to several percent of the amount of ADAMTS13 present in the blood of healthy individuals), it is very short, for example, within 20 minutes, preferably from 3 minutes to 15 Enables measurement with minute contact time.

  In the step (A2), after performing the step (A1), the substrate fragment cleaved by the enzyme activity of ADAMTS13 and the substrate that has not been cleaved each have at least a sequence or structure that can be recognized by other substances. Since it has an end on one side, it can bind to an insoluble carrier carrying a partner that specifically recognizes the end. Since the uncleaved substrate bound to the insoluble carrier has a recognizable end for the labeling partner, the labeling partner is further bound. On the other hand, the cleaved substrate fragment bound to the insoluble carrier does not have a recognizable end for the labeled partner, and therefore the labeled partner is not bound. The other fraction (ie, the liquid fraction containing the free substrate fragment and the test sample) is separated in step (A2). This separation operation can be performed by a conventional method. For example, when magnetic particles are used as the insoluble carrier, the separation can be performed, for example, by using a magnet or by a centrifugal operation. When latex particles are used as the insoluble carrier, they can be separated by, for example, centrifugation or filtration. When analyzing the labeling substance contained in the insoluble carrier in the next step (A3), it is preferable to sufficiently remove the free substrate fragment by washing the insoluble carrier after the separation.

In the step (A3), a substrate that is captured by an insoluble carrier without being cleaved, or a substrate fragment in a solution that has been cleaved and released without being captured by an insoluble carrier, or both can be analyzed.
When analyzing a substrate fragment in a solution released after being cleaved and not captured by an insoluble carrier, the amount of the labeling substance contained in the labeling partner capable of recognizing the free substrate fragment is measured. The amount of fragments can be determined, and furthermore, the amount or activity of ADAMTS13 contained in the test sample can be determined, for example, by preparing a calibration curve. When ADAMTS13 is contained in the test sample, the higher the amount or activity of the enzyme, the more labeled substance brought into the analysis.
On the other hand, when analyzing a substrate that is captured without being cleaved by an insoluble carrier, it is possible to determine the amount of the substrate that has not been cleaved by measuring the amount of the labeling substance contained in the bound labeling partner. Furthermore, for example, the amount or activity of ADAMTS13 contained in the test sample can be determined by preparing a calibration curve. When ADAMTS13 is contained in the test sample, the higher the amount or activity of the enzyme, the less the labeled substance brought into the analysis.

In the embodiment in which the substrate is directly labeled with a labeling substance, which is an embodiment of the analysis method of the present invention, the substrate is not limited to the following steps.
(B1) A test sample possibly containing ADAMTS13 and a labeled substrate are interacted with the substrate in the presence of a cationic water-soluble polysaccharide capable of forming a complex. Contacting,
(B2) a step of mixing the liquid and the insoluble carrier, and further separating the insoluble carrier and the other liquid; and (B3) a substrate that is captured by the insoluble carrier without being cleaved, and / or subjected to cleavage. A step of analyzing a substrate fragment in a solution released without being captured by an insoluble carrier.
The insoluble carrier used in the step (B2) carries a partner that specifically recognizes the end opposite to the labeled end of the substrate.

  The substrate used in the step (B1) is a substrate whose end is labeled with a labeling substance. This substrate has a specific cleavage site of ADAMTS13, one of which is labeled with a labeling substance and the other has a sequence or structure that can be recognized by another substance. In the step (B1), when the cationic water-soluble polysaccharide capable of interacting with the substrate and forming a complex, the substrate and the test sample are contacted at the same time, ADAMTS13 is usually released as a single substrate. The substrate can be cleaved at the specific cleavage site more efficiently than when it is present. The cationic water-soluble polysaccharide capable of interacting with the substrate to form a complex and the substrate are mixed in advance to form a complex of the cationic water-soluble polysaccharide and the substrate. When the test sample containing ADAMTS13 is brought into contact with, the substrate can be cleaved more efficiently at the specific cleavage site. The contact time between ADAMTS13 and the substrate throughout this step is not particularly limited, but in the method of the present invention, it is desirable to add a cationic water-soluble polysaccharide that can interact with the substrate and form a complex. In addition to having reactivity (amount corresponding to several percent of the amount of ADAMTS13 present in the blood of healthy persons), it is very short, for example, within 20 minutes, preferably from 3 minutes to 15 Enables measurement with minute contact time.

  In the step (B2), after performing the step (B1), each of the substrate fragment cleaved by the enzyme activity of ADAMTS13 and the substrate that has not been cleaved has at least a sequence or structure that can be recognized by other substances. Since it has at one end (namely, the end opposite to the labeled end), it can bind to an insoluble carrier carrying a partner that specifically recognizes the end. Further, the substrate bound to the insoluble carrier without being cleaved is labeled with a labeling substance at one end. The other fraction (that is, the liquid fraction containing the free substrate fragment and the test sample) is separated in step (B2). This separation operation can be performed by a conventional method. For example, when magnetic particles are used as the insoluble carrier, the separation can be performed, for example, by using a magnet or by a centrifugal operation. When latex particles are used as the insoluble carrier, they can be separated by, for example, centrifugation or filtration. When analyzing the labeling substance contained in the insoluble carrier in the next step (B3), it is preferable to sufficiently remove the free substrate fragment by washing the insoluble carrier after the separation.

In the step (B3), a substrate that is captured by an insoluble carrier without being cleaved, or a substrate fragment in a solution that has been cleaved and released without being captured by an insoluble carrier, or both can be analyzed.
When analyzing substrate fragments in a solution released after being cleaved and not captured by an insoluble carrier, determine the amount of free substrate fragments by measuring the amount of labeling substance contained in the free substrate fragments. Furthermore, for example, by creating a calibration curve, the amount or activity of ADAMTS13 contained in the test sample can be determined. When ADAMTS13 is contained in the test sample, the higher the amount or activity of the enzyme, the more labeled substance brought into the analysis.
On the other hand, when analyzing a substrate that is captured without being cleaved by an insoluble carrier, the amount of the substrate that has not been cleaved can be determined by measuring the amount of the labeled substance labeled at one end, Furthermore, for example, by creating a calibration curve, the amount or activity of ADAMTS13 contained in the test sample can be determined. When ADAMTS13 is contained in the test sample, the higher the amount or activity of the enzyme, the less the labeled substance brought into the analysis.

In an embodiment of the analysis method of the present invention, an embodiment using a labeling partner that specifically binds to a substrate neoantigen cleaved by ADAMTS13 and is labeled with a labeling substance is limited to the following steps. For example,
(C1) a cation capable of forming a complex by interacting with a substrate a test sample that may contain ADAMTS13 and a substrate having a sequence or structure that can be recognized by another substance at the terminal. A step of contacting in a liquid in the presence of a water-soluble polysaccharide,
(C2) a step of mixing the liquid, the insoluble carrier and the labeling partner, and further separating the insoluble carrier and the other liquid; and (C3) a substrate that is captured by the insoluble carrier without being cleaved, and / or Alternatively, it may include a step of analyzing a substrate fragment in a solution that has been cleaved and released without being captured by an insoluble carrier. The insoluble carrier used in the step (C2) carries a partner that specifically recognizes the end opposite to the neoantigen in the cleaved substrate fragment.

  In the present specification, “neoantigen” means a sequence or structure newly generated when a substrate is cleaved by a degrading enzyme, and is appropriately performed on the N-terminal side or C-terminal side of the cleaved amino acid sequence, or both. Is possible.

  For example, in the step (C2), the labeling partner specifically binds to the neoantigen produced when the substrate is cleaved by ADAMTS13, so it does not bind to the substrate before enzymatic cleavage, and the ADAMTS13 itself enzyme reaction is labeled. It progresses regardless of the presence or absence of a partner. Here, if it has a sequence or structure that can be recognized by other substances on the opposite side of the cleavage site and a substance that can recognize this is bound to an insoluble carrier, the substrate fragment and the labeling partner A complex (preferably an immune complex) with (preferably a labeled antibody) can be captured on an insoluble carrier.

  In the step (C3) in this embodiment, the amount of the cleaved substrate can be determined by measuring the amount of the labeling substance of the labeling partner specifically bound to the cleaved substrate fragment captured by the insoluble carrier. Furthermore, for example, by creating a calibration curve, the amount or activity of the enzyme to be analyzed contained in the test sample can be determined. When ADAMTS13 is contained in the test sample, the higher the amount or activity of the enzyme, the more labeled substance brought into the analysis.

The analysis kit of the present invention comprises at least a substrate that can be cleaved by von Willebrand factor-degrading enzyme and a cationic water-soluble polysaccharide that can interact with the substrate to form a complex, and a labeling substance and a detection system For example, a substrate, a washing solution, a specimen dilution solution, and / or a reaction amplification agent can be further included. For example, the first reagent can contain a substrate, the second reagent can immobilize the substrate, the third reagent can contain a labeling partner, and the fourth reagent can contain a substance that expresses the signal of the labeling substance. Specifically, for example,
First reagent: a solution containing a substrate and a water-soluble polymer Second reagent: a magnetic particle with a surface treatment that can capture the substrate, etc. Third reagent: a label that can specifically recognize a cleaved fragment or recognize a substrate Antibody etc. (for example, label is alkaline phosphatase)
Fourth reagent: chemiluminescent substrate (eg, AMPPD)
The composition of the reagent can be exemplified, and the composition can be changed as appropriate depending on the labeling substance and the detection system, and various structures can be obtained.

  Mixing after the addition of each reagent needs to be performed sufficiently, but after mixing uniformly, the mixing may be stopped and allowed to react. The reaction is usually carried out at pH 5-10, preferably pH 6-8. About temperature, although it can implement normally in the range of 2-50 degreeC, it is made to react desirably at room temperature or 30-45 degreeC. The reaction time is arbitrary from immediately after the reaction to 1 day and night, but in the present invention, by adding a water-soluble polymer, the desired reactivity (amount corresponding to several% of the amount of ADAMTS13 present in the blood of healthy humans). Can be detected (within 20 minutes as the contact time between the substrate and the test sample, and within 30 minutes as the time required for all measurements).

  In order to maintain the target pH, a buffer solution is usually used. As the buffer, for example, Tris, MES (2-Morpholinoethansulphonic Acid), phosphoric acid and the like are used, but most buffers that are commonly used from weakly acidic to neutral can be used. In many cases, salts (such as sodium chloride) and proteins (such as bovine serum albumin) are added to avoid non-specific reactions. In addition, a drug containing a divalent metal ion (for example, calcium, barium, etc.) essential for the expression of the enzyme activity of ADAMTS13 and further inhibiting an enzyme activity other than metalloprotease may be added. In addition to this, additives and the like can be added for the purpose of sensitizing the immune reaction.

  Insoluble magnetic particles are usually 0.0001 to 1% by weight, preferably 0.001 to 0.3% by weight, based on the reaction solution, and enzyme-labeled antibodies and the like are usually 0.00001 to 10 mAbs, preferably It is used so that it may become 0.01-1 mAbs.

EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.
<< Example 1: Effect of DEAE-dextran in measurement of ADAMTS13 enzyme activity by SDS-agarose gel electrophoresis >>
Normal human pooled plasma containing ADAMTS13 and dilution series thereof are mixed with an equal volume of Tris buffer (pH 7.4; containing 1.5 mol / L urea and 0.1 mol / L barium chloride), and further a final concentration of 2.4 mmol / L. 4- [2-Aminoethyl] -benzenesulfonyl fluoride hydrochloride (Pefabloc; Roche) was added so that The sample solution thus treated was treated with 3 μg / mL of vWF (purified from human plasma by the method described in Non-Patent Document 3) and DEAE-dextran [Diethylaminoethyl-Dextran (GE Healthcare Bioscience)] (final Mixed with Tris buffer (concentration 0.1% or 0.2%) (pH 7.4, 1.5 mol / L urea) at a volume ratio of 1: 5, incubated at 37 ° C. for 2 hours, and vWF was added to the sample solution It decomposed | disassembled with ADAMTS13 in the inside. The DEAE-dextran used in this example has an average molecular weight of 500,000 and an N content of about 3.2%. From this value, a substituent that exhibits a cationic property for approximately three glucoses. Is equivalent to one introduced. The decomposition reaction was stopped by adding EDTA to a final concentration of 40 mmol / L. The sample thus prepared was separated by SDS-agarose gel electrophoresis (1.4% agarose gel) under non-reducing conditions, and then transferred to a PVDF (polyvinylidene difluoride) membrane by Western blotting. After blocking with a commercially available blocking agent (Block Ace; Dainippon Pharmaceutical) at room temperature, it was washed with Tris buffer (pH 7.4). After reacting for 1 hour at room temperature with an HRP (horseradish peroxidase) -labeled anti-vWF antibody (DAKO) diluted 1/1000 in Tris buffer (pH 7.4) / 10% Block Ace, Tris buffer (pH 7.4) ), And the color was developed with a commercially available color development kit (Immunostein HRP-1000; Konica).

  The result of electrophoresis is shown in FIG. The NHP (Normal human plasma) and the numerical values in each lane shown in FIG. 1 are the contents of normal human pool plasma and the pool plasma included in the dilution series when normal human pool plasma is 1 (= 100%). It is a ratio. The vWF is decomposed by ADAMTS13 in the sample, and the length of the vWF band according to the multimer size is detected. Here, in the case where DEAE-dextran was added (0.1% and 0.2%), vWF multimer was significantly degraded as compared with the case where this was not added (control; symbol “ctrl”), and the vWF It became clear that the length of the band became extremely short.

Example 2: Analysis of interaction between DEAE-dextran and vWF
The interaction between vWF and DEAE-dextran used in Example 1 was observed and the dissociation constant was measured. In the measurement, BIACORE J (Biacore) was used.

The vWF was immobilized on the sensor chip CM5 (Biacore). In the immobilization method, the carboxyl group in CM dextran is activated with a mixed solution of NHS (N-hydroxysuccinimide) / EDC [1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride] and bonded to the amino group in vWF. The amine coupling method was used. Here, the amount of vWF immobilized on the chip was about 1000 RU (Resonance Unit) = 1 ng / mm ² .

Next, interaction with DEAE-dextran was measured using a vWF-immobilized chip. As a running buffer used for the measurement, one containing 5 mmol / L Bis-Tris (pH 6) / 20 mmol / L CaCl ₂ /0.01% Tween 20 was used. DEAE-dextran was dissolved in this running buffer so as to have a concentration of several to several tens of μg / mL, and the interaction between vWF and DEAE-dextran was measured at a flow rate of 30 μL / min. The interaction results of DEAE-dextran and vWF are shown in FIG. The binding phase is 0 to 180 sec, and the dissociation phase is 180 to 360 sec. The concentration dependency of DEAE-dextran to be interacted here was obtained, and the interaction curve obtained from this was subjected to linear or nonlinear analysis using an analysis program BIAevaluation version 3.0 (Biacore). As a result, the calculated dissociation constant of DEAE-dextran and vWF was 3.21 × 10 ⁻⁹ mol / L.

  From this dissociation constant, it was found that the substrate and DEAE-dextran can form a complex under the conditions in which the substrate and DEAE-dextran coexist in Example 1 or Example 3. From this result, it is presumed that the cleavage enhancing effect obtained when DEAE-dextran is added forms a form that is easily cleaved by ADAMTS13 when the substrate and DEAE-dextran form a complex.

Example 3: Automated measurement of ADAMTS13 activity in the presence of DEAE-dextran
(1) Preparation of Substrate As a substrate of ADAMTS13, a partial fragment of vWF (fragment consisting of amino acids 1596 to 1668, hereinafter referred to as vWF73) was designated as Koichi Kokame, Masanori Matsumoto, Yoshihiro Fujimura, and Toshiyuki Miyata, VWF73, a region from D1596 to R1668 of von Willebrand factor, provides a minimal substrate for ADAMTS-13. Blood, Jan 2004; 103: 607-612. Here, the one used in the actual assay was a 73-amino acid N-terminal glutathione S-transferase (hereinafter referred to as GST) and a C-terminal introduced 6XHis (hereinafter referred to as GST-vWF73-H). Using.

(2) Binding of antibody to magnetic particles A 1% solution of magnetic latex (manufactured by JSR) having a particle size of 2.4 μm and an anti-GST antibody (GE Healthcare Biosciences) (about 200 μg) were mixed with 50 mmol / L MES. React in buffer solution (pH 6) at room temperature for 1 hour, wash with the buffer solution, block with 0.3% bovine albumin / 0.1 mol / L Tris buffer solution (pH 8), and anti-GST antibody binding magnetism Particles were prepared.

(3) Measurement of ADAMTS13 activity Measurement was performed using a small automatic immunoassay device (PATHHFAST; manufactured by Mitsubishi Chemical Yatron Corp.). Specifically, 60 μL of diluted sample of healthy human plasma and GST-vWF73-H were added with 1 mAbs and DEAE-dextran (0.05%, 0.1%, 0.2%, 0.4%) and protease inhibitor cocktail (Complete Buffer solution containing a suitable amount of Mini EDTA-free (Roche) [5 mmol / L Bis-Tris (pH 6) / 20 mmol / L CaCl ₂ /0.01% Tween 20 / 0.5% bovine serum albumin (Sigma): reaction below (Referred to as buffer)] 50 μL was reacted at 37 ° C. for 3.3 minutes. Further, 60 μL of a solution in which 0.03% of the anti-GST antibody-bound magnetic particles prepared in (2) were suspended in the reaction buffer was added, and reacted at 37 ° C. for 2.5 minutes. To this was further added 60 μL of a HRP-labeled anti-6XHi antibody (Roche) diluted 1/500 in the reaction buffer, followed by reaction at 37 ° C. for 4 minutes. After washing magnetic particles with 0.1 mol / L Tris buffer (pH 8) /0.15 mol / L NaCL / 0.1% Triton X-100, a chemiluminescence substrate solution [ELISA Femto Maximum Sensitivity Substrate (PIERCE Solution) 100 μL was mixed, reacted at 37 ° C. for 2 minutes, and the amount of luminescence was counted. As a blank, healthy human plasma that was inactivated at 56 ° C. and inactivated ADAMTS13 activity was used.

  The results are shown in FIG. In this embodiment, the higher the amount or activity of the enzyme, the less the labeled substance brought into the analysis. Here, it was cleaved by taking the ratio (S / N) of the luminescence count (S) of the blank (immobilized plasma sample at 56 ° C.) and the luminescence count (N) when using the plasma sample. The amount of substrate, ie the degree of enzyme activity, was compared. When DEAE-dextran was added (especially 0.1% and 0.2%), an increase of 10 times or more was observed in the ratio compared to the case where it was not added (ctrl). This is because, as in the previous results, DEAE-dextran and GST-vWF73-H formed a complex that was easily cleaved by ADAMTS13 when a complex was formed, and GST-vWF73-H was efficiently cleaved. It seemed.

(4) Measurement of ADAMTS13 activity under various dextran addition conditions Measurement under the same conditions as the measurement of ADAMTS13 activity carried out in Example 3 (3) above, but under conditions containing dextran or dextran sulfate instead of DEAE-dextran Went. The results are shown in FIG. As shown in FIG. 4, when the ratio (S / N) of the luminescence count (S) of the blank and the luminescence count (N) when using the plasma sample was compared as described above, dextran and dextran sulfate were found to be the enzyme. There was no increase in activity. Further, in the measurement by BIACORE performed under the same conditions as in Example 2, it was not recognized that dextran and dextran sulfate interact with the vWF-immobilized chip.

(5) ADAMTS13 activity measurement (correlation with known methods)
In order to verify that the automated activity measurement in the presence of DEAE-dextran [compact automated immunoassay device (PATHHFAST; manufactured by Mitsubishi Chemical Yatron)] is specific as a measurement of ADAMTS13 activity. Clinical specimens with ADAMTS13 activity measurement values distributed in the range of 15 to 75% (with healthy human pool plasma as 100%) were measured by the above method, and the consistency with the values calculated by the existing method was compared. As an existing method for measuring ADAMTS13 activity, FRETS-vWF73 [Peptide Institute, Inc. (Non-patent Document 4)] was used.

  The results are shown in FIGS. In this embodiment, the higher the amount or activity of the enzyme, the less the labeled substance brought into the analysis. A standard curve for quantifying clinical specimens was prepared by preparing a dilution series of healthy human plasma and measuring this (FIG. 5). Using this standard curve, the luminescence count obtained by measuring the clinical specimen was recalculated to% display when the healthy human pool plasma was taken as 100%, and used as the ADAMTS13 activity measurement value of the clinical specimen. As a result, a very good correlation coefficient (r = 0.848) with the existing method was obtained, and what was measured by this method was considered to be specific as ADAMTS13 activity (FIG. 6 and Table 1).

  The analysis method of the present invention can be applied to clinical diagnostic tests.

It is a photograph instead of a drawing showing the result of electrophoresis of recombinant vWF degraded with ADAMTS13 in the presence or absence of DEAE-dextran. It is a graph which shows the result of the interaction of DEAE-dextran and vWF measured with the Biacore system. It is a graph which shows the result of having measured ADAMTS13 activity in presence of DEAE-dextran using the small automatic immunoassay apparatus (PATHHFAST; Mitsubishi Chemical Yatron make). It is a graph which shows the result of having compared DEAE-dextran with dextran or dextran sulfate about ADAMTS13 activity improvement effect. It is a graph which shows the standard curve created using the small automatic immunoassay apparatus (PATHHFAST; Mitsubishi Chemical Yatron company make). It is a graph which shows the correlation with the measurement result of a small-sized automatic immunoassay apparatus (PATHHFAST; Mitsubishi Chemical Yatron Co., Ltd.) and the measurement result by the existing method.

Claims (12)

  In the analysis method of von Willebrand factor degrading enzyme using a substrate cleavable by von Willebrand factor degrading enzyme, the contact between the test sample and the substrate in the liquid can interact with the substrate to form a complex. A method for analyzing von Willebrand factor-degrading enzyme, which is carried out in the presence of various cationic water-soluble polysaccharides.   The analysis method according to claim 1, wherein the cationic water-soluble polysaccharide is a polysaccharide having glucose as a structural unit.   The analysis according to claim 1, wherein the cationic water-soluble polysaccharide is a polysaccharide having glucose as a structural unit and having a tertiary amino group and / or a quaternary ammonium group as a functional group exhibiting cationic properties. Method.   The analysis method according to claim 1, wherein the cationic water-soluble polysaccharide is DEAE-dextran.   The analysis method according to claim 1, wherein the analysis is performed using a fully automatic analyzer.   The analysis according to any one of 1 to 5, wherein an insoluble carrier capable of capturing a substrate fragment generated by cleaving the substrate with a von Willebrand factorase and / or an uncleaved substrate is used. Method.   The analysis method according to claim 6, wherein the insoluble carrier is a particle.   The analysis method according to claim 7, wherein the particles are latex particles or magnetic particles.   9. The substrate according to any one of claims 6 to 8, wherein the substrate is labeled with a labeling substance on the side of a substrate fragment that is cleaved and released by von Willebrand factorase, or has an amino acid sequence that can be specifically recognized. The analysis method according to claim 1.   The analysis method according to claim 9, wherein the labeling substance is a fluorescent substance, a luminescent substance, a coloring substance, or an enzyme.   The analysis method according to any one of claims 6 to 8, wherein a partner that specifically binds to a neoantigen generated when the substrate is cleaved by a von Willebrand factorase is used.   A von Willebrand factor comprising: (1) a substrate cleavable by a von Willebrand factor degrading enzyme; and (2) a cationic water-soluble polysaccharide capable of interacting with the substrate to form a complex. Degradation enzyme analysis kit.

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