Von Willebrand factor lyase 13 activity detection kit and preparation and application thereof

Abstract

The invention relates to a von willebrand factor lyase 13 activity detection kit and preparation and application thereof. The detection method is a magnetic particle chemiluminescence immunoassay method, and the kit comprises a reagent M, a reagent R1 and a reagent R2. The reagent M is a magnetic particle coated with an antibody and is combined with one end of the VWF substrate protein in the reaction, and the coated antibody is an anti-MBP tag antibody; the reagent R1 contains a VWF73 substrate protein, one end of the protein contains an MBP label, the other end of the protein contains a His label, and the protein can be respectively combined with antibodies with the two labels to form a sandwich compound; the reagent R2 is an enzyme-labeled antibody reagent, and an enzyme-labeled antibody is an anti-His label antibody. According to the present disclosure, high-speed and high-sensitivity detection is achieved, and the diagnosis and monitoring of thrombotic thrombocytopenic purpura (TTP) are facilitated.

Description

Technical Field

[0001] The present disclosure relates to a von Willebrand factor cleaving enzyme 13 activity detection kit based on a magnetic particle chemiluminescence immunoassay, and belongs to the fields of biomedicine and detection technology. Background Art

[0002] Von Willebrand factor cleaving enzyme (ADAMTS13), also known as VWFCP, is a zinc-containing metalloproteinase.

[0003] Under physiological conditions, ADAMTS13 is in a potentially closed conformation. When blood vessels are subjected to shear forces, von Willebrand factor (VWF) unwinds and exposes its A1 domain. Subsequently, the A2 domain of VWF is elongated, exposing the ADAMTS13 binding site and cleavage site Tyr1605-Met1606. When ADAMTS13 binds to VWF, the conformation changes, causing the activity of ADAMTS13 to be activated, thereby cleaving VWF. This cleavage can maintain the stability of blood circulation. If the activity of ADAMTS13 is lost or the concentration is reduced, it leads to the formation of microthrombi in blood vessels. This situation is mainly related to some thrombotic diseases such as thrombotic thrombocytopenic purpura (TTP).

[0004] ADAMTS13 kits in the prior art include:

[0005] 1. Enzyme-linked immunosorbent assay (ELISA), such as Technoclone (Technozym ADAMTS13 antigen), Invitrogen (Human ADAMTS13 ELISA Kit), etc. The double antibody sandwich method is used to capture the antigen in the sample by coating the anti-ADAMTS13 antibody, and then adding the enzyme-labeled secondary antibody and the colorimetric substrate, and finally quantifying the antigen concentration by absorbance (450nm). Advantages: relatively simple operation, suitable for large-scale screening, and low cost. Disadvantages: The detection time is long (usually 4-5 hours), and it is easily interfered by rheumatoid factors, anti-mouse antibodies, etc. in the sample, resulting in false positives. In addition, the ELISA method cannot distinguish between active enzymes and inactive enzymes, and may overestimate the actual functional level.

[0006] 2. Fluorescence resonance energy transfer (FRET) method, such as 520 ADAMTS13 Activity Assay Kit. Using a fluorescently labeled VWF73 peptide as a substrate, ADAMTS13 cleavage releases a fluorescent signal whose intensity is proportional to enzyme activity. Advantages: High sensitivity (detection limit up to 0.31 ng / mL) and short operation time (approximately 2 hours). Disadvantages: Avoid hemolysis of the sample due to interference from hemoglobin, bilirubin, and other substances in the sample.

[0007] In view of this, there is a need in the art for a chemiluminescent immunoassay kit for detecting ADAMTS13 activity with excellent performance in terms of accuracy, precision, and stability. Summary of the Invention

[0008] In view of the above needs, a von Willebrand factor cleaving enzyme (ADAMTS13) detection kit is provided, which comprises: a first reagent, a second reagent and a third reagent.

[0009] The components of the kit are divided into the first reagent, the second reagent and the third reagent, which is only used to clearly define the different component combinations and their technical characteristics. There is no limitation on the order of use, quantity or priority between them.

[0010] In some embodiments, the first reagent comprises:

[0011] 0.1mg / ml to 1.0mg / ml magnetic beads coated with the primary antibody,

[0012] 10 mM to 100 mM first buffer, pH 6.0 to 8.0,

[0013] 10mM to 300mM sodium chloride,

[0014] 1mM to 50mM calcium chloride,

[0015] 10mg / ml to 200mg / ml trehalose,

[0016] 0.01% to 0.5%(w / v)Tween 20,

[0017] 0.05% to 5% (w / v) BSA,

[0018] 0mg / ml to 1.0mg / ml blocking agent,

[0019] 0% to 1.0% (w / v) preservative.

[0020] In some embodiments, for "magnetic beads coated with a first antibody", the magnetic beads serve as a carrier, and their surface properties are specifically modified (such as with carboxyl, streptavidin, or p-toluenesulfonyl modification, preferably carboxyl modification) so that they can bind to the first antibody. The first antibody can specifically bind to the first tag on the target substance (such as GST, MBP, FLAG, His, etc., preferably MBP, His). During the preparation process, the first antibody is fixed to the surface of the magnetic beads through a chemical coupling reaction (such as using carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the carboxyl group and react with the antibody amino group for carboxyl-modified magnetic beads), so that the magnetic beads have the ability to recognize and capture the target substance.

[0021] In some embodiments, the second reagent comprises:

[0022] 200ng / ml to 1000ng / ml VWF73 short peptide,

[0023] 10 mM to 100 mM second buffer, pH 6.0 to 8.0,

[0024] 10mM to 300mM sodium chloride,

[0025] 0.05% to 5% (w / v) casein,

[0026] 0.05% to 5% (w / v) BSA,

[0027] 0% to 1.0% (w / v) preservative.

[0028] In some embodiments, the third reagent comprises:

[0029] 0.5 μg / ml to 2.5 μg / ml (preferably 1 μg / ml to 2 μg / ml) enzyme-labeled secondary antibody,

[0030] 10 mM to 100 mM third buffer, pH 6.0 to 8.0,

[0031] 10mM to 300mM sodium chloride,

[0032] 1mM to 100mM magnesium chloride,

[0033] 1mM to 100mM zinc chloride,

[0034] 0.01% to 0.5%(w / v)Tween 20,

[0035] 0.05% to 5% (w / v) BSA,

[0036] 0% to 10% (w / v) protective agent,

[0037] 0% to 1.0% (w / v) preservative.

[0038] In some embodiments, the first antibody specifically binds to a first tag; and the second antibody specifically binds to a second tag.

[0039] In some embodiments, the first tag and the second tag are each independently selected from the group consisting of: GST, MBP, FLAG, His; preferably MBP, His.

[0040] In some embodiments, the first tag is different from the second tag.

[0041] In some embodiments, the first label and the second label are interchangeable.

[0042] The VWF73 peptide is naturally a 73-amino acid fragment whose amino acid sequence corresponds to the region from D1596 to R1668 of von Willebrand factor. The VWF73 peptide is bound to a first tag at the N-terminus and a second tag at the C-terminus. This unique tag design, as disclosed herein, provides the basis for its specific recognition and binding during detection. The first tag differs from the second tag, and the two are theoretically interchangeable at the N-terminus and C-terminus, providing greater flexibility for VWF73 peptides in diverse experimental requirements and detection systems. In a more preferred embodiment, the applicant unexpectedly discovered that placing one tag only at the N-terminus and the other only at the C-terminus is more advantageous; without being limited by theory, this may be due to potential steric hindrance or antibody recognition. For example, when MBP is selected as the first tag at the N-terminus and His is selected as the second tag at the C-terminus, the VWF73 peptide can be represented as MBP-VWF73-His, in order from N-terminus to C-terminus. For preparation, the synthesis of VWF73 short peptides is usually carried out using solid phase peptide synthesis.

[0043] In some embodiments, the VWF73 short peptide is selected from the group consisting of GST-VWF73-His, MBP-VWF73-His, FLAG-VWF73-His, GST-VWF73-FLAG, MBP-VWF73-FLAG, and GST-VWF73-MBP in order from N-terminus to C-terminus.

[0044] In some embodiments, the first buffer, the second buffer, and the third buffer are each independently selected from the group consisting of: phosphate buffer, carbonate buffer, and Tris buffer.

[0045] In some embodiments, the blocker is selected from the group consisting of: MARK33, HBR, TRUBLOCK, CHEMIBLOCK, and HIER. MARK33 is a commonly used immunoassay blocker that binds to sites in samples and reagents that may trigger nonspecific binding (such as free sites on the surface of magnetic beads with unbound antibodies, impurity proteins in the sample, etc.), occupies these sites, and prevents subsequent erroneous binding of non-target substances. HBR can quickly cover various surfaces (including magnetic beads, container walls, etc.) that may produce non-specific adsorption in the reaction system. Its mechanism of action relies on its own chemical structure to form a dense and stable barrier layer, making it difficult for non-target proteins and molecules to attach, thereby reducing background noise and ensuring that the specific recognition and binding of the first antibody to the target is not interfered with. TRUBLOCK is a specific blocking reagent designed to address the common non-specific binding problems in immunoassays. It can accurately identify and bind to cross-reacting substances in the sample, free active groups in the reagent, etc., and "shield" these interference sources by specifically binding to them, allowing the first antibody-magnetic bead complex to bind only to the target tag (such as the N-terminal tag of the VWF73 short peptide). CHEMIBLOCK interacts with the various components in the reaction system (magnetic beads, buffer salts, proteins, etc.) to form a dynamically balanced blocking network. On the one hand, it can fill the non-specific sites on the surface of the magnetic beads and in the solution that may adsorb the target or antibody; on the other hand, it can adjust the microenvironment of the reaction system to keep the first antibody in a suitable conformation. HIER is usually used for antigen repair in pre-treatment of immunoassays, but in the first reagent of this kit, it can block non-specific binding by changing the microscopic state of the antigen in the sample (if there are related interfering antigens) and the reagent components.

[0046] In some embodiments, the enzyme is selected from the group consisting of: alkaline phosphatase, horseradish peroxidase.

[0047] In some embodiments, the protective agent is selected from the group consisting of: sucrose, trehalose, dextran, and dextran sulfate.

[0048] In some embodiments, the preservative is selected from the group consisting of sodium azide (such as PC300), isothiazolinone, and gentamicin.

[0049] In some embodiments, the alkaline phosphatase is a high-sugar alkaline phosphatase or a low-sugar alkaline phosphatase. The high-sugar alkaline phosphatase has a high degree of glycosylation and has more sugar residues connected in its molecular structure. These sugar chains change the spatial conformation and surface charge distribution of the enzyme, giving it unique solubility and stability in solution. In a third reagent buffer system containing multiple ions and proteins (such as 20mM tris hydroxymethylaminomethane buffer, containing sodium chloride, magnesium chloride, zinc chloride, etc.), high sugar chains can enhance the hydration of the enzyme molecules with the surrounding environment, allowing the enzyme to disperse more stably and reduce aggregation and inactivation caused by intermolecular interactions. Low-sugar alkaline phosphatase has a low degree of glycosylation and relatively fewer sugar chain modifications, which exposes the active center of the enzyme more fully, making it easier for the substrate to contact the active center during the catalytic reaction. However, its ability to resist the influence of environmental factors (such as changes in ionic strength and protein interference) in complex buffer systems is slightly weaker than that of high-sugar alkaline phosphatase.

[0050] In some embodiments, the surface of the magnetic beads is modified with carboxyl groups, streptavidin, or p-toluenesulfonyl groups.

[0051] In some embodiments, the magnetic beads have carboxyl groups on their surfaces.

[0052] In some embodiments, the average particle size of the magnetic beads is 1.0 μm, 1.6 μm, or 3.0 μm.

[0053] In some embodiments, the average particle size of the magnetic beads is 1.6 μm.

[0054] In some embodiments, the ADAMTS13 detection kit of the present disclosure further comprises: a calibrator, and / or a quality control substance.

[0055] In some embodiments, the calibrator or the control comprises:

[0056] One or more ADAMTS13 of known concentration,

[0057] 10 mM to 100 mM buffer, pH 6.0 to 8.0;

[0058] 10 mM to 500 mM sodium chloride, preferably 50 mM sodium chloride;

[0059] 1 mM to 100 mM calcium chloride, preferably 10 mM calcium chloride;

[0060] 0.05% to 5% (w / v) BSA, preferably 1.0% (w / v) BSA;

[0061] 0% to 1.0% (w / v) preservative, preferably 0.1% (w / v) PC300;

[0062] Preferably, the buffer is 20 mM Tris pH 7.2.

[0063] In some embodiments, in the first reagent, the concentration of the first antibody relative to the magnetic beads is 5 μg / mg to 40 μg / mg.

[0064] In some embodiments, in the third reagent, the mass ratio of the enzyme to the second antibody is 1:2.

[0065] In a specific embodiment, the ADAMTS13 detection kit of the present disclosure, wherein:

[0066] The first reagent comprises or consists of:

[0067] 0.3mg / ml magnetic beads coated with anti-MBP antibody,

[0068] 50 mM Tris buffer, pH 6.5,

[0069] 300mM sodium chloride,

[0070] 10mM calcium chloride,

[0071] 100mg / ml trehalose,

[0072] 0.05% (w / v) Tween 20,

[0073] 0.5% (w / v) BSA,

[0074] 0.05mg / ml MARK33,

[0075] 0.1% (w / v) PC300;

[0076] Wherein, the second reagent comprises or consists of the following:

[0077] 200ng / ml VWF73 short peptide with MBP tag at N-terminus and 6×His tag at C-terminus,

[0078] 20 mM Tris buffer, pH 7.0,

[0079] 150mM sodium chloride,

[0080] 0.5% (w / v) casein,

[0081] 0.5% (w / v) BSA,

[0082] 0.1% (w / v) PC300;

[0083] Wherein, the third reagent comprises or consists of the following:

[0084] 1.0 μg / ml alkaline phosphatase-labeled anti-His antibody,

[0085] 20 mM Tris buffer, pH 7.2,

[0086] 150mM sodium chloride,

[0087] 1mM magnesium chloride,

[0088] 10mM zinc chloride,

[0089] 0.05% (w / v) Tween 20,

[0090] 0.5% (w / v) BSA,

[0091] 0.2% (w / v) dextran sulfate,

[0092] 0.1% (w / v) PC300.

[0093] The present disclosure also provides a method for using the aforementioned ADAMTS13 detection kit, comprising the steps of:

[0094] 1) Provide samples;

[0095] 2) contacting the sample with the first reagent and the second reagent at 37° C. for 5 to 20 minutes (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably 8 to 15 minutes) to obtain an incubation;

[0096] 3) contacting the incubated substance with the third reagent at 37° C. for 3 to 10 minutes (e.g., 3, 4, 5, 6, 7, 8, 9, 10; preferably 3 to 6 minutes) to obtain a complex;

[0097] 4) recovering the complex by magnetic separation and washing;

[0098] 5) A chemiluminescent substrate (substrate of alkaline phosphatase) is added to the complex, and the light signal is measured.

[0099] The methods of use disclosed herein are not directed to the diagnosis or treatment of disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 .Schematic diagram of the reaction process.

[0101] Figure 2 : Reactivity at different dilutions.

[0102] Figure 3 : Linear. DETAILED DESCRIPTION

[0103] The following describes in detail the embodiments of the present disclosure, but it should be understood that the embodiments are only for illustration and explanation, and should not be understood as limiting the present disclosure.

[0104] Example 1. Preparation of ADAMTS13 activity detection kit

[0105] 1. Reagent M (first reagent), comprising:

[0106] 0.3mg / ml magnetic beads coated with anti-MBP antibody,

[0107] 50 mM Tris buffer (pH = 6.5),

[0108] 300mM sodium chloride,

[0109] 10mM calcium chloride,

[0110] 100mg / ml trehalose,

[0111] 0.05% (w / v) Tween 20,

[0112] 0.5% (w / v) BSA,

[0113] 0.05mg / ml MARK33,

[0114] 0.1% (w / v) PC300.

[0115] 2. Reagent R1 (second reagent), comprising:

[0116] 200ng / ml VWF73 short peptide (MBP tag at N-terminus and 6×His tag at C-terminus),

[0117] 20 mM Tris buffer (pH = 7.0),

[0118] 150mM sodium chloride,

[0119] 0.5% (w / v) casein,

[0120] 0.5% (w / v) BSA,

[0121] 0.1% (w / v) PC300.

[0122] 3. Reagent R2 (third reagent), comprising:

[0123] 1.0 μg / ml alkaline phosphatase-labeled anti-His antibody

[0124] 20 mM Tris buffer (pH = 7.2),

[0125] 150mM sodium chloride,

[0126] 1mM magnesium chloride,

[0127] 10mM zinc chloride,

[0128] 0.05% (w / v) Tween 20,

[0129] 0.5% (w / v) BSA,

[0130] 0.2% (w / v) dextran sulfate,

[0131] 0.1% (w / v) PC300.

[0132] 4. Calibrators and quality control products (optional)

[0133] Commercially available calibrators and quality control products can be used, as well as homemade calibrators and quality control products. In this embodiment, the calibrators and quality control products are diluted from international standards, and their values ​​can be traced back to the international standard WHO 1st International Standard ADAMTS13 Plasma NIBSC code: 12 / 252.

[0134] The activity range of calibrator 1 point is 5%-25%, and the activity range of calibrator 2 point is 70%-90%;

[0135] The activity range of quality control product 1 point is 5%-25%, and the activity range of quality control product 2 point is 35%-55%.

[0136] Buffers in calibrators and controls include:

[0137] 20 mM Tris buffer (pH = 7.2),

[0138] 50mM sodium chloride,

[0139] 10mM calcium chloride,

[0140] 1% (w / v) BSA,

[0141] 0.1% (w / v) PC300.

[0142] 5. Detection principle of the kit:

[0143] The sample reacts with reagent M and reagent R1. The ADAMTS13 in the sample will cleave the tagged VWF73 short peptide, and the cleaved VWF73 short peptide will expose the cleavage site. Reagent M carries an anti-MBP antibody, so it can capture the MBP-VWF73 short peptide. After 10 minutes of incubation, reagent R2 is added. The enzyme-labeled antibody in R2 specifically recognizes VWF73-His and can form a "sandwich" complex of magnetic beads-antibody-VWF73-enzyme-labeled antibody with the uncut MBP-VWF73-His. After 5 minutes of incubation, the unreacted substances can be removed by washing. Add chemiluminescent substrate, collect photon signals through the enzymatic reaction of alkaline phosphatase on the substrate, and calculate the ADAMTS13 activity in the sample from the standard curve. The chemiluminescent reaction is measured in relative light units (RLUs). RLU is negatively correlated with the ADAMTS13 activity in the sample ( Figure 1 ).

[0144] Test Example 1. Comparison of substrates MBP-VWF73-His and GST-VWF73-His

[0145] The performance of two different tag combinations, MBP-VWF73-His and GST-VWF73-His, was compared. The reactivity of serially diluted samples was tested, and the results are shown below.

[0146] Table 1

[0147]

[0148] Using MBP-VWF73-His as the substrate of ADAMTS13 has a higher signal value and a better reaction gradient ( Figure 2 ).

[0149] Test Example 2: One-step delayed detection method

[0150] The instrument used in this test example is a Gi2000 fully automatic chemiluminescence immunoassay analyzer, which performs a delayed one-step detection mode.

[0151] 1. The specific testing process is as follows:

[0152] (1) Take 15 μl of sample or calibrator and add it to the reaction cup. Then add 50 μl of reagent M and 100 μl of reagent R1, mix well and incubate at 37°C for 10 min.

[0153] (2) Add 50 μl of reagent R2 to (1), mix well, incubate at 37°C for 5 min, and then perform magnetic separation and washing;

[0154] 200 μl of substrate AMPPD was added to (2), and the photon signal was collected. The ADAMTS13 activity in the sample was calculated from the standard curve (for ADAMTS13 activity, IU / ml=100%).

[0155] 2. Reaction mode grouping:

[0156] One-step method, two-step method and delayed one-step method refer to the differences in reaction modes.

[0157] One-step method usually means that no matter how many reagent components are added to the reaction cup at the same time (or almost at the same time), they react almost at the same time.

[0158] The two-step method involves reacting the sample with several reagent components. After a period of incubation, magnetic separation and cleaning are required before adding other reagent components. The biggest difference from the one-step method is that the instrument needs to be cleaned midway;

[0159] The delayed one-step method means that due to competition (or other reasons), the sample needs to react with a certain reagent component for a period of time to make the process more complete before adding other reagents. It is worth noting that the delayed one-step method is different from the two-step method in that no magnetic separation and cleaning operation is performed in the middle.

[0160] Table 2

[0161]

[0162]

[0163] 2. The reaction mode of the kit disclosed herein has a suitable signal gradient.

[0164] Test Example 3. Comparison with ELISA method

[0165] The method in Test Example 2 was used to test 40 sodium citrate plasma samples and compared with the commercially available ELISA kit ( ADAMTS-13Activity) were compared, and the results are shown in the following table.

[0166] Table 3

[0167]

[0168] The linear regression equation of the detection results of the kit disclosed herein and the detection results of the commercially available ELISA kit is y=0.9839x+1.0554, and the correlation coefficient R 2 =0.9484. It can be seen that the two detection methods have a good correlation.

[0169] Test Example 4. Repeatability

[0170] Use the method in Test Example 2 to test the repeatability of high-value and low-value samples.

[0171] Table 4

[0172]

[0173] The disclosed kit has good repeatability performance in detecting high and low value samples, both within 5%.

[0174] Test Example 5. Stability

[0175] 1. Use the method in Test Example 2 to detect the accelerated stability of the reagent.

[0176] Table 5

[0177]

[0178]

[0179] 2. Real-time stability of detection reagents.

[0180] Table 6

[0181]

[0182] The reagents were tested using the disclosed kit for accelerated stability. After seven days of testing at 37°C, the reagents showed good stability, with a deviation of less than 10% compared to the values ​​measured at 4°C. After testing for six months, the reagents showed good real-time stability, with a deviation of less than 5%.

[0183] Test Example 6. Linearity

[0184] The linearity of the reagent was detected using the method in Test Example 2.

[0185] Table 7

[0186]

[0187] The linearity test was performed using the kit disclosed herein. Within the linear range, the correlation coefficient r was ≥ 0.990 (r 2 =0.9993), good linear performance ( Figure 3 ).

[0188] Advantages of the disclosed kit:

[0189] ① The first use of the fusion protein MBP-VWF73-His containing MBP and His tags, which has higher signal values ​​and better reaction gradients than the conventional GST-VWF73-His protein;

[0190] ② A delayed one-step method (first react the sample and reagents M and R1 for 10 minutes without washing, then add the enzyme-labeled reagent for 5 minutes, perform magnetic separation, wash, and add the luminescent substrate) is used to allow ADAMTS13 in the sample to more completely cleave the substrate protein and form a complex of magnetic bead-coated antibody-substrate protein-enzyme-labeled antibody, achieving a suitable signal gradient;

[0191] ③ Confirm the appropriate reagent components and their ratios, and ensure good reactions in reagent comparison, stability, and linearity;

[0192] ④ Compared with the two-step detection mode, the reagent disclosed in the present invention greatly improves the detection speed and shortens the sample turnover time when using a high-speed chemiluminescence instrument.

Claims

1. A von Willebrand factor cleaving enzyme (ADAMTS13) detection kit, comprising: a first reagent, a second reagent, and a third reagent; in, The first reagent comprises: 0.1mg / ml to 1.0mg / ml magnetic beads coated with the primary antibody, 10 mM to 100 mM first buffer, pH 6.0 to 8.0, 10mM to 300mM sodium chloride, 1mM to 50mM calcium chloride, 10mg / ml to 200mg / ml trehalose, 0.01% to 0.5%(w / v)Tween 20, 0.05% to 5% (w / v) BSA, 0mg / ml to 1.0mg / ml blocking agent, 0% to 1.0% (w / v) preservatives; Wherein, the second reagent comprises: 200ng / ml to 1000ng / ml VWF73 short peptide, 10 mM to 100 mM second buffer, pH 6.0 to 8.0, 10mM to 300mM sodium chloride, 0.05% to 5% (w / v) casein, 0.05% to 5% (w / v) BSA, 0% to 1.0% (w / v) preservatives; Wherein, the third reagent comprises: 0.5 μg / ml to 2.5 μg / ml, preferably 1 μg / ml to 2 μg / ml, enzyme-labeled secondary antibody, 10 mM to 100 mM third buffer, pH 6.0 to 8.0, 10mM to 300mM sodium chloride, 1mM to 100mM magnesium chloride, 1mM to 100mM zinc chloride, 0.01% to 0.5%(w / v)Tween 20, 0.05% to 5% (w / v) BSA, 0% to 10% (w / v) protective agent, 0% to 1.0% (w / v) preservatives; in: The VWF73 short peptide is a short peptide having a first tag bound to the N-terminus of VWF73 and a second tag bound to the C-terminus; The first antibody specifically binds to the first tag; The second antibody specifically binds to the second tag; The first tag and the second tag are each independently selected from: GST, MBP, FLAG, His; preferably MBP, His; the first label is different from the second label; The first label and the second label are interchangeable; The first buffer, the second buffer, and the third buffer are each independently selected from: phosphate buffer, carbonate buffer, and tris buffer; The blocker is selected from the group consisting of: MARK33, HBR, TRUBLOCK, CHEMIBLOCK, and HIER; The enzyme is selected from the group consisting of alkaline phosphatase and horseradish peroxidase; Preferably, the alkaline phosphatase is high-sugar alkaline phosphatase or low-sugar alkaline phosphatase; The protective agent is selected from the group consisting of sucrose, trehalose, dextran, and dextran sulfate; The preservative is selected from the group consisting of sodium azide, isothiazolinone, and gentamicin.

2. The ADAMTS13 detection kit according to claim 1, wherein: The surface of the magnetic beads is modified with carboxyl, streptavidin, or p-toluenesulfonyl groups; Preferably, the surface of the magnetic beads has carboxyl groups.

3. The ADAMTS13 detection kit according to claim 1, wherein: The average particle size of the magnetic beads is 1.0 μm, 1.6 μm or 3.0 μm, preferably 1.6 μm.

4. The ADAMTS13 detection kit according to claim 1, further comprising: Calibrators, and / or Quality control products; The calibrator or the quality control product comprises: One or more ADAMTS13 of known concentration, 10 mM to 100 mM buffer, pH 6.0 to 8.0; 10 mM to 500 mM sodium chloride, preferably 50 mM sodium chloride; 1 mM to 100 mM calcium chloride, preferably 10 mM calcium chloride; 0.05% to 5% (w / v) BSA, preferably 1.0% (w / v) BSA; 0% to 1.0% (w / v) preservative, preferably 0.1% (w / v) PC300; Preferably, the buffer is 20 mM Tris pH 7.

2.

5. The ADAMTS13 detection kit according to claim 1, wherein: In order from N-terminus to C-terminus, the VWF73 short peptide is selected from: GST-VWF73-His, MBP-VWF73-His, FLAG-VWF73-His, GST-VWF73-FLAG, MBP-VWF73-FLAG, GST-VWF73-MBP; Preferred are MBP-VWF73-His and FLAG-VWF73-His. The ADAMTS13 detection kit according to claim 1 , wherein in the first reagent, the concentration of the first antibody relative to the magnetic beads is 5 μg / mg to 40 μg / mg. The ADAMTS13 detection kit according to claim 1 , wherein in the third reagent, the mass ratio of the enzyme to the second antibody is 1:

2.

8. The ADAMTS13 detection kit according to claim 1, wherein: The first reagent comprises: 0.3mg / ml magnetic beads coated with anti-MBP antibody, 50 mM Tris buffer, pH 6.5, 300mM sodium chloride, 10mM calcium chloride, 100mg / ml trehalose, 0.05% (w / v) Tween 20, 0.5% (w / v) BSA, 0.05mg / ml MARK33, 0.1% (w / v) PC300; Wherein, the second reagent comprises: 200ng / ml VWF73 short peptide with MBP tag at N-terminus and 6×His tag at C-terminus, 20 mM Tris buffer, pH 7.0, 150mM sodium chloride, 0.5% (w / v) casein, 0.5% (w / v) BSA, 0.1% (w / v) PC300; Wherein, the third reagent comprises: 1.0 μg / ml alkaline phosphatase-labeled anti-His antibody, 20 mM Tris buffer, pH 7.2, 150mM sodium chloride, 1mM magnesium chloride, 10mM zinc chloride, 0.05% (w / v) Tween 20, 0.5% (w / v) BSA, 0.2% (w / v) dextran sulfate, 0.1% (w / v) PC300.

9. A method for using an ADAMTS13 detection kit, comprising the steps of: 1) Provide samples; 2) contacting the sample with the first reagent and the second reagent as defined in claim 1 at 37° C. for 5 to 20 minutes (preferably 8 to 15 minutes) to obtain an incubation; 3) contacting the incubated substance with the third reagent as defined in claim 1 at 37° C. for 3 to 10 minutes (preferably 3 to 6 minutes) to obtain a complex; 4) recovering the complex by magnetic separation and washing; 5) Add chemiluminescent substrate to the complex and measure the light signal.

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