Preparation method of t-pai·c detection kit and application thereof

Abstract

This invention relates to the field of biological detection, and more particularly to the preparation method and application of the t-PAI·C detection kit. The invention provides a detection reagent comprising: antibody-coated magnetic microparticles and enzyme-labeled antibodies; the antibodies include: tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody. In the detection reagent provided by this invention, when the sample contains the tissue plasminogen activator-plasminogen activator inhibitor-1 complex, the antibody coated with the magnetic microparticles and the enzyme-labeled antibody undergo an immune reaction with the complex, forming a sandwich-like immune complex consisting of the antibody coated with the magnetic microparticles, the complex, and the enzyme-labeled antibody; the enzyme on this complex acts on a luminescent substrate and emits light, and the concentration of the complex in the sample is calculated by detecting the luminescence signal.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more particularly to the preparation method and application of t-PAI·C detection kit. Background Technology

[0002] Tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) form a 1:1 ratio to form the tissue-type plasminogen activator-plasminogen activator inhibitor-1 complex (t-PAI·C), which is an important factor in regulating coagulation and fibrinolysis in the body.

[0003] Tissue plasminogen activator is a glycoprotein mainly synthesized by vascular endothelial cells. It can activate plasminogen, converting it into plasmin, which degrades fibrin.

[0004] Plasminogen activator inhibitors include plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2). PAI-1 is a glycoprotein mainly synthesized by vascular endothelial cells and plays a major role in inactivating tissue-type plasminogen activator and inhibiting fibrinolysis.

[0005] The site of action for t-PA and PAI-1 is between lysine 296 and aspartic acid 304 in their light chain. Under normal circumstances, most t-PA and PAI-1 in the bloodstream form a complex in a 1:1 ratio, with a small amount remaining in a free state. Platelet α-granules store a large amount of PAI-1. When vascular endothelial cells are damaged, platelets are activated by thrombin, releasing PAI-1 onto the surface of platelet α-granules, causing changes in t-PA expression and resulting in abnormal t-PAI·C concentrations. Many risk factors for coronary heart disease, such as smoking, hypertension, diabetes, and obesity, which are associated with elevated blood insulin, elevated blood triglycerides, decreased high-density lipoprotein, and hyperglycemia, can directly stimulate the transcription and secretion of PAI-1 in vascular endothelial cells. Furthermore, elevated PAI-1 levels are also common in cancer patients, all of which can lead to abnormal t-PAI·C concentrations.

[0006] The tissue plasminogen activator-plasminogen activator inhibitor-1 complex can serve as a reference indicator for early detection and prognosis analysis of cardiovascular diseases, tumors, and other diseases. Combined detection with TAT, TM, and other markers can enhance the early warning efficacy for venous thromboembolism, disseminated intravascular coagulation, and other conditions.

[0007] Currently, most methods for detecting tissue plasminogen activator-plasminogen activator inhibitor-1 (TPA-I) complexes on the market use ELISA plates. However, there are fewer reagent options, more complex procedures, and longer processing times available for fully automated analyzers (chemiluminescence immunoassay) to detect TPA-I. As this diagnostic test gains increasing importance, market demand is rapidly growing. Summary of the Invention

[0008] In view of this, the present invention provides a method for preparing a t-PAI·C detection kit and its application. In the detection reagent provided by this invention, when the sample contains a tissue plasminogen activator-plasminogen activator inhibitor-1 complex, the antibody coated with magnetic microparticles and the enzyme-labeled antibody react with the tissue plasminogen activator-plasminogen activator-1 complex to form a sandwich-type immune complex consisting of the antibody coated with magnetic microparticles, the tissue plasminogen activator-plasminogen activator-1 complex, and the enzyme-labeled antibody. Using magnetic microparticles as a carrier, the immune complex is adsorbed by a magnetic field, and unreacted substances are washed away with a washing solution to obtain the sandwich-type immune complex consisting of the antibody coated with magnetic microparticles, the tissue plasminogen activator-plasminogen activator-1 complex, and the enzyme-labeled antibody. By adding a chemiluminescent substrate solution, the enzyme on the sandwich-type immune complex acts on the luminescent substrate and emits light. By detecting the luminescent signal, the concentration of the tissue plasminogen activator-plasminogen activator-1 complex in the sample is calculated. The reagent components of this invention are relatively stable and have good repeatability. It adopts a one-step method and is simple to operate.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] The present invention provides a detection reagent comprising: antibody-coated magnetic microparticles and enzyme-labeled antibodies;

[0011] The antibody includes: tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody.

[0012] In some embodiments of the present invention, the magnetic microparticles in the above-mentioned detection reagent include: amino magnetic microparticles or carboxyl magnetic microparticles.

[0013] In some embodiments of the present invention, the magnetic microparticles in the above-mentioned detection reagent are carboxyl magnetic microparticles.

[0014] In some embodiments of the present invention, the enzyme in the above-mentioned detection reagent includes: horseradish peroxidase or alkaline phosphatase.

[0015] In some embodiments of the present invention, the enzyme in the above-mentioned detection reagent is alkaline phosphatase.

[0016] In some embodiments of the present invention, in the above-mentioned detection reagent, the final concentration of the antibody-coated magnetic microparticles is 5-10 mg / mL; and the final concentration of the enzyme-labeled antibody is 0.5-1 μg / mL.

[0017] In some embodiments of the present invention, the final concentration of the antibody-coated magnetic microparticles in the above-mentioned detection reagent is 5 mg / mL and / or 10 mg / mL.

[0018] In some embodiments of the present invention, the final concentration of the enzyme-labeled antibody in the above-mentioned detection reagent is 0.5 μg / mL and / or 1 μg / mL.

[0019] In some embodiments of the present invention, the above-mentioned detection reagent further includes: 0.01 to 200 mM metal ions and 0.05% to 15% (w / v) stabilizer.

[0020] In some embodiments of the present invention, the above-mentioned detection reagent further includes: 0.5 to 150 mM metal ions and 0.5% to 2% (w / v) stabilizer.

[0021] In some embodiments of the present invention, the metal ions in the above-mentioned detection reagent include one or more of magnesium ions, zinc ions, aluminum ions, manganese ions, sodium ions, potassium ions and / or copper ions.

[0022] The stabilizer includes one or more of bovine serum albumin, amino acids, polyhydroxy compounds, and / or polymers.

[0023] In some embodiments of the present invention, the polymer in the above-mentioned detection reagent includes one or more of the following: polyvinylpyrrolidone-10, polyvinylpyrrolidone K30, polyvinylpyrrolidone P5288, polyvinyl alcohol, polyethylene oxide, polyethylene glycol monomethyl ether, Triton X-100, polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 12000, and polypropylene glycol 2000.

[0024] In some embodiments of the present invention, the metal ions in the above-mentioned detection reagent include one or more of aluminum ions, sodium ions, and / or copper ions;

[0025] The stabilizer includes: bovine serum albumin and / or polymers;

[0026] In some embodiments of the present invention, the polymer in the above-mentioned detection reagent includes one or more of the following: polyvinylpyrrolidone K30, polyvinyl alcohol, Triton X-100, and polyethylene glycol 8000.

[0027] In some embodiments of the present invention, the metal ions in the above-mentioned detection reagent include: 120 mM sodium ions, 0.5 mM aluminum ions, and 1 mM magnesium ions; or

[0028] 136 mM sodium ions, 2 mM aluminum ions, and 0.8 mM copper ions; or

[0029] 150 mM sodium ions, 5 mM zinc ions and 2 mM aluminum ions.

[0030] In some embodiments of the present invention, the stabilizer in the above-mentioned detection reagent comprises: 3% bovine serum albumin, 2% polyvinylpyrrolidone P5288, and 0.5% Triton X-100; or

[0031] 2% bovine serum albumin, 1% polyvinylpyrrolidone K30 and 2% polyethylene glycol 8000; or

[0032] 1% bovine serum albumin, 2% polyvinyl alcohol.

[0033] In some embodiments of the present invention, the above-mentioned detection reagent further includes: a magnetic microparticle buffer system and / or an enzyme labeling buffer system.

[0034] In some embodiments of the present invention, the magnetic particle buffer system in the above-mentioned detection reagent includes one or more of the following: MOPS buffer system, Tris-HCl buffer system, PBS buffer system, HEPES buffer system and / or glycine buffer system;

[0035] The enzyme-labeled buffer system includes one or more of the following: MES buffer system, Tris-HCl buffer system, PBS buffer system, HEPES buffer system, glycine buffer system, and / or borate buffer system.

[0036] In some embodiments of the present invention, the above-mentioned test reagent further includes 0.02% (v / v) preservative.

[0037] In some embodiments of the present invention, the preservative in the above-mentioned test reagent includes one or more of Proclin 300 and / or Proclin 600.

[0038] In some embodiments of the present invention, the preservative in the above-mentioned test reagent is Proclin 300.

[0039] In some embodiments of the present invention, the magnetic particle buffer system in the above-mentioned detection reagent is a PBS buffer system;

[0040] The enzyme labeling buffer system is a Tris-HCl buffer system.

[0041] In some embodiments of the present invention, the pH value of the magnetic microparticle buffer system and the enzyme labeling buffer system in the above-mentioned detection reagent is 7.1 to 7.3.

[0042] The present invention also provides a method for preparing the above-mentioned detection reagent, comprising the following steps:

[0043] S1: Mix the magnetic microparticle buffer system with magnetic microparticles, wash, and then activate the magnetic microparticles to obtain a magnetic microparticle solution;

[0044] S2: Mix the magnetic microparticle solution with the antibody, incubate, and obtain the antibody-coated magnetic microparticles;

[0045] S3: Take the activated antibody and the activated enzyme, mix them, purify them, and then mix them with the metal ions, the stabilizer and the enzyme label buffer system to obtain the enzyme-labeled antibody.

[0046] In some embodiments of the present invention, in the above preparation method, the mass ratio of the magnetic microparticle solution to the antibody is (80-100):1; and the molar ratio of the activated antibody to the activated enzyme is 1:(1-2).

[0047] In some embodiments of the present invention, in the above preparation method, the volume ratio of the magnetic microparticle buffer system to the magnetic microparticles in S1 is 3:1.

[0048] In some embodiments of the present invention, in the above preparation method, the antibody in S3 is activated using 10-20 mg / mL 2-Imino thiolane hydrochloride for 5-10 min.

[0049] In some embodiments of the present invention, in the above preparation method, the enzyme in S3 is activated using 5-10 mg / mL SMCC for 5-10 min.

[0050] In some embodiments of the present invention, in the above preparation method, the time for mixing the activated antibody with the activated enzyme is 18-24 hours.

[0051] The present invention also provides the application of the above-described detection reagents and / or the detection reagents obtained by the above-described preparation method in the preparation of a kit for detecting tissue plasminogen activator-plasminogen activator inhibitor-1 complex.

[0052] The present invention provides a kit comprising the above-described detection reagents and / or detection reagents obtained by the above-described preparation method, as well as acceptable adjuvants or carriers.

[0053] The present invention also provides a method for using the kit, wherein the sample to be tested is mixed with the detection reagent in the kit and then tested.

[0054] The present invention provides a detection reagent comprising: antibody-coated magnetic microparticles and enzyme-labeled antibodies;

[0055] The antibody includes: tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody.

[0056] The beneficial effects of this invention include:

[0057] (1) In the detection reagent provided by the present invention, when the sample contains a tissue plasminogen activator-plasminogen activator inhibitor-1 complex, the antibody coated with magnetic microparticles and the antibody labeled with the enzyme react with the tissue plasminogen activator-plasminogen activator-1 complex to form a sandwich-type immune complex consisting of an antibody coated with magnetic microparticles, a tissue plasminogen activator-plasminogen activator-1 complex, and an antibody labeled with the enzyme. Using magnetic microparticles as a carrier, the immune complex is adsorbed by a magnetic field, and unreacted substances are washed away with a washing solution to obtain a sandwich-type immune complex consisting of an antibody coated with magnetic microparticles, a tissue plasminogen activator-plasminogen activator-1 complex, and an antibody labeled with the enzyme. By adding a chemiluminescent substrate solution, the enzyme on the sandwich-type immune complex acts on the luminescent substrate and emits light. By detecting the luminescent signal, the concentration of the tissue plasminogen activator-plasminogen activator-1 complex in the sample is calculated.

[0058] (2) The components of the reagent of the present invention are relatively stable and have good repeatability. The one-step method is simple to operate.

[0059] (3) The addition of polymers and metal ions to the detection reagents provided by the present invention can make the enzyme markers dispersed evenly, prevent aggregation, and maintain their activity. Attached Figure Description

[0060] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0061] Figure 1 Example 2 shows the Bland-Altman concordance limit analysis (Bland-Altman) between the reagent and Sysmex reagent in clinical samples.

[0062] Figure 2 Example 2 shows the regression analysis (Deming) comparing clinical samples of the reagent and Sysmex reagent; where: regression line (solid line), label line (y=x, dashed line). Detailed Implementation

[0063] This invention discloses a method for preparing a t-PAI·C detection kit and its application.

[0064] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0065] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0066] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0067] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0068] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0069] This invention provides a reagent for detecting the concentration of tissue plasminogen activator-plasminogen activator inhibitor-1 complex (t-PAI·C), the reagent comprising:

[0070] Reagent 1 includes antibody-coated magnetic microparticles and magnetic microparticle buffer, wherein the antibody-coated magnetic microparticles are obtained by covalently binding the antibody to the magnetic microparticles, and the magnetic microparticle buffer includes a buffer system, a stabilizer, and a preservative;

[0071] Reagent 2 includes an enzyme-labeled antibody and an enzyme-labeled buffer solution, wherein the enzyme-labeled buffer solution includes a buffer system, preservatives, stabilizers, and metal ions.

[0072] Optionally, the magnetic particles include either amino magnetic particles or carboxyl magnetic particles.

[0073] Optionally, the enzyme includes one of horseradish peroxidase or alkaline phosphatase.

[0074] Optionally, the magnetic microparticle buffer solution may include one of the following buffer systems: MOPS buffer system, Tris-HCl buffer system, PBS buffer system, HEPES buffer system, glycine buffer system, etc., preferably the PBS buffer system.

[0075] Optionally, the stabilizer included in the magnetic microparticle buffer may be one or more of bovine serum albumin, amino acids, polyhydroxy compounds, polymers, etc.

[0076] Optionally, the preservative included in the magnetic microparticle buffer may be one or more of Proclin 300 and Proclin 600.

[0077] Optionally, the magnetic microparticle buffer solution comprises a buffer system with a pH range of 7.2 ± 0.1.

[0078] Optionally, the enzyme labeling buffer may include one of the following buffer systems: MES buffer system, Tris-HCl buffer system, PBS buffer system, HEPES buffer system, glycine buffer system, borate buffer system, etc., preferably the Tris-HCl buffer system.

[0079] Optionally, the preservative included in the labeling buffer may be one or more of Proclin 300 and Proclin 600, preferably Proclin 300.

[0080] The polymers and metal ions included in the enzyme labeling buffer can ensure uniform dispersion of the enzyme labeling, prevent aggregation, and maintain its activity.

[0081] Optionally, the stabilizer included in the label buffer may be one or more of bovine serum albumin, amino acids, polyhydroxy compounds, polymers, etc., preferably bovine serum albumin or polymers.

[0082] Optionally, the polymer may be one or more of the following: polyvinylpyrrolidone-10, polyvinylpyrrolidone K30, polyvinylpyrrolidone P5288, polyvinyl alcohol, polyethylene oxide, polyethylene glycol monomethyl ether, Triton X-100, polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 12000, and polypropylene glycol 2000, preferably one or more of the following: polyvinylpyrrolidone K30, polyvinyl alcohol, Triton X-100, and polyethylene glycol 8000.

[0083] Optionally, the concentration of the polymer in the enzyme-labeled buffer may be 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 7.5%, 10%, 12.5%, or 15%, preferably 0.5% to 2%.

[0084] Optionally, the metal ions included in the labeling buffer may be one or more of magnesium ions, zinc ions, aluminum ions, manganese ions, sodium ions, potassium ions, and copper ions, preferably aluminum ions, sodium ions, and copper ions.

[0085] Optionally, the concentration of metal ions in the enzyme-labeled buffer solution can be 0.01mM, 0.02mM, 0.05mM, 0.1mM, 0.2mM, 0.25mM, 0.5mM, 1mM, 2mM, 5mM, 10mM, 20mM, 50mM, 100mM, 150mM, or 200mM, preferably 0.5mM to 150mM.

[0086] Optionally, the labeling buffer solution comprises a buffer system with a pH range of 7.2 ± 0.1.

[0087] The present invention further proposes a reagent preparation method as described above, comprising the following steps:

[0088] The antibody was incubated with activated magnetic microparticles to covalently bind them. After removing the unbound antibody, the mixture was diluted with magnetic microparticle buffer to obtain reagent one.

[0089] The activated enzyme was covalently linked to the antibody. After removing the free antibody and enzyme, it was diluted with enzyme-labeled buffer to obtain reagent two.

[0090] The present invention further proposes a reagent detection method as described above, comprising the following steps:

[0091] First, take 10 μL of the sample to be tested, add 50 μL of reagent one and 50 μL of reagent two, mix gently, and incubate at 37°C for 15 min.

[0092] The second step is to remove the reaction solution, add 500μL of cleaning solution, and wash 3 times.

[0093] Third, add 200 μL of substrate solution, mix well, and detect the luminescence intensity;

[0094] The fourth step is to calculate the concentration of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex based on the luminescence intensity and the standard curve.

[0095] In Examples 1 to 5 and the effect examples of this invention, all raw materials and reagents used can be purchased from the market.

[0096] The present invention will be further illustrated below with reference to the embodiments:

[0097] Example 1: A reagent for detecting the concentration of tissue plasminogen activator-plasminogen activator inhibitor-1 complex (t-PAI·C) in plasma.

[0098] 1. Reagents include:

[0099] Reagent 1: Carboxyl magnetic microparticles (hereinafter referred to as magnetic microparticles) coated with commercially available tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody at a concentration of 5 mg / mL and magnetic microparticle buffer.

[0100] Reagent 2: Alkaline phosphatase-labeled tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody and enzyme-labeled buffer at a concentration of 1 μg / mL.

[0101] 2. The preparation method of the magnetic microparticle reagent coated with tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody is as follows:

[0102] 1) Place the thoroughly mixed magnetic microparticles into a reaction flask, place the flask in a magnetic field for 5 minutes, and after all the magnetic microparticles are adsorbed to one side, remove the supernatant. Add magnetic microparticle buffer solution with a volume of 3 times that of the magnetic microparticles to the reaction flask, and gently shake and wash for 3 minutes. Place the reaction flask in a magnetic field for another 5 minutes and remove the supernatant. Repeat the washing process 3 times. Finally, dilute the magnetic microparticle solution to 5 mg / mL, add 0.15 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.6 mg of N-hydroxysuccinimide (NHS) to activate the magnetic beads and obtain the magnetic microparticle solution.

[0103] 2) According to the mass ratio of magnetic microparticle solution: tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody = 80:1, add tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody to the magnetic microparticle solution prepared in step 1), and incubate at room temperature for 4 hours.

[0104] 3) Place the reaction flask in a magnetic field for 3 minutes. After the magnetic microparticles settle, wash 3 times with magnetic microparticle buffer, then dilute to 5 mg / mL and store at 2-8℃ to obtain antibody-coated magnetic microparticles.

[0105] The magnetic microparticle buffer solution includes: a 0.1M PBS magnetic microparticle buffer system, pH 7.2±0.1.

[0106] raw materials concentration Sodium hydrogen phosphate 72mM Sodium dihydrogen phosphate 28mM Sodium chloride 150mM Polyvinylpyrrolidone K30 1％ Bovine serum albumin 2％ Proclin300 0.02％

[0107] 3. The preparation method of alkaline phosphatase-labeled tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody is as follows:

[0108] 1) Take 1 mg (1.0 mg / mL) of tissue-type plasminogen activator-plasminogen activator inhibitor-1 complex antibody, add 5 μL of 20 mg / mL activator 2-Imino thiolane hydrochloride (2-IT) solution, incubate at room temperature for 60 minutes, then add glycine, incubate at room temperature for 10 minutes to terminate the activation reaction, and obtain the activated antibody.

[0109] 2) Take 1 mg of alkaline phosphatase and add it to 12 μL of Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) solution with a concentration of 10 mg / mL. Let it stand at room temperature for 60 minutes, then add glycine and let it stand at room temperature for 10 minutes to terminate the activation reaction and obtain activated alkaline phosphatase.

[0110] 3) Mix the activated tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody with the activated alkaline phosphatase at a molar ratio of 1:2 and let it react at 2-8°C for 24 hours.

[0111] 4) Select an ultrafiltration column with an appropriate molecular weight cutoff to purify the above-mentioned ligation product. The alkaline phosphatase-labeled tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody (enzyme-labeled antibody) is stored at 2-8℃ for later use.

[0112] 5) Add the alkaline phosphatase-labeled tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody prepared in step 4) to an enzyme-labeled buffer containing sodium, aluminum, and magnesium ions to a final concentration of 1 μg / mL.

[0113] The enzyme labeling buffer was a 0.05M Tris-HCl buffer with pH 7.2±0.1, and its components are shown in the table below.

[0114] raw materials concentration Tris(hydroxymethyl)aminomethane 50mM Sodium chloride (sodium ions) 120mM Bovine serum albumin 3％ Polyvinylpyrrolidone P5288 2％ Triton X-100 0.5％ Aluminum chloride hexahydrate (aluminum ions) 0.5mM Anhydrous magnesium chloride (magnesium ions) 1mM Proclin300 0.02％

[0115] Example 2: A reagent for detecting the concentration of tissue plasminogen activator-plasminogen activator inhibitor-1 complex (t-PAI·C) in plasma.

[0116] 1. Reagents include:

[0117] Reagent 1: Carboxyl magnetic microparticles (hereinafter referred to as magnetic microparticles) coated with tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody at a concentration of 10 mg / mL and magnetic microparticle buffer.

[0118] Reagent 2: Alkaline phosphatase-labeled tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody and enzyme-labeled buffer at a concentration of 1 μg / mL.

[0119] 2. The preparation method of the magnetic microparticle reagent coated with tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody is as follows:

[0120] 1) The cleaning and activation steps for carboxyl magnetic microparticles are described in step 2(1) of Example 1;

[0121] 2) According to the mass ratio of magnetic microparticle solution: tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody = 100:1, add tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody to the magnetic microparticle solution prepared in step 1), and incubate at room temperature for 4 hours.

[0122] 3) Place the reaction flask in a magnetic field for 5 minutes. After the magnetic microparticles settle, wash them 3 times with magnetic microparticle buffer, then dilute to 10 mg / mL and store at 2-8℃ to obtain antibody-coated magnetic microparticles.

[0123] The magnetic microparticle buffer solution was a 0.05M PBS magnetic microparticle buffer system with a pH of 7.2±0.1.

[0124] raw materials concentration Sodium hydrogen phosphate 36mM Sodium dihydrogen phosphate 14mM Sodium chloride 150mM Polyvinylpyrrolidone K30 1％ Bovine serum albumin 2％ Proclin300 0.02％

[0125] 3. The preparation method of alkaline phosphatase-labeled tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody is as follows:

[0126] 1) Take 1 mg (1.0 mg / mL) of tissue-type plasminogen activator-plasminogen activator inhibitor-1 complex antibody, add 5 μL of 10 mg / mL activator 2-Imino thiolane hydrochloride (2-IT) solution, incubate at room temperature for 60 minutes, then add glycine, incubate at room temperature for 5 minutes to terminate the activation reaction, and obtain the activated antibody.

[0127] 2) Take 1 mg of alkaline phosphatase and add it to 12 μL of Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) solution with a concentration of 5 mg / mL. Let it stand at room temperature for 60 minutes, add glycine, and let it stand at room temperature for 5 minutes to terminate the activation reaction and obtain activated alkaline phosphatase.

[0128] 3) Mix the activated tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody with the activated alkaline phosphatase at a molar ratio of 1:2 and let it react at 2-8°C for 20 hours.

[0129] 4) Select an ultrafiltration column with an appropriate molecular weight cutoff to purify the above-mentioned ligation product. The alkaline phosphatase-labeled tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody (enzyme-labeled antibody) is stored at 2-8°C for later use.

[0130] 5) Add the prepared alkaline phosphatase-labeled tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody to an enzyme-labeled buffer containing sodium, aluminum, and copper ions to a final concentration of 1 μg / mL.

[0131] The enzyme labeling buffer is a 0.1M Tris-HCl buffer with pH 7.2±0.1, and its components are shown in the table below.

[0132] raw materials concentration Tris(hydroxymethyl)aminomethane 100mM Sodium chloride (sodium ions) 136mM Bovine serum albumin 2％ Polyvinylpyrrolidone K30 1％ Polyethylene glycol 8000 2％ Aluminum chloride hexahydrate (aluminum ions) 2mM Copper chloride dihydrate (copper ions) 0.8mM Proclin300 0.02％

[0133] Example 3: A reagent for detecting the concentration of tissue plasminogen activator-plasminogen activator inhibitor-1 complex (t-PAI·C) in plasma.

[0134] 1. Reagents include:

[0135] Reagent 1: Carboxyl magnetic microparticles (hereinafter referred to as magnetic microparticles) coated with tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody at a concentration of 10 mg / mL and magnetic microparticle buffer.

[0136] Reagent 2: Alkaline phosphatase-labeled tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody and enzyme labeling buffer at a concentration of 0.5 μg / mL.

[0137] 2. The preparation method of the magnetic microparticle reagent coated with tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody is as follows:

[0138] 1) The cleaning and activation steps for carboxyl magnetic microparticles are described in step 2(1) of Example 1;

[0139] 2) According to the mass ratio of magnetic microparticle solution: tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody = 100:1, add tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody to the magnetic microparticle solution prepared in step 1), and incubate at room temperature for 2 hours.

[0140] 3) Place the reaction flask in a magnetic field for 3 minutes. After the magnetic microparticles settle, wash them 3 times with magnetic microparticle buffer. Then dilute to 10 mg / mL and store at 2-8℃ to obtain antibody-coated magnetic microparticles.

[0141] The magnetic microparticle buffer solution is the same as in Example 2.

[0142] 3. The preparation method of alkaline phosphatase-labeled tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody is as follows:

[0143] 1) Take 1 mg (1.0 mg / mL) of tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody, add 5 μL of 10 mg / mL activator 2-Imino thiolane hydrochloride (2-IT) solution, incubate at room temperature for 45 minutes, add glycine, incubate at room temperature for 5 minutes to terminate the activation reaction, and obtain the activated antibody.

[0144] 2) Take 1 mg of alkaline phosphatase and add it to 12 μL of Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) solution with a concentration of 10 mg / mL. Let it stand at room temperature for 60 minutes, add glycine, and let it stand at room temperature for 5 minutes to terminate the activation reaction and obtain activated alkaline phosphatase.

[0145] 3) Mix the activated tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody with the activated alkaline phosphatase at a molar ratio of 1:1 and let it react at 2-8°C for 18 hours.

[0146] 4) Select an ultrafiltration column with an appropriate molecular weight cutoff to purify the above-mentioned ligation product. The alkaline phosphatase-labeled tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody (enzyme-labeled antibody) is stored at 2-8°C for later use.

[0147] 5) The prepared alkaline phosphatase-labeled tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody was added to an enzyme-labeled buffer containing sodium, zinc, magnesium and aluminum ions to achieve a final concentration of 0.5 μg / mL.

[0148] The enzyme labeling buffer was a 0.1M Tris-HCl buffer with pH 7.2±0.1, and its components are shown in the table below.

[0149] raw materials concentration Tris(hydroxymethyl)aminomethane 100mM Sodium chloride (sodium ions) 150mM Bovine serum albumin 1％ Polyvinyl alcohol 2％ Zinc chloride (zinc ions) 5mM Anhydrous magnesium chloride (magnesium ions) 3mM Aluminum chloride hexahydrate (aluminum ions) 2mM Proclin300 0.02％

[0150] Example 4: A reagent for detecting the concentration of tissue plasminogen activator-plasminogen activator inhibitor-1 complex (t-PAI·C) in plasma.

[0151] 1. Reagents include:

[0152] Reagent 1: Carboxyl magnetic microparticles (hereinafter referred to as magnetic microparticles) coated with tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody at a concentration of 10 mg / mL and magnetic microparticle buffer.

[0153] Reagent 2: Alkaline phosphatase-labeled tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody and enzyme-labeled buffer at a concentration of 1 μg / mL.

[0154] 2. The preparation method of the magnetic microparticle reagent coated with tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody is described in step 2 of Example 2.

[0155] 3. The preparation method of alkaline phosphatase-labeled tissue plasminogen activator-plasminogen activator inhibitor-1 complex antibody is described in step 3 of Example 2. The enzyme labeling buffer is 0.1M Tris-HCl buffer with pH 7.2±0.1, and the components are shown in the table below.

[0156] raw materials concentration Tris(hydroxymethyl)aminomethane 100mM Sodium chloride (sodium ions) 136mM Bovine serum albumin 2％ Proclin300 0.02％

[0157] Example 5: Detection method for tissue plasminogen activator-plasminogen activator inhibitor-1 complex assay reagent.

[0158] Includes the following steps:

[0159] First, take 10 μL of the sample to be tested, add 50 μL of reagent one and 50 μL of reagent two, mix gently, and incubate at 37°C for 15 min.

[0160] The second step is to remove the reaction solution and add 500 μL of Tris-HCl cleaning solution to wash three times.

[0161] Third, add 200 μL of substrate solution containing AMPPD, mix well, and detect the luminescence intensity;

[0162] The fourth step is to calculate the concentration of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex based on the luminescence intensity and the standard curve.

[0163] Effect Example Testing and Evaluation

[0164] 1. Repeatability testing

[0165] The same normal sample was tested 10 times using the reagents from Examples 1 to 4 and Sysmex reagent, respectively, according to the detection method of Example 5. The repeatability test results are shown in Table 1.

[0166] Table 1. Repeatability test results of Examples 1-4 and commercially available reagents.

[0167]

[0168]

[0169] As shown in Table 1, the coefficients of variation of the detection results in Examples 1-4 were all below 8%, which meets the standard, indicating good repeatability of Examples 1-4. Furthermore, the measured values ​​in Example 2 were closest to those of commercially available reagents. Compared to Example 2, Example 4 had lower measured values ​​and a larger coefficient of variation because it did not contain aluminum ions, copper ions, polyvinylpyrrolidone, and polyethylene glycol 8000. This may be due to reduced enzyme label activity and uneven dispersion.

[0170] 2. Clinical sample comparison

[0171] The samples were blood samples from 200 randomly selected inpatients and outpatients at Longhua Hospital affiliated with Shanghai University of Traditional Chinese Medicine. The blood was mixed with 0.109 mol / L sodium citrate anticoagulant at a ratio of 9:1 and centrifuged at 2500 rpm for 15 minutes to obtain plasma.

[0172] The reagents from Example 2 and Sysmex reagents were selected, and the detection method from Example 5 was used to test 200 samples simultaneously.

[0173] The test results were subjected to Bland-Altman consistency limit analysis, and the results are shown below. Figure 1 See Table 2. In detail, a deviation graph was plotted with the mean values ​​of the Sysmex reagent and the reagent of Example 2 as the x-axis and the percentage of the difference between the Sysmex reagent and the reagent of Example 2 as the y-axis. At the same time, the mean d of the percentage difference and the 95% distribution interval of the percentage difference were calculated to determine the consistency limit (LoA) graphically.

[0174] Table 2. Bland-Altman concordance limit analysis between clinical samples of the reagents in Example 2 and Sysmex reagents.

[0175]

[0176] Depend on Figure 1 As shown in Table 2, 96.0% of the samples were within the LoA, and the LoA was included in the clinically acceptable range (±10.0%), indicating that the sample test results of Example 2 were highly consistent with the sample test results of Sysmex reagent.

[0177] The test results were subjected to regression analysis, and the results are shown below. Figure 2 Compared with Table 3, in detail, the measurements were fitted with the Sysmex reagent values ​​on the x-axis and the measurements with the reagent from Example 2 on the y-axis.

[0178] Table 3. Regression analysis comparing clinical samples of reagents from Example 2 and Sysmex reagents (Deming)

[0179]

[0180] The fitted linear equation is y = -0.0886 + 1.005x, with a slope 95% CI including 1 and an intercept 95% CI including 0. Statistically, there is no significant difference between the two systems. Since the results are within the clinically acceptable range and have clinical significance, the sample test results of Example 2 are highly consistent with the sample test results of Sysmex reagent.

[0181] 3. Accelerate stability

[0182] The reagents from Example 2 were placed in a 37°C incubator for accelerated stability testing. The reagents were removed on days 0, 1, 2, 5, 7, and 9, and five samples containing different concentrations of tissue plasminogen activator-plasminogen activator inhibitor-1 complex were tested. The relative deviations of the luminescence values ​​on accelerated days 1, 2, 5, 7, and 9 and the unaccelerated day (day 0) were calculated. The results are shown in Tables 4 and 5 below.

[0183] Table 4. Accelerated stability test results of Example 2

[0184]

[0185] Table 5. Relative deviation (%) of accelerated stability test in Example 2

[0186] Sample number Concentration (ng / mL) Day 1 Day 2 Day 5 Day 7 Day 9 1 1.0 0.12 0.03 -0.05 -0.14 -0.12 2 2.5 -0.13 -0.63 -1.36 -1.94 -2.49 3 10.0 -0.04 -0.90 -1.47 -1.55 -1.97 4 50.0 0.02 -0.42 -0.91 -1.34 -2.57 5 80.0 -0.02 -0.27 -0.59 -1.26 -2.79

[0187] As shown in Tables 4 and 5, the luminescence values ​​of the reagents at 37℃ on days 1, 2, 5, 7, and 9 were all within 10% of those of the unaccelerated reagents, indicating good reagent stability.

[0188] In summary, the reagent components of this invention are relatively stable and have good reproducibility. It adopts a one-step method, is simple to operate, and can be applied to the detection of tissue-type plasminogen activator-plasminogen activator inhibitor-1 complex.

[0189] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A test reagent, characterized by The application relates to a detection reagent for detecting tissue-type plasminogen activator-plasminogen activator inhibitor-1 complex, and a preparation method thereof. The detection reagent comprises antibody-coated magnetic particles, enzyme-labeled antibodies, a magnetic particle buffer and an enzyme label buffer. The enzyme label buffer comprises 0.01-200 mM metal ions and 0.05%-15% (w / v) stabilizers. The antibody comprises a tissue-type plasminogen activator-plasminogen activator inhibitor-1 complex antibody. The metal ions comprise aluminum ions, sodium ions and copper ions. The stabilizers comprise bovine serum albumin, amino acids, polyhydroxyl compounds and polymers. The polymers comprise polyvinylpyrrolidone K30 and polyethylene glycol 8000.

2. The detection reagent of claim 1, wherein The final concentration of the antibody-coated magnetic particles is 5-10 mg / mL, and the final concentration of the enzyme-labeled antibodies is 0.5-1 ug / mL.

3. The test reagent according to claim 1 or 2, wherein The magnetic particle buffer and the enzyme label buffer respectively comprise a magnetic particle buffer system and an enzyme label buffer system.

4. The detection reagent of claim 3, wherein The magnetic particle buffer system comprises one or more of a MOPS buffer system, a Tris-HCl buffer system, a PBS buffer system, a HEPES buffer system and a glycine buffer system. The enzyme label buffer system comprises one or more of a MES buffer system, a Tris-HCl buffer system, a PBS buffer system, a HEPES buffer system, a glycine buffer system and a boric acid buffer system.

5. The method for preparing the detection reagent as described in claim 4, characterized in that, The application further relates to a preparation method of the detection reagent. S1: mixing the magnetic particle buffer system with magnetic particles, cleaning the magnetic particles, activating the magnetic particles and obtaining a magnetic particle solution; S2: mixing the magnetic particle solution with the antibody, incubating the mixture and obtaining the antibody-coated magnetic particles; S3: mixing the activated antibody with activated enzyme, purifying the mixture, mixing the purified mixture with the metal ions, the stabilizers and the enzyme label buffer and obtaining the enzyme-labeled antibodies.

6. The production method according to claim 5, wherein The mass ratio of the magnetic particle solution to the antibody is (80-100):1, and the molar ratio of the activated antibody to the activated enzyme is 1:(1-2).

7. The detection reagent according to any one of claims 1-4 or obtained by the preparation method according to claim 5 or 6 is used for preparing a kit for detecting tissue-type plasminogen activator-plasminogen activator inhibitor-1 complex.

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