Plasmin-alpha 2 plasmin inhibitor complex assay reagent and use thereof

Abstract

The present application relates to the technical field of in vitro diagnosis, in particular to plasmin-alpha2 plasmin inhibitor complex determination reagent and application thereof.The present application provides plasmin-alpha2 plasmin inhibitor complex determination reagent, which comprises magnetic particle suspension and labeled antibody mixture.The magnetic particle suspension comprises plasmin-alpha2 plasmin inhibitor complex antibody coated magnetic particles and magnetic particle buffer.The labeled antibody mixture comprises tracer labeled plasmin-alpha2 plasmin inhibitor complex antibody and labeled antibody buffer.The magnetic particle buffer and the labeled antibody buffer comprise buffer system, stabilizer and preservative.The reagent combination provided by the present application has high accuracy and good repeatability, and is suitable for popularization and application.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, specifically to a reagent for the assay of plasmin-α2 plasmin inhibitor complex and its application. Background Technology

[0002] The plasmin-α2-plasmin inhibitor complex (PIC) is a complex formed by the 1:1 combination of plasmin and α2-plasmin inhibitor, and is a key factor in regulating the fibrinolytic and coagulation processes in the human body.

[0003] Plasmin is formed by the hydrolysis of plasminogen (Plg) by tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA), and is an essential serine protease for fibrinolysis. The main inhibitors of plasmin are α2-plasmin inhibitors.

[0004] α2-plasmin inhibitor (α2-PI), also known as anti-plasmin (α2-AP), is a glycoprotein synthesized in the liver. Human α2-plasmin inhibitor belongs to the serine protease family and can inactivate plasmin.

[0005] In blood circulation, the inactivation of plasmin can be divided into two steps: ① Lysine 452 at the carboxyl-terminal accessory lysine binding site of α2-PI binds to the K1 region of plasmin A chain, forming a reversible but inactive PIC. ② The peptide bond between arginine 354 and methionine 355 of α2-PI breaks under the action of plasmin, releasing a peptide with a molecular weight of 11 kDa at the carboxyl terminus. Arginine 354 of α2-PI covalently binds to serine 740 of plasmin, forming a new stable complex with a molecular weight of 150 kDa, which does not have plasmin activity.

[0006] Under normal circumstances, plasmin in the bloodstream is quickly bound by α2-PI and loses its activity. When vascular endothelial cells are damaged, fibrin in the blood vessels interacts with platelets to form blood clots. α2-PI can bind directly to plasmin, or it can cross-link with fibrin through FVIII and then bind to plasmin to form PIC, inhibiting plasmin activity, preventing the clot from dissolving, and forming a thrombus.

[0007] PIC diagnostic results have high clinical value in the diagnosis of bleeding and thrombosis, as well as in the treatment of thrombosis. Combined detection with TAT, TM, and tPAI·C can improve the early warning efficacy of venous thromboembolism.

[0008] Currently, most PIC assays on the market use ELISA plates. There are relatively few reagents available for detecting plasmin-α2 plasmin inhibitor complexes using fully automated analyzers (chemiluminescence immunoassay), and the procedures are numerous and time-consuming. As this diagnostic tool gains increasing importance, market demand is rapidly growing. Summary of the Invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide a plasmin-α2 plasmin inhibitor complex assay reagent and its application. The plasmin-α2 plasmin inhibitor complex assay reagent provided by the present invention has high accuracy and good repeatability, is suitable for fully automated detection and analysis instruments (chemiluminescence method), and is simple to operate.

[0010] This invention provides a reagent combination comprising a magnetic microparticle suspension and a labeled antibody mixture;

[0011] The magnetic microparticle suspension comprises magnetic microparticles coated with a plasmin-α2 plasmin inhibitor complex antibody and a magnetic microparticle buffer, wherein the magnetic microparticle buffer comprises buffer system A, stabilizer A and preservative A;

[0012] The labeled antibody mixture includes a plasmin-α2 plasmin inhibitor complex antibody labeled with a tracer marker and a labeled antibody buffer, wherein the labeled antibody buffer includes buffer system B, stabilizer B and preservative B;

[0013] The buffer systems A and B are independently selected from at least one of MOPS, Tris-HCl, MES, PBS, HEPES, glycine, and boric acid.

[0014] The stabilizers A and B are independently selected from at least one of sodium chloride, bovine serum albumin, polyhydroxy compounds, and nonionic polymeric surfactants. The polyhydroxy compounds include at least one of trimethylolpropane, hydroxypropyl methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, sodium alginate, glycerol, mannitol, and sorbitol. The nonionic polymeric surfactants include at least one of nonylcyclohexanol polyoxyethylene ether, isotridecyl alcohol polyoxyethylene ether, dodecyl alcohol polyoxyethylene ether, secondary alcohol polyoxyethylene ether, polyvinylpyrrolidone K30, and polyethylene oxide-polypropylene oxide.

[0015] The preservatives A and B are independently selected from at least one of NaN3, Proclin 300, and Proclin 600.

[0016] Compared with other reagent combinations, the reagent combination provided by this invention has good suspension, solubilization, and dispersion properties in the magnetic microparticle buffer and labeled antibody buffer, as well as in the components of the nonionic polymeric surfactant, preventing antibody aggregation and precipitation. At the same time, due to the close cooperation between the components of the reagent combination, the capture accuracy and repeatability of the plasmin-α2 plasmin inhibitor complex are high, thus achieving better technical results.

[0017] In some embodiments, the magnetic microparticle suspension comprises 5–10 g / L magnetic microparticles coated with a plasmin-α2 plasmin inhibitor complex antibody, 12.114 g / L buffer system A, 25.884–126.844 g / L stabilizer A, and 0.01–0.1 g / L preservative A;

[0018] The labeled antibody mixture comprises 0.5–1 mg / L of a plasmin-α2 plasmin inhibitor complex antibody labeled with a tracer marker, 13–13.58 g / L of buffer system B, 14.766–26.38 g / L of stabilizer B, and 0.01–0.1 g / L of preservative B.

[0019] Within the above concentration range, the components of the magnetic microparticle suspension and the labeled antibody mixture are closely coordinated, exhibiting strong antibody compatibility. The reagent combination demonstrates high accuracy and repeatability in capturing the plasmin-α2 plasmin inhibitor complex, thereby achieving better technical results.

[0020] Preferably, the magnetic microparticles include amino magnetic microparticles or carboxyl magnetic microparticles, and the tracer label includes horseradish peroxidase or alkaline phosphatase.

[0021] Preferably, buffer system A is Tris-HCl, and buffer system B is PBS.

[0022] Preferably, stabilizer A comprises sodium chloride, bovine serum albumin, hydroxypropyl methylcellulose, nonylcyclohexanol polyoxyethylene ether, and polyvinylpyrrolidone K30, and stabilizer B comprises sodium chloride, bovine serum albumin, hydroxypropyl methylcellulose, and polyvinylpyrrolidone K30.

[0023] Experiments show that the reagent combination using the above components has higher sensitivity, stronger specificity, and better repeatability in capturing the plasmin-α2 plasmin inhibitor complex, thus achieving better technical results.

[0024] Preferably, preservative A and preservative B are NaN3.

[0025] Experiments show that the reagent combination using the above components has higher sensitivity, stronger specificity, and better repeatability in capturing the plasmin-α2 plasmin inhibitor complex, thus achieving better technical results.

[0026] In some specific embodiments, the magnetic microparticle suspension comprises 10 g / L plasmin-α2 plasmin inhibitor complex antibody-coated magnetic microparticles, 12.114 g / L Tris-HCl, 5.844 g / L sodium chloride, 10 g / L bovine serum albumin, 5 g / L hydroxypropyl methylcellulose, 10 g / L nonylcyclohexanol polyoxyethylene ether, 50 g / L polyvinylpyrrolidone K30, and 0.1 g / L NaN3;

[0027] The labeled antibody mixture comprises 0.5 mg / L of a tracer-labeled plasmin-α2 plasmin inhibitor complex antibody, 13.58 g / L PBS, 8.766 g / L sodium chloride, 5 g / L bovine serum albumin, 1 g / L hydroxypropyl methylcellulose, 0.5 g / L polyvinylpyrrolidone K30, and 0.1 g / L NaN3.

[0028] Experiments show that, under the above-mentioned components and concentrations, the reagent combination exhibits the highest sensitivity, strongest specificity, and best repeatability in capturing the plasmin-α2 plasmin inhibitor complex, thus achieving better technical results.

[0029] Furthermore, the preparation method of the magnetic microparticle suspension includes: after the magnetic microparticles are washed with the magnetic microparticle buffer, the magnetic microparticles are activated with EDC and NHS to obtain a magnetic microparticle solution, a plasmin-α2 plasmin inhibitor complex antibody is added to the magnetic microparticle solution, and after incubation, the solution is washed with the magnetic microparticle buffer to obtain the magnetic microparticle suspension;

[0030] The preparation method of the labeled antibody mixture includes: activating the plasmin-α2 plasmin inhibitor complex antibody with 2-IT solution, terminating the activation with glycine to obtain the activated plasmin-α2 plasmin inhibitor complex antibody; activating the tracer marker with SMCC solution, terminating the activation with glycine to obtain the activated tracer marker; mixing the activated plasmin-α2 plasmin inhibitor complex antibody and the activated tracer marker, purifying, and adding the labeled antibody buffer to obtain the labeled antibody mixture.

[0031] In some embodiments, the magnetic microparticles are washed three times with the magnetic microparticle buffer solution, the concentration of the magnetic microparticle solution is 10 mg / mL, and the mass ratio of the magnetic microparticle solution to the plasmin-α2 plasmin inhibitor complex antibody is (80-100):1.

[0032] In some embodiments, the mass ratio of the plasmin-α2 plasmin inhibitor complex antibody to the 2-IT is 1:(0.05-0.1), the mass ratio of the tracer label to the SMCC is 1:0.12, and after the activation reaction, the mass ratio of the plasmin-α2 plasmin inhibitor complex antibody to the tracer label is 1:(1.5-3).

[0033] This invention provides the application of the aforementioned reagent combination in the preparation of a kit for the assay of plasmin-α2 plasmin inhibitor complex.

[0034] This invention provides a kit for the assay of plasmin-α2 plasmin inhibitor complex, comprising the aforementioned reagent combination.

[0035] This invention provides a method for detecting plasmin-α2 plasmin inhibitor complex, comprising: detecting a sample using the reagent combination described in this invention or a plasmin-α2 plasmin inhibitor complex assay kit.

[0036] This invention provides a reagent for the assay of plasmin-α2 plasmin inhibitor complex, comprising a magnetic microparticle suspension and a labeled antibody mixture. The magnetic microparticle suspension includes magnetic microparticles coated with plasmin-α2 plasmin inhibitor complex antibody and a magnetic microparticle buffer. The labeled antibody mixture includes plasmin-α2 plasmin inhibitor complex antibody labeled with a tracer and a labeled antibody buffer. Both the magnetic microparticle buffer and the labeled antibody buffer include a buffer system, a stabilizer, and a preservative. The reagent combination provided by this invention has high accuracy and good repeatability. This reagent combination uses a "sandwich" immunoassay to detect the plasmin-α2 plasmin inhibitor complex and is suitable for fully automated analyzers (chemiluminescence method), with simple operation. The magnetic microparticle buffer of this invention contains a combination of the polyhydroxy compound hydroxypropyl methylcellulose and the nonionic polymeric surfactants nonylcyclohexanol polyoxyethylene ether and polyvinylpyrrolidone K30, exhibiting excellent suspension, solubilization, and dispersion properties, preventing antibody aggregation and precipitation. Attached Figure Description

[0037] Figure 1 Example 3 shows the Bland-Altman limit of conformity analysis (BLA) between the reagent and Sysmex reagent in clinical samples. Detailed Implementation

[0038] This invention provides a reagent for the assay of plasmin-α2 plasmin inhibitor complex and its application. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0039] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0040] Example 1

[0041] A reagent for detecting the concentration of plasmin-α2 plasmin inhibitor complex (PIC) in plasma, the reagent comprising:

[0042] Reagent 1 (magnetic microparticle suspension, the same below): magnetic microparticles and magnetic microparticle buffer coated with plasmin-α2 plasmin inhibitor complex antibody (commercially available) at a concentration of 5 mg / mL.

[0043] Reagent 2 (labeled antibody mixture, the same below): 1 μg / mL alkaline phosphatase-labeled plasmin-α2 plasmin inhibitor complex antibody (commercially available) and labeled antibody buffer.

[0044] The preparation method of magnetic microparticle reagent coated with plasmin-α2 plasmin inhibitor complex antibody is as follows:

[0045] 1) Place the thoroughly mixed carboxyl magnetic microparticles (hereinafter referred to as magnetic microparticles) into a reaction flask, place the reaction 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 5 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 10 mg / mL, add 0.4 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.6 mg of N-hydroxysuccinimide (NHS) to activate the magnetic beads.

[0046] 2) Add the plasmin-α2 plasmin inhibitor complex antibody to the magnetic microparticle solution prepared in step 1) at a mass ratio of 100:1, and incubate at room temperature for 4 hours.

[0047] 3) Place the reaction flask in a magnetic field for 5 minutes. After the magnetic particles are adsorbed, remove the supernatant, wash 3 times with magnetic particle buffer, then dilute to 5 mg / mL and store at 2-8℃ to obtain the required reagent one.

[0048] The magnetic microparticle buffer is a Tris-HCl buffer with pH 7.3±0.1, and its components are shown in Table 1 below.

[0049] Table 1

[0050] raw materials Mass (g / L) Tris(hydroxymethyl)aminomethane 12.114 Sodium chloride 5.844 Bovine serum albumin 10.000 Carboxymethyl cellulose 1.000 Sorbitol 25.000 Secondary alcohol polyoxyethylene ether 5.000 Polyethylene oxide-polypropylene oxide 80.000 Proclin300 0.010

[0051] The preparation method of alkaline phosphatase-labeled plasmin-α2 plasmin inhibitor complex antibody is as follows:

[0052] 1) Take 1 mg (1.0 mg / mL) of plasmin-α2 plasmin inhibitor 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.

[0053] 2) Take 1 mg of alkaline phosphatase and add it to 12 μL of Succinimidyl4-(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.

[0054] 3) Mix the activated plasmin-α2 plasmin inhibitor complex antibody with the activated alkaline phosphatase at a ratio of 1:2 and let it react at 2-8°C for 24 hours.

[0055] 4) Select an ultrafiltration column with an appropriate molecular weight cutoff to purify the above-mentioned ligation product. The obtained alkaline phosphatase-labeled plasmin-α2 plasmin inhibitor complex antibody is stored at 2-8℃ for later use.

[0056] 5) Add the prepared alkaline phosphatase-labeled plasmin-α2 plasmin inhibitor complex antibody to the labeled antibody buffer to a final concentration of 1 μg / mL to obtain the required reagent two.

[0057] The labeling antibody buffer solution is a PBS solution with pH 7.2, and its components are shown in Table 2 below.

[0058] Table 2

[0059] raw materials Mass (g / L) disodium hydrogen phosphate 10.221 Sodium dihydrogen phosphate 3.359 Sodium chloride 8.766 Bovine serum albumin 5.000 Mannitol 1.000 Polyethylene oxide-polypropylene oxide 1.000 Proclin300 0.010

[0060] Example 2

[0061] A reagent for detecting the concentration of plasmin-α2 plasmin inhibitor complex (PIC) in plasma, the reagent comprising:

[0062] Reagent 1: Magnetic microparticles coated with plasmin-α2 plasmin inhibitor complex antibody at a concentration of 10 mg / mL and magnetic microparticle buffer.

[0063] Reagent 2: Alkaline phosphatase-labeled plasmin-α2 plasmin inhibitor complex antibody and labeled antibody buffer at a concentration of 1 μg / mL.

[0064] The preparation method of magnetic microparticle reagent coated with plasmin-α2 plasmin inhibitor complex antibody is as follows:

[0065] 1) The cleaning and activation steps for carboxyl magnetic microparticles are described in Example 1.

[0066] 2) Add the plasmin-α2 plasmin inhibitor complex antibody to the magnetic microparticle solution prepared in step 1) at a mass ratio of 80:1 (magnetic microparticle solution: plasmin-α2 plasmin inhibitor complex antibody) and incubate at room temperature for 2 hours.

[0067] 3) Place the reaction flask in a magnetic field for 5 minutes. After the magnetic particles settle, wash it 3 times with magnetic particle buffer solution, then dilute it to 10 mg / mL and store it at 2-8℃ to obtain the required magnetic particle reagent.

[0068] The magnetic microparticle buffer is a Tris-HCl buffer with pH 7.3±0.1, and its components are shown in Table 3 below.

[0069] Table 3

[0070] raw materials Mass (g / L) Tris(hydroxymethyl)aminomethane 12.114 Sodium chloride 5.844 Bovine serum albumin 10.000 Hydroxyethyl cellulose 5.000 Isomeric tridecyl alcohol polyoxyethylene ether 5.000 Sodium azide 0.100

[0071] The preparation method of alkaline phosphatase-labeled plasmin-α2 plasmin inhibitor complex antibody is as follows:

[0072] 1) Take 1 mg (1.0 mg / mL) of plasmin-α2 plasmin inhibitor complex antibody, add 5 μL of 20 mg / mL activator 2-Imino thiolane hydrochloride (2-IT) solution, let stand at room temperature for 30 minutes, add glycine, let stand at room temperature for 5 minutes to terminate the activation reaction, and obtain the activated antibody.

[0073] 2) The alkaline phosphatase activation steps are described in Example 1.

[0074] 3) Mix the activated plasmin-α2 plasmin inhibitor complex antibody with the activated alkaline phosphatase at a ratio of 1:3 and let it react at 2-8℃ for 20 hours.

[0075] 4) Select an ultrafiltration column with an appropriate molecular weight cutoff to purify the above-mentioned ligation product. The obtained alkaline phosphatase-labeled plasmin-α2 plasmin inhibitor complex antibody is stored at 2-8℃ for later use.

[0076] 5) Add the prepared alkaline phosphatase-labeled plasmin-α2 plasmin inhibitor complex antibody to the labeled antibody buffer to a final concentration of 1 μg / mL to obtain the required reagent two.

[0077] The labeling antibody buffer is a HEPES buffer with pH 7.3±0.1, and its components are shown in Table 4 below.

[0078] Table 4

[0079] raw materials Mass (g / L) HEPES 13.000 Sodium chloride 16.000 Sodium hydrogen phosphate heptahydrate 0.380 Bovine serum albumin 5.000 Hydroxypropyl methylcellulose 5.000 Sodium azide 0.100

[0080] Example 3

[0081] A reagent for detecting the concentration of plasmin-α2 plasmin inhibitor complex (PIC) in plasma, the reagent comprising:

[0082] Reagent 1: Magnetic microparticles coated with plasmin-α2 plasmin inhibitor complex antibody at a concentration of 10 mg / mL and magnetic microparticle buffer.

[0083] Reagent 2: Alkaline phosphatase-labeled plasmin-α2 plasmin inhibitor complex antibody and labeled antibody buffer at a concentration of 0.5 μg / mL.

[0084] The preparation method of magnetic microparticle reagent coated with plasmin-α2 plasmin inhibitor complex antibody is as follows:

[0085] 1) The cleaning and activation steps for carboxyl magnetic microparticles are described in Example 1.

[0086] 2) According to the mass ratio of magnetic microparticle solution: plasmin-α2 plasmin inhibitor complex antibody = 80:1, add plasmin-α2 plasmin inhibitor complex antibody to the magnetic microparticle solution prepared in step 1) and incubate at room temperature for 4 hours;

[0087] 3) Place the reaction flask in a magnetic field for 5 minutes. After the magnetic particles settle, wash it 3 times with magnetic particle buffer solution, then dilute it to 10 mg / mL and store it at 2-8℃ to obtain the required magnetic particle reagent.

[0088] The magnetic microparticle buffer is a Tris-HCl buffer with pH 7.3±0.1, and its components are shown in Table 5 below.

[0089] Table 5

[0090] raw materials Mass (g / L) Tris(hydroxymethyl)aminomethane 12.114 Sodium chloride 5.844 Bovine serum albumin 10.000 Hydroxypropyl methylcellulose 5.000 Nonylcyclohexanol polyoxyethylene ether 10.000 Polyvinylpyrrolidone K30 50.000 Sodium azide 0.100

[0091] The preparation method of alkaline phosphatase-labeled plasmin-α2 plasmin inhibitor complex antibody is as follows:

[0092] 1) Take 1 mg (1.0 mg / mL) of plasmin-α2 plasmin inhibitor complex antibody, add 5 μL of 10 mg / mL activator 2-Imino thiolane hydrochloride (2-IT) solution, let stand at room temperature for 30 minutes, add glycine, let stand at room temperature for 5 minutes to terminate the activation reaction, and obtain the activated antibody.

[0093] 2) The alkaline phosphatase activation steps are described in Example 1.

[0094] 3) Mix the activated plasmin-α2 plasmin inhibitor complex antibody with the activated alkaline phosphatase at a ratio of 1:1.5 and let it react at 2-8℃ for 16 hours.

[0095] 4) Select an ultrafiltration column with an appropriate molecular weight cutoff to purify the above-mentioned ligation product. The obtained alkaline phosphatase-labeled plasmin-α2 plasmin inhibitor complex antibody is stored at 2-8℃ for later use.

[0096] 5) Add the prepared alkaline phosphatase-labeled plasmin-α2 plasmin inhibitor complex antibody to the labeled antibody buffer to a final concentration of 0.5 μg / mL to obtain the required reagent two.

[0097] The labeling antibody buffer solution is a PBS solution with pH 7.2, and its components are shown in Table 6 below.

[0098] Table 6

[0099] raw materials Mass (g / L) disodium hydrogen phosphate 10.221 Sodium dihydrogen phosphate 3.359 Sodium chloride 8.766 Bovine serum albumin 5.000 Hydroxypropyl methylcellulose 1.000 Polyvinylpyrrolidone K30 0.500 Sodium azide 0.100

[0100] Example 4

[0101] A reagent for detecting the concentration of plasmin-α2 plasmin inhibitor complex (PIC) in plasma, the reagent comprising:

[0102] Reagent 1: Magnetic microparticles coated with plasmin-α2 plasmin inhibitor complex antibody at a concentration of 10 mg / mL and magnetic microparticle buffer.

[0103] Reagent 2: Alkaline phosphatase-labeled plasmin-α2 plasmin inhibitor complex antibody and labeled antibody buffer at a concentration of 0.5 μg / mL.

[0104] The preparation method of the magnetic microparticle reagent coated with the plasmin-α2 plasmin inhibitor complex is described in Example 3.

[0105] The magnetic microparticle buffer is a Tris-HCl buffer with pH 7.3±0.1, and its components are shown in Table 7 below.

[0106] Table 7

[0107]

[0108]

[0109] The preparation method of the alkaline phosphatase-labeled plasmin-α2 plasmin inhibitor complex antibody is described in Example 3.

[0110] The buffer system for the labeled antibody buffer is PBS with pH 7.2, and the components are shown in Table 8 below.

[0111] Table 8

[0112] raw materials Mass (g / L) disodium hydrogen phosphate 10.221 Sodium dihydrogen phosphate 3.359 Sodium chloride 8.766 Bovine serum albumin 5.000 Hydroxypropyl methylcellulose 0.500 Polyvinylpyrrolidone K30 0.500 Sodium azide 0.100

[0113] Example 5

[0114] A reagent for detecting the concentration of plasmin-α2 plasmin inhibitor complex (PIC) in plasma, the reagent comprising:

[0115] Reagent 1: Magnetic microparticles coated with plasmin-α2 plasmin inhibitor complex antibody at a concentration of 10 mg / mL and magnetic microparticle buffer.

[0116] Reagent 2: Alkaline phosphatase-labeled plasmin-α2 plasmin inhibitor complex antibody and labeled antibody buffer at a concentration of 0.5 μg / mL.

[0117] The preparation method of the magnetic microparticle reagent coated with the plasmin-α2 plasmin inhibitor complex is described in Example 3.

[0118] The magnetic microparticle buffer is a MOPS buffer with pH 7.3±0.1, and its components are shown in Table 9 below.

[0119] Table 9

[0120]

[0121]

[0122] The preparation method of the alkaline phosphatase-labeled plasmin-α2 plasmin inhibitor complex antibody is described in Example 3.

[0123] The labeling antibody buffer is a PBS solution with pH 7.2, and its components are shown in Table 10 below.

[0124] Table 10

[0125] raw materials Mass (g / L) disodium hydrogen phosphate 10.221 Sodium dihydrogen phosphate 3.359 Sodium chloride 8.766 Bovine serum albumin 5.000 Hydroxypropyl methylcellulose 0.200 Sodium azide 0.100

[0126] Example 6

[0127] Further explanation of the detection method for plasmin-α2 plasmin inhibitor complex (PIC) assay reagent (chemiluminescence method), including the following steps:

[0128] 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.

[0129] The second step is to remove the reaction solution, add 500μL of cleaning solution, and wash 3 times.

[0130] Third, add 200 μL of substrate solution, mix well, and detect the luminescence intensity;

[0131] The fourth step is to calculate the concentration of the plasmin-α2 plasmin inhibitor complex based on the luminescence intensity and the standard curve.

[0132] Example of effect

[0133] 1. Repeatability testing

[0134] The same normal sample was tested 10 times using the reagents from Examples 1-5 and the Sysmex reagent, respectively. The coefficient of variation and relative deviation were calculated. The repeatability results are shown in Table 11, and the relative deviations between the values ​​measured by Examples 1-5 and the Sysmex reagent are shown in Table 12. Wherein, difference = Sysmex reagent value - Example reagent value; mean = (mean of Sysmex reagent value + mean of Example reagent value) / 2; relative deviation = 100% * difference / mean.

[0135] Table 11. Repeatability test results of Examples 1-5 with Sysmex reagent

[0136]

[0137] As shown in Table 11, the CVs of the test results in Examples 1 to 5 are all below 8%, which meets the standard, and the repeatability of Examples 1 to 5 is good. Among them, Example 3 is better.

[0138] Table 12 Relative deviations between the values ​​measured by Examples 1-5 and Sysmex reagent

[0139]

[0140] From the relative deviations of each embodiment (Table 12), the relative deviation of Example 3 is smaller, that is, the detection results of Example 3 are closer to those of Sysmex reagent.

[0141] 2. Clinical sample comparison

[0142] 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.

[0143] Example 3 was selected, and 200 samples were tested simultaneously with Sysmex reagent.

[0144] The test results were subjected to Bland-Altman consistency limit analysis, and the results are shown below. Figure 1 See Table 13. In detail, a deviation graph was plotted with the mean of the Sysmex reagent and the reagent of Example 3 as the x-axis and the percentage of the difference between the measured values ​​of the Sysmex reagent and the reagent of Example 3 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.

[0145] Table 13. Bland-Altman concordance limit analysis between clinical samples of the reagents in Example 3 and Sysmex reagents.

[0146]

[0147] Depend on Figure 1 As shown in Table 3, 95.5% 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 3 were in good agreement with the sample test results of Sysmex reagent.

[0148] In summary, the reagents of this invention have good repeatability, use a one-step method, are suitable for fully automated detection and analysis instruments (chemiluminescence method), are simple to operate, and can be widely used in the detection of plasmin-α2 plasmin inhibitor complexes.

[0149] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications 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 reagent combination, characterized in that The magnetic particle suspension and the labeled antibody mixture are mixed to form a magnetic particle suspension solution. The magnetic particle suspension is composed of 5-10 g / L fibrinolysin-α2 plasmin inhibitor complex antibody coated magnetic particles, 12.114 g / L buffer system A, 25.884-126.844 g / L stabilizer A, and 0.01-0.1 g / L preservative A. The labeled antibody mixture is composed of 0.5-1 mg / L tracer labeled fibrinolysin-α2 plasmin inhibitor complex antibody, 13-13.58 g / L buffer system B, 14.766-26.38 g / L stabilizer B, and 0.01-0.1 g / L preservative B. The buffer system A and B are independently selected from at least one of MOPS, Tris-HCl, MES, PBS, HEPES, glycine, and boric acid. The stabilizer A includes sodium chloride, bovine serum albumin, hydroxypropyl methyl cellulose, nonylcyclohexanol polyoxyethylene ether, and polyvinylpyrrolidone K30, and the stabilizer B includes sodium chloride, bovine serum albumin, hydroxypropyl methyl cellulose, and polyvinylpyrrolidone K30. The preservative A and B are independently selected from at least one of NaN3, Proclin 300, and Proclin 600.

2. The combination of agents according to claim 1, characterized in that, The magnetic particles include amino magnetic particles or carboxyl magnetic particles, and the tracer label includes horseradish peroxidase or alkaline phosphatase.

3. The combination of agents according to claim 1 or 2, characterized in that, The buffer system A is Tris-HCl, and the buffer system B is PBS.

4. The combination of agents according to claim 1 or 2, characterized in that, The preservative A and the preservative B are NaN3.

5. The reagent combination according to claim 4, wherein The magnetic particle suspension is composed of 10 g / L fibrinolysin-α2 plasmin inhibitor complex antibody coated magnetic particles, 12.114 g / L Tris-HCl, 5.844 g / L sodium chloride, 10 g / L bovine serum albumin, 5 g / L hydroxypropyl methyl cellulose, 10 g / L nonylcyclohexanol polyoxyethylene ether, 50 g / L polyvinylpyrrolidone K30, and 0.1 g / L NaN3. The labeled antibody mixture is composed of 0.5 mg / L tracer labeled fibrinolysin-α2 plasmin inhibitor complex antibody, 13.58 g / L PBS, 8.766 g / L sodium chloride, 5 g / L bovine serum albumin, 1 g / L hydroxypropyl methyl cellulose, 0.5 g / L polyvinylpyrrolidone K30, and 0.1 g / L NaN3.

6. The reagent combination according to claim 1 or 2, wherein The preparation method of the magnetic particle suspension includes: after the magnetic particles are washed by a magnetic particle buffer, the magnetic particles are activated by EDC and NHS to obtain a magnetic particle solution, the fibrinolysin-α2 plasmin inhibitor complex antibody is added to the magnetic particle solution, and after incubation, the magnetic particle solution is washed by the magnetic particle buffer to obtain the magnetic particle suspension. The preparation method of the labeled antibody mixture comprises: taking the activated plasmin-α2 plasmin inhibitor complex antibody after plasmin-α2 plasmin inhibitor complex antibody is activated with 2-IT solution and terminated with glycine, taking the activated tracer label after the tracer label is activated with SMCC solution and terminated with glycine, mixing and reacting the activated plasmin-α2 plasmin inhibitor complex antibody and the activated tracer label, purifying, and adding the labeled antibody buffer to obtain the labeled antibody mixture.

7. Use of the reagent combination according to any one of claims 1-6 in the preparation of a plasmin-α2 plasmin inhibitor complex assay kit.

8. A plasmin-α2 plasmin inhibitor complex assay kit characterized in that, The reagent combination according to any one of claims 1-6 is included.

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