Thrombomodulin chemiluminescence detection kit

Abstract

The present application relates to the technical field of in vitro detection, in particular to a thrombomodulin chemiluminescence detection kit. The present application specifically provides a preparation method of carboxyl magnetic beads and application thereof, and the carboxyl magnetic beads prepared by the method have high carboxyl content on the surface and high coupling rate with antigens or antibodies. The thrombomodulin chemiluminescence detection kit prepared by the carboxyl magnetic beads provided by the present application has stable properties, good accuracy and repeatability of measurement, and can be applied to clinical use.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of in vitro detection technology, in particular to a thrombomodulin chemiluminescence detection kit. BACKGROUND

[0002] Thrombomodulin (TM) is a transmembrane glycoprotein existing on the cell membrane surface, which is synthesized by megakaryocytes and endothelial cells and widely distributed on the surface of vascular endothelial cells. After TM binds with thrombin, it reduces the coagulation activity of thrombin and enhances the activity of activated protein C. Since activated protein C has an anticoagulant effect, TM is an important coagulation inhibitor. Under normal physiological conditions, TM is distributed on the surface of the cytoplasmic membrane. When the vascular endothelial cells are damaged, it often causes abnormal expression, secretion and release of TM into the blood, causing changes in the content of thrombomodulin in plasma. Clinically, the increase of blood TM content is seen in disseminated intravascular coagulation, acute myocardial infarction and cerebral thrombosis, etc. Therefore, the determination of the content of thrombomodulin is of great significance for the diagnosis and treatment of some diseases.

[0003] At present, the methods for determining thrombomodulin mainly include enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). However, both of them have some defects in practical application. ELISA is complicated to operate, time-consuming, unstable in test results, poor in repeatability, inconvenient for on-site testing and hospital emergency; RIA has radioactive contamination, unstable test results, and the required instrument is expensive. Therefore, it is necessary to develop a reagent kit that is simple to operate and can quickly and accurately detect thrombomodulin. Magnetic nanoparticles have the advantages of large specific surface area, fast separation and washing speed, good reproducibility, and can be coupled with labels. Some researchers have applied magnetic microparticles as detection carriers to clinical project detection. Magnetic microparticle chemiluminescence immunoassay (CLIA) based on magnetic nanoparticles combines the high sensitivity of chemiluminescence detection technology and the high specificity of immune response, and has the characteristics of high sensitivity, wide linear range, high specificity, stable measurement value, high automation, etc. Magnetic microparticle chemiluminescence method combines magnetic separation technology, immunoassay technology and chemiluminescence technology to complete the quantitative determination of specific antigens. Antigen first binds with antibody 1 coated on the surface of magnetic beads to form a stable antigen-antibody 1 complex, enzyme-labeled antibody then binds with other sites of antigen to form a double antibody sandwich complex, and alkaline phosphatase labeled on the antibody catalyzes the hydrolysis of luminescent substrate to emit light signal. The light signal value is positively correlated with the concentration of alkaline phosphatase, and the quantitative test of double antibody sandwich complex (antigen) is realized by detecting and analyzing the chemiluminescence instrument.

[0004] The carboxyl magnetic beads prepared by the prior method have poor process repeatability, unstable product quality, poor antigen or antibody coupling rate and poor detection result; and the TM magnetic microparticle chemiluminescence detection kit usually uses streptavidin-biotin or radioactive element labeling, and the detection result is easily interfered by excessive label and has radioactive hazard. SUMMARY

[0005] Therefore, the present application aims to provide a thrombomodulin chemiluminescence detection kit, and specifically provide a preparation method of core-shell type superparamagnetic magnetic microparticles, and preparation of magnetic microparticle suspension (immunomagnetic beads) coated with thrombomodulin antibody 1 and alkaline phosphatase labeled thrombomodulin antibody 2 (enzyme labeled antibody).

[0006] The present application provides a preparation method of carboxyl magnetic beads, which comprises the following steps: preparing a nano microparticle core from a soluble trivalent iron ion salt, coating the nano microparticle core with silicon dioxide, and reacting with carboxylated silane after magnetic separation.

[0007] Specifically, the step of preparing a nano microparticle core from a soluble trivalent iron ion salt comprises the following steps: mixing and reacting a soluble trivalent iron ion salt, sodium acetate, a high molecular polymer and a mixed solution of ethylene glycol and ethylenediamine, and obtaining Fe3O4 nano microparticles after magnetic separation.

[0008] The high molecular polymer is selected from any one of polyoxyethylene, polylactic acid, polyvinylpyrrolidone and sodium carboxymethyl cellulose, and in the embodiment of the present application, the high molecular polymer is sodium carboxymethyl cellulose.

[0009] The reaction temperature is 180-300℃, and the reaction time is 6-12h, and in the embodiment of the present application, the reaction temperature is 220℃, and the reaction time is 8h.

[0010] Specifically, the step of coating the nano microparticle core with silicon dioxide comprises the following steps: resuspending the Fe3O4 nano microparticles with an alkali solution, and reacting with tetraethyl orthosilicate, and obtaining silicon dioxide coated Fe3O4 nano microparticles after magnetic separation.

[0011] The mass ratio of the tetraethyl orthosilicate to the Fe3O4 nano microparticles is 1:(10-30).

[0012] The resuspension liquid of the resuspended Fe3O4 nano microparticles has a pH value of 10-12, and preferably a pH value of 10.5.

[0013] The tetraethyl orthosilicate is dissolved in ethanol, and the concentration of the tetraethyl orthosilicate is 10%.

[0014] The reaction temperature is 25-50℃, and preferably 25℃, and the reaction time is 3-6h, and preferably 5h.

[0015] Specifically, the preparation method of the carboxylated silane comprises reacting an amino silane with an acid anhydride in a water-free solvent for 1-2 hours to obtain;

[0016] The molar ratio of the amino silane to the acid anhydride is 1:(2-30), preferably 1:10;

[0017] The amino silane is selected from any one of vinyltriethoxysilane, vinyltrimethoxysilane, vinyltri(β-methoxyethoxy)silane, (3-mercaptopropyl)trimethoxysilane, and preferably vinyltri(β-methoxyethoxy)silane;

[0018] The acid anhydride is selected from any one of hexanoic anhydride, phenylthioformic anhydride, benzenesulfonic anhydride, ethyl propionic anhydride, cyclohexanecarboxylic anhydride, and ethyl chloroacetate anhydride, and preferably ethyl chloroacetate anhydride.

[0019] Under the same molar ratio, the type of the amino silane and the acid anhydride affects the number and site of the carboxyl modification on the surface of the silica-coated Fe3O4 nanoparticle, and further affects the coupling rate of the antibody and the detection of the kit, so the present application screens different amino silanes and acid anhydrides, and the results show that the coupling rate of the carboxyl magnetic bead prepared by using vinyltri(β-methoxyethoxy)silane and ethyl chloroacetate anhydride is the highest, and the coefficient of variation is the smallest, which is superior to the magnetic beads prepared by using other amino silanes and acid anhydrides.

[0020] Specifically, the step of reacting the silica-coated Fe3O4 nanoparticle with the carboxylated silane comprises dissolving the carboxyl silane in anhydrous ethanol and then reacting with the silica-coated Fe3O4 nanoparticle for 2-6 hours to obtain;

[0021] The reaction temperature is 25-50°C, and preferably 25°C, and the pH value is 10-20, and preferably 11.

[0022] The present application provides a carboxyl magnetic bead obtained by the preparation method.

[0023] Compared with the immunomagnetic beads prepared from the commercially available magnetic beads, the immunomagnetic beads prepared from the carboxyl magnetic beads have high and stable coupling rate, and can be widely applied to the preparation of a chemiluminescence detection kit, so the present application provides the application of the carboxyl magnetic bead in the preparation of a chemiluminescence detection kit.

[0024] The present application further provides a thrombomodulin detection kit comprising the immunomagnetic beads prepared from the carboxyl magnetic bead and an enzyme-labeled TM antibody;

[0025] The immunomagnetic beads are coupled with a TM antibody 1, and the enzyme-labeled TM antibody is an alkaline phosphatase-labeled TM antibody 2.

[0026] Specifically, the TM antibodies 1 and 2 include monoclonal antibodies or modified antibody fragments with Fab activity, antibodies, antibody fragment multimers, can specifically bind to human TM surface antigen determinants, and can be derived from mice, rabbits, sheep, dogs, etc., preferably mouse-derived monoclonal antibodies;

[0027] The TM antibodies 1 and 2 are commercially available antibodies;

[0028] The mass ratio of the carboxyl magnetic beads to the TM antibody 1 is (10-200):1, preferably 50:1;

[0029] The mass ratio of the alkaline phosphatase to the TM antibody 2 is (0.5-10):1, preferably 1:1.

[0030] In some specific embodiments, the carboxyl magnetic beads coupled with the TM antibody 1 can be coupled by EDC, EDC / NHS, EDC / Sulfo-NHS, etc. activators, preferably EDC / Sulfo-NHS. The coupling steps are as follows:

[0031] a) uniformly disperse the magnetic microparticles in the activation buffer;

[0032] b) dissolve the activator using the activation buffer, add it to the magnetic microparticle suspension and mix uniformly, incubate at room temperature (10-30°C) for 30 min-2 h to obtain activated magnetic beads;

[0033] c) remove the residual activator by magnetic separation, and add the cross-linking buffer to disperse the magnetic microparticles again;

[0034] d) dilute the TM antibody 1 using the cross-linking buffer, add it to the activated magnetic microparticle suspension, mix uniformly, and incubate at 4-30°C for 0.5-12 h;

[0035] e) magnetic separation, add the quenching buffer to the immunomagnetic beads, and incubate at 4-30°C for 0.5-3 h;

[0036] f) disperse the immunomagnetic beads using the storage buffer after magnetic separation, and store for later use.

[0037] The mass ratio of the magnetic beads to the activator is 0.1:1-20:1, preferably 20:1.

[0038] The blocking agent can be one or more of ethanolamine, bovine serum albumin, casein, amino acids, etc. containing free amino groups.

[0039] The activation and cross-linking buffers can be one of MES buffer, boric acid buffer, PBS buffer, MOPS buffer, HEPES buffer, etc. without amino and carboxyl groups, preferably MES buffer;

[0040] The activation and cross-linking buffer has a pH range of 5.5-7.0, preferably 6.5.

[0041] The storage buffer buffer system can be one of MOPS buffer, Tris-HCl buffer, PBS buffer, HEPES buffer, glycine buffer, etc., preferably Tris-HCl buffer.

[0042] The storage buffer contains bovine serum albumin (BSA) and preservative NaN3.

[0043] The storage buffer has a pH range of 6.0-9.0, preferably 8.0.

[0044] In other specific embodiments, the enzyme-labeled antibody can be prepared by glutaraldehyde method, sodium periodate method or heterobifunctional reagent cross-linking method, preferably Sulfo-SMCC cross-linking method. The preparation steps are as follows:

[0045] a) Dissolve / dilute alkaline phosphatase with enzyme-labeled buffer, add a certain amount of Sulfo-SMCC, and incubate at 4-30°C for 0.5-3h, then desalt the product and store it for later use;

[0046] b) Dissolve / dilute TM antibody 2 with enzyme-labeled buffer, add a certain amount of Traut's reagent, and incubate at 4-30°C for 0.5-3h, then add 0.5%-2.5% glycine solution to quench for 5min, desalt the product and store it for later use;

[0047] c) Mix the activated alkaline phosphatase with the thiolated antibody, and incubate at 4-30°C with gentle stirring for 2-30h, then add 0.5-2.5% cysteine solution to block for 0.5-2h;

[0048] d) Desalt the product and store it in storage buffer for later use.

[0049] The mass ratio of alkaline phosphatase / antibody to Sulfo-SMCC is 2-30:1, preferably 15:1.

[0050] The mass ratio of alkaline phosphatase / antibody to Traut's is 20-250:1, preferably 20:1.

[0051] The enzyme-labeled buffer can be one of MES buffer, boric acid buffer, PBS buffer, etc., which do not contain amino and carboxyl groups, preferably Tris-HCl buffer.

[0052] The pH of the enzyme-labeled buffer is 7.0-9.0, the spatial structure of the antibody is different under different pH conditions, the site coupled with alkaline phosphatase is different, different stereoscopic enzyme-labeled antibody complexes are formed in the preparation process, the recognition and combination of the antigen are affected, and then the detection of thrombomodulin is affected, and the pH is preferably 7.5.

[0053] The storage buffer buffer system can be one of MOPS buffer, Tris-HCl buffer, PBS buffer, HEPES buffer, glycine buffer and the like, and preferably Tris-HCl buffer.

[0054] The storage buffer contains bovine serum albumin (BSA) and preservative NaN3; the pH of the storage buffer is 6.0-9.0, preferably 8.0.

[0055] The application also provides a method for detecting the content of thrombomodulin, comprising using the kit of the application.

[0056] The method provided by the application can be for diagnostic purposes or for non-diagnostic purposes. For example, it includes a detection method for diagnostic purposes on a human or animal body, or a sample from a human or animal body ex vivo; it also includes a detection method for scientific research or other non-diagnostic purposes on an environmental sample or a simulated sample.

[0057] The application prepares a carboxyl magnetic bead with stable product quality and high antigen or antibody coupling rate, and uses the carboxyl magnetic bead as a carrier, labels the antibody with alkaline phosphatase, uses TM paired antibodies to capture antigens in a plasma sample, and completes quantitative detection by using a full-automatic immune test system with a substrate liquid containing AMPPD or APS-5 and the like. The core-shell superparamagnetic magnetic microparticle antibody coupling rate is high, the kit is stable in quality, and the measured value is good in repeatability. Specifically, the carboxyl magnetic bead prepared by the application has stable product quality, high antigen or antibody coupling rate, simple preparation process, safe and reliable operation, easy control, and can quickly capture antigens in a sample, the kit is stable in quality, and the measured value is good in repeatability.

[0058] Drawings of the specification

[0059] Figure 1 Different enzyme-labeled antibody group calibration curve graphs are shown. DETAILED DESCRIPTION

[0060] The present application provides a thrombomodulin chemiluminescence detection kit, and those skilled in the art can improve the process parameters according to the content herein. It should be particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0061] The test materials used in the present application are all ordinary commercially available products and can be purchased in the market.

[0062] The present application will be further described below in conjunction with examples:

[0063] Example 1 Preparation of carboxyl magnetic beads

[0064] 1.1 Preparation of carboxyl magnetic beads

[0065] Dissolve 10 mg of soluble iron salt FeCl3·6H2O and 25 mg of sodium acetate in 100 mL of a mixed solution of ethylene glycol and ethylenediamine, add 0.1 mg of sodium carboxymethyl cellulose, mix well, and react at 220°C for 8 hours. After the reaction is completed, the Fe3O4 nanoparticles, i.e. the core of the carboxyl magnetic beads, are obtained by magnetic separation.

[0066] Resuspend the Fe3O4 nanoparticles in 60 mL of lye, adjust the pH to 10.5, add 0.5 mL of tetraethyl orthosilicate (10%) dissolved in ethanol, mix well, and stir at 25°C for 5 hours. After the reaction is completed, the Fe3O4 nanoparticles coated with silica with a core-shell structure are obtained by magnetic separation.

[0067] Dissolve 2.85 g of vinyltriethoxysilane (0.015 mol of aminosilane) and 3.57 g of cyclohexanecarboxylic anhydride (0.15 mol of anhydride) in 50 mL of anhydrous ethanol, and stir at room temperature for 2 h to prepare carboxylated silane for modification of the carboxyl groups on the surface of the Fe3O4 nanoparticles coated with silica.

[0068] Add the Fe3O4 nanoparticles coated with silica to the carboxylated silane, and stir at a pH of 11 and a temperature of 25°C for 4 hours. After the reaction is completed, the carboxyl superparamagnetic microparticles with a core-shell structure are obtained by magnetic separation.

[0069] Dilute the prepared magnetic microparticles to 50 mg / mL with a magnetic bead preservation solution for storage and standby use.

[0070] 1.2 Optimization of aminosilane and anhydride

[0071] The kind of aminosilane and acid anhydride can affect the number and site of carboxyl modification on the surface of silica-coated Fe3O4nanoparticles, and further affect the coupling rate of antibody, and affect the detection of the kit. Therefore, different aminosilanes and acid anhydrides were selected for investigation. The aminosilanes vinyltriethoxysilane, vinyltrimethoxysilane, and vinyltris(β-methoxyethoxy)silane were combined with the acid anhydrides cyclohexanecarbonyl anhydride and ethylchloroacetate, as shown in Table 1. The carboxyl magnetic beads were prepared according to the experimental procedure of Example 1.1 and stored for determination of the antibody coupling rate. After the antibody was coupled to the magnetic beads, the supernatant was separated by magnetic separation, and the antibody concentration in the supernatant was determined by the BCA method. The antibody coupling rate of the different groups of magnetic beads was calculated based on the volume of the supernatant and the amount of antibody fed. By comparing the antibody coupling rates of the different groups of immunomagnetic beads, the best aminosilane and acid anhydride in the preparation process were determined.

[0072] Table 1 Aminosilane and acid anhydride for preparing carboxyl magnetic beads in different groups

[0073] Group Aminosilane Anhydride 1 Vinyltriethoxysilane Cyclohexanecarboxylic anhydride 2 Vinyltrimethoxysilane Cyclohexanecarboxylic anhydride 3 Vinyltri(β-methoxyethoxy)silane Cyclohexanecarboxylic anhydride 4 Vinyltriethoxysilane Ethylchloroacetate anhydride 5 Vinyltrimethoxysilane Ethylchloroacetate anhydride 6 Vinyltri(β-methoxyethoxy)silane Ethylchloroacetate anhydride

[0074] 1.3 Determination of carboxyl content

[0075] The carboxyl content on the surface of the carboxyl magnetic beads was tested by acid-base chemical titration. 0.2 mg of carboxyl magnetic beads were weighed into a 100 mL conical flask, 10 mL of methanol was added, and the mixture was ultrasonically dispersed for 2 min. 2-3 drops of an acid-base indicator, phenolphthalein, were added, and the mixture was shaken well. Titration was performed using 0.01 mol / L NaOH until the solution turned light pink and did not fade within 30 seconds. The volume of the base used during titration, V1, was recorded. The blank sample was determined in the same way, and the volume of the base used during titration, V2, was recorded after titration. The carboxyl content on the surface of the carboxyl magnetic beads (nmol / mg) was calculated based on the difference between the amounts of base consumed in the two experiments (V1-V2) and the concentration of the NaOH base used in the experiment.

[0076] Example 2 Preparation of the kit

[0077] 2.1 Preparation of immunomagnetic beads

[0078] The carboxyl magnetic beads were mixed well using a vortex mixer. 10 mg of the magnetic bead solution was taken into a centrifuge tube, and 1 mL of activation buffer (0.1 M MES, pH 6.5) was added to make up the volume. After mixing well, the mixture was magnetically separated for 1 min, and the supernatant was removed. Another 1 mL of activation buffer was added, the mixture was mixed well, and then magnetically separated for 1 min. The supernatant was removed, and 1 mL of activation buffer was added to the mixture, which was mixed well and ready for use.

[0079] After adding 100 μL of activator (5 mg / mL EDC & 5 mg / mL Sulfo-NHS, prepared fresh) and mixing well, incubate at room temperature for 30 min with rotation.

[0080] After magnetic separation, remove the supernatant, take 200 μg of TM antibody 1, add cross-linking buffer to 1 mL, and add to the activated magnetic beads. After mixing well, incubate at room temperature for 4 h with rotation.

[0081] Magnetic separation, take the supernatant for determination of the coupling rate of the immunomagnetic beads antibody;

[0082] Add 1 mL of cross-linking buffer containing 1% BSA, block at room temperature for 1 h with rotation, and after magnetic separation, remove the supernatant.

[0083] Use 1 mL of storage buffer to disperse the coupled immunomagnetic beads, store at 2-8°C, and before testing, dilute to 0.5 mg / mL using the storage buffer.

[0084] 2.2, Enzyme-labeled antibody preparation

[0085] Dissolve / dilute 1 mg of alkaline phosphatase to 10 mg / mL of alkaline phosphatase solution using 1 mL of enzyme-labeled buffer (0.1 M PBS, 0.15 M NaCl, pH 7.5), add 16.7 μL of Sulfo-SMCC (4 mg / mL), and incubate gently at room temperature for 1 h. After desalting the product, disperse it in enzyme-labeled buffer for use;

[0086] Dilute 1 mg of TM antibody 2 to 10 mg / mL of antibody solution using 1 mL of enzyme-labeled buffer, add 5 μL of Traut's (10 mg / mL), and incubate gently at room temperature for 1 h. After mixing well with 20 μL (5 mg / mL) of glycine solution, continue to incubate for 5 min. After desalting the product, disperse it in enzyme-labeled buffer for use;

[0087] After mixing well with activated alkaline phosphatase and thiolated TM antibody 2, incubate gently at 4°C for 24 h. After mixing well with 50 μL (10 mg / mL) of cysteine, continue to incubate for 30 min. After desalting the product, dilute it with enzyme-labeled storage buffer. Before testing, dilute to 0.5 μg / mL using enzyme-labeled storage buffer.

[0088] 2.3, Determination of antibody coupling rate

[0089] Carboxyl magnetic beads were prepared using different amino silane and acid anhydride respectively, and the antibody coupling rate of the immunomagnetic beads was tested after coupling with antibodies. The antibody concentration in the supernatant was determined by BCA method, and the antibody coupling rate of the immunomagnetic beads was calculated according to the antibody feed amount, the volume of supernatant after magnetic separation, and the protein concentration. The antibody coupling rates of three different batches of magnetic beads were tested, and the average value and coefficient of variation CV of the antibody coupling rate were calculated. The results are shown in Table 2. The antibody coupling rate of the magnetic beads prepared using vinyl tri(β-methoxyethoxy) silane and ethyl chloroacetate was the highest, and the coefficient of variation CV was the smallest, indicating that the quality of the carboxyl magnetic beads was stable, and vinyl tri(β-methoxyethoxy) silane and ethyl chloroacetate were determined to be suitable for preparing carboxyl magnetic beads.

[0090] Table 2 Comparison of antibody coupling rates of different carboxyl magnetic beads

[0091]

[0092] Immunomagnetic beads were prepared by coupling antibodies with carboxyl magnetic beads prepared by the present application and carboxyl magnetic beads from Merck respectively, and the antibody coupling rate of the immunomagnetic beads was determined after antibody coupling. The average value and coefficient of variation were calculated by repeating the test three times. The results are shown in Table 3. The antibody coupling rate of the carboxyl magnetic beads prepared by the present application was high and stable, and the carboxyl magnetic beads could be applied to the preparation of thrombomodulin chemiluminescence detection reagent kit.

[0093] Table 3 Comparison of antibody coupling rates of carboxyl magnetic beads of the present application and carboxyl magnetic beads from Merck

[0094] Name Test 1 Test 2 Test 3 Average Merck carboxyl beads 80.93 74.97 76.63 77.51 Carboxyl beads of the invention 94.12 91.82 89.07 91.67

[0095] 2.4, Determination of carboxyl content

[0096] Carboxyl magnetic beads were prepared by the method optimized by experiments, and the carboxyl content on the surface of the magnetic beads was determined. The carboxyl content on the surface of the carboxyl magnetic beads was tested by chemical titration method, and the results are shown in Table 4. The carboxyl content on the surface of the carboxyl magnetic beads prepared by the present application was higher, which was beneficial to the coupling of antibodies on the surface of the magnetic beads, and thus the antibody coupling rate was high.

[0097] Table 4 Comparison of carboxyl contents of carboxyl magnetic beads of the present application and carboxyl magnetic beads from Merck

[0098]

[0099]

[0100] 2.5, Determination of calibration curve

[0101] The pH of the enzyme-labeled buffer solution can affect the stereostructure of the prepared enzyme-labeled antibody, and further affect the detection of thrombomodulin antigen. Enzyme-labeled antibodies were prepared using enzyme-labeled buffer solutions with pH values of 7.0, 7.5, 8.0, 8.5, and 9.0 respectively, and were tested on the machine by matching the prepared optimized immunomagnetic beads, and the calibration curve was fitted. The results are shown inFigure 1 The highest photon value was measured when the pH was 7.5, the linear relationship was the best, and R 2 was 0.9849, so the enzyme-labeled antibody prepared with the enzyme-labeled buffer at pH 7.5 was selected for the preparation of the thrombomodulin detection kit.

[0102] Example 3 Stability Test

[0103] The kit was stored at 37°C, and on days 0, 1, 3, 5, 7, 9, and 12, the immunomagnetic beads, enzyme-labeled antibody, and sample were placed in the designated positions of the chemiluminescence instrument, and the following procedures were followed for testing: 1) 50 μL of immunomagnetic beads, 50 μL of enzyme-labeled antibody, and 10 μL of sample were uniformly mixed and incubated at 37°C for 30 min;

[0104] 2) Magnetic separation, the complex was washed with chemiluminescence instrument cleaning solution;

[0105] 3) 200 μL of substrate solution for the full-automatic immunological test system was added to the washed magnetic bead complex, and incubated at 37°C in the dark for 5 min;

[0106] 4) The photon value of the substrate solution after reaction was measured using a photoelectric reactor;

[0107] 5) The sample concentration was calculated by fitting the calibration curve.

[0108] The sample test results are shown in Table 5. The subsequent test results were compared with the test results on day 0, and the relative deviations were calculated, as shown in Table 6. The relative deviations of the test results of the kit on days 1, 3, 5, 7, 9, and 12 from the test results on day 0 were all within ±10%, indicating that the kit had good stability.

[0109] Table 5, Kit Stability Test Results

[0110]

[0111]

[0112] Table 6, Kit Stability Test Result Relative Deviation Analysis

[0113]

[0114] Example 4 Reproducibility Test

[0115] The kit of the present application, the kit of Xisenmeikang, TM low value quality control and TM high value quality control were respectively placed in the designated position of the chemiluminescence instrument, and the test was carried out, the test was repeated for 10 times, the average value and the coefficient of variation CV of the test results were calculated, and the results were shown in Table 7. The coefficient of variation of the test results of the kit of the present application was smaller, indicating that the repeatability of the test value was better.

[0116] Table 7 repeatability test results

[0117]

[0118]

[0119] The above is only the preferred embodiment of the present application, it should be pointed out that, for the ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing carboxyl magnetic beads, characterized in that, This includes preparing a nanoparticle core from a soluble ferric ion salt, coating it with silica, reacting it with carboxylated silane, and then obtaining it through magnetic separation; The method for preparing the carboxylated silane includes reacting an aminosilane with an acid anhydride in an anhydrous solvent for 1-2 hours; the molar ratio of the aminosilane to the acid anhydride is 1:(2-30), the aminosilane is vinyltris(β-methoxyethoxy)silane, and the acid anhydride is ethylchloroacetic anhydride.

2. The preparation method according to claim 1, characterized in that, The step of preparing the nanoparticle core from the soluble ferric ion salt includes reacting the soluble ferric ion salt, sodium acetate, polymer with a mixed solution of ethylene glycol and ethylenediamine, and obtaining Fe3O4 nanoparticles after magnetic separation. The polymer is selected from any one of polyoxyethylene, polylactic acid, polyvinylpyrrolidone, and sodium carboxymethyl cellulose. The reaction temperature is 180~300°C, and the reaction time is 6~12h.

3. The preparation method according to claim 1, characterized in that, The step of coating the nanoparticle core with silica includes resuspending Fe3O4 nanoparticles and reacting them with tetraethyl orthosilicate, followed by magnetic separation to obtain Fe3O4 nanoparticles coated with silica. The mass ratio of tetraethyl orthosilicate to Fe3O4 nanoparticles is 1:(10~30); The pH value of the resuspended Fe3O4 nanoparticles was 10-12. The tetraethyl orthosilicate is soluble in ethanol; The reaction temperature is 25~50°C, and the reaction time is 3~6 hours.

4. The preparation method according to claim 1, characterized in that, The step of reacting the silica-coated Fe3O4 nanoparticles with carboxylated silanes includes dissolving the carboxylated silanes in anhydrous ethanol and reacting them with the silica-coated Fe3O4 nanoparticles for 2-6 hours, followed by magnetic separation to obtain the final product. The reaction is carried out at a temperature of 25-50°C and a pH value of 10-12.

5. Carboxyl magnetic beads, characterized in that, Obtained by the preparation method described in any one of claims 1 to 4.

6. A thrombomodulin detection kit, characterized in that, This includes immunomagnetic beads prepared using the carboxyl magnetic beads described in claim 5 and enzyme-labeled™ antibodies; The immunomagnetic bead is conjugated with TM antibody 1; the enzyme-labeled TM antibody is alkaline phosphatase-labeled TM antibody 2; The TM antibody 1 and antibody 2 include monoclonal antibodies or modified antibody fragments with Fab activity, or antibody fragment polymers.

7. The reagent kit according to claim 6, characterized in that, The mass ratio of the carboxyl magnetic beads to TM antibody 1 is (10~200):1; The mass ratio of alkaline phosphatase to TM antibody 2 is (0.5~10):

1.

8. A method for detecting thrombomodulin levels for non-diagnostic purposes, characterized in that, This includes the kit described in claim 6 or 7.

Images