11-dehydrothromboxane B2-BSA conjugate, magnetic particle complex, kit and application thereof

Abstract

The present invention provides an 11-dehydrothromboxane B2-BSA conjugate, a magnetic particle complex, a kit and its application, belonging to the field of immunological detection technology. The 11-dehydrothromboxane B2-BSA conjugate is prepared by modifying 11-dehydrothromboxane B2 with a straight-chain alkyl hydrocarbon ether and then coupling it with bovine serum albumin; the 11-dehydrothromboxane B2-BSA conjugate is used to prepare a magnetic particle complex, and then the magnetic particle complex is used to prepare a kit; the kit is used to detect the concentration of 11-dehydrothromboxane B2 in a sample to be tested. The kit prepared by the 11-dehydrothromboxane B2-BSA conjugate of the present invention has the advantages of strong specificity, high sensitivity, good repeatability, etc., and presents a certain linear range, which can meet a wide range of clinical diagnosis needs.

Description

Technical Field

[0001] The invention relates to immunological detection technology, in particular to an 11-dehydrothromboxane B2-BSA conjugate, a magnetic particle complex, a kit and applications thereof. Background Art

[0002] At present, the burden of cardiovascular and cerebrovascular diseases in the middle-aged and elderly population in my country is increasing, and it has become an important public health issue. Aspirin, as an antithrombotic drug with sufficient evidence in evidence-based medicine, wide indications and the best cost-effectiveness ratio, has been widely used. It is the basic drug for antithrombotic treatment and the basic drug for the prevention and treatment of cardiovascular and cerebrovascular diseases. Due to the widespread and irregular use of this drug, about 30% of patients in my country have drug resistance. However, due to insufficient understanding of the clinical status of aspirin and considering its possible adverse reactions, some patients with cardiovascular and cerebrovascular diseases who can benefit from aspirin treatment still fail to take aspirin or take aspirin for a long time. This phenomenon is particularly common at the grassroots level. For the clinical application of low-dose aspirin, we should focus on the principle of combining evidence-based medicine with individualized medication, explore the dosage of aspirin for Chinese patients, solve the problem of patients' medication dosage, and maximize the therapeutic benefits of the drug. However, due to differences in aspirin metabolism among patients, it is necessary to analyze the metabolism of aspirin in patients to help clinicians determine whether to improve medication measures.

[0003] After platelets are activated, arachidonic acid (AA) is released from their membrane phospholipids, which generate prostaglandins G2 and H2 under the action of cyclooxygenase COX-1, and further catalyze the generation of thromboxane A2 (TXA2) under the action of platelet thromboxane synthase. TXA2 is the strongest platelet aggregation and activation factor. TXA2 is highly unstable and will be rapidly hydrolyzed into the more stable thromboxane B2 (TXB2). TXB2 is then converted into 11-dehydrothromboxane B2 (11-DH-TXB2) with a longer half-life in the liver and excreted in the urine. Therefore, the detection of 11-dehydrothromboxane B2 can reflect the real-time excretion rate of thromboxane A2, thereby quantifying the degree of platelet activation. Since 11-dehydrothromboxane B2 (11-DH-TXB2) is a stable final metabolite of TXA2, it can be used as a quantitative indicator of the degree of platelet activation in vivo. Aspirin irreversibly binds to the acetyl group on platelet cyclooxygenase-1 (COX-1), which inhibits its activity and reduces the activity of platelets. The therapeutic effect of aspirin can be directly reflected by measuring the concentration of 11-dehydrothromboxane B2 in the patient's urine sample. Therefore, how to effectively and accurately measure the concentration of 11-dehydrothromboxane B2 has become a standard for analyzing the degree of aspirin metabolism in patients. Therefore, developing an 11-dehydrothromboxane B2 detection kit that is easy to market and has high accuracy and sensitivity has become a technical problem that needs to be solved urgently. Summary of the invention

[0004] In view of the above problems, the present invention provides an 11-dehydrothromboxane B2-BSA conjugate, a magnetic particle complex, a kit and applications thereof.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] A 11-dehydrothromboxane B2-BSA conjugate, wherein the 11-dehydrothromboxane B2-BSA conjugate is prepared by modifying 11-dehydrothromboxane B2 with a straight-chain alkyl hydrocarbon ether and then coupling it with bovine serum albumin;

[0007] The structural formula of the 11-dehydrothromboxane B2-BSA conjugate is:

[0008]

[0009] Wherein, x and y are each an integer greater than or equal to 1;

[0010] BSA is bovine serum albumin;

[0011] The coupling process is:

[0012] 11-dehydrothromboxane B2 is modified with a straight-chain alkyl hydrocarbon ether, and the resulting modified compound solution is placed in an organic solvent DMSO. The resulting solution is added to an activation buffer for dissolution, and after stirring at 450-550 rpm for 18-22 minutes, an activator EDC and an activator Surfo-NHS are added, and stirring is continued for more than 30 minutes. The resulting activated reaction solution is added to a coupling buffer containing BSA, and stirring is continued for more than 12 hours. Then, ethanolamine is added to terminate the reaction. The resulting reaction solution is placed in a dialysis bag and dialyzed in a PBS buffer for more than 12 hours to obtain the 11-dehydrothromboxane B2-BSA conjugate.

[0013] A magnetic particle complex coated with a 11-dehydrothromboxane B2-BSA conjugate, wherein the magnetic particle complex coated with a 11-dehydrothromboxane B2-BSA conjugate is prepared by coating the above-mentioned 11-dehydrothromboxane B2-BSA conjugate with magnetic particles;

[0014] The particle size of the magnetic particle complex is 100 to 200 μm;

[0015] The magnetic particles are superparamagnetic microspheres;

[0016] The density of the magnetic particle surface is 20.0-50.0 μmol / g;

[0017] The coating process is as follows:

[0018] Add the magnetic particles to the activation buffer, stir at 450-550 rpm for 18-22 min, add activator EDC and activator Surfo-NHS, continue stirring for 25-35 min, and obtain the activated magnetic particle reaction solution;

[0019] Take 11-dehydrothromboxane B2-BSA conjugate and add it to the coupling buffer, stir at 500-600 rpm for more than 10 minutes, add the activated magnetic particle reaction solution, continue stirring for more than 3 hours, then add the sodium caseinate aqueous solution blocking agent, continue stirring for more than 2 hours, and the resulting system is separated into solid and liquid by a magnetic separator, and then washed with purified water to obtain the magnetic particle complex.

[0020] A kit for detecting 11-dehydrothromboxane B2 based on acridinium ester chemiluminescence method, comprising reagent I, reagent II, calibrants and quality control products prepared by using a magnetic particle complex coated with the above-mentioned 11-dehydrothromboxane B2-BSA conjugate and a phosphate buffer containing an anti-interference agent and a stabilizer.

[0021] Furthermore, the concentration of the magnetic particle complex contained in the reagent I is 0.5-1.0 mg / mL.

[0022] Furthermore, the anti-interference agent in the reagent I is at least one of phenylmethylsulfonyl fluoride, pepsin inhibitor, trypsin inhibitor, leupeptin and disodium ethylenediaminetetraacetate;

[0023] The stabilizer in the reagent I is at least one of PEG200, PA1080, calf serum, sodium caseinate and fish skin gelatin;

[0024] The molar number of the phosphate buffer is 100-200 mmol / L.

[0025] Furthermore, the kit also includes a reagent II prepared by using an acridinium ester conjugate labeled with 11-dehydrothromboxane B2 polyclonal antibody and a phosphate buffer containing a stabilizer.

[0026] Furthermore, the acridinium ester conjugate is prepared by reacting acridinium ester with 11-dehydrothromboxane B2 polyclonal antibodies responsive to 11-dehydrothromboxane B2-BSA produced by immunizing animals with 11-dehydrothromboxane B2-BSA conjugate;

[0027] The specific preparation method of the acridinium ester conjugate is:

[0028] Preparation of 11-dehydrothromboxane B2 polyclonal antibody using 11-dehydrothromboxane B2-BSA conjugate:

[0029] First immunization: Inoculate 11-dehydrothromboxane B2-BSA conjugate into host rabbits: Use PBS buffer to dilute 11-dehydrothromboxane B2-BSA conjugate to 0.5-2.0 mg / mL, add an equal volume of Freund's complete adjuvant, and after complete emulsification, perform the first immunization on the host rabbits at a dose of 0.2-0.5 mg / rabbit, and obtain host rabbits that have completed the first immunization;

[0030] Enhanced immunization: After an interval of 4 weeks, weigh 0.5-2.0 mg of 11-dehydrothromboxane B2-BSA conjugate and 0.5-2.0 mg of Freund's incomplete adjuvant, mix evenly and fully emulsify, and perform enhanced immunization at a dose of 0.2-0.5 mg / rabbit to the host rabbits that have completed the first immunization;

[0031] The booster immunization is repeated 3-4 times, and the interval between each booster immunization is not less than 4 weeks. During this period, serum is collected for titer evaluation, and the number of booster immunizations is confirmed based on the titer results;

[0032] Dissolve the 11-dehydrothromboxane B2-BSA conjugate polyclonal antibody in a labeling buffer, stir at 450-550 rpm for 18-22 min, add an organic solution containing acridinium ester, stir at 500-600 rpm in the dark for more than 2 h, then add ethanolamine to terminate the reaction, and then dialyze in PBS buffer for more than 12 h through a dialysis bag to obtain the acridinium ester conjugate;

[0033] The acridinium ester is NSP-SA-NHS or / and NSP-DMAE-NHS;

[0034] The stabilizer in the reagent II is at least one of BSA, PA1080, calf serum, sodium caseinate and yellow sucrose.

[0035] Furthermore, the kit also includes calibration products and quality control products prepared using 11-dehydrothromboxane B2-BSA conjugate and tris-hydroxyaminomethane buffer containing a stabilizer.

[0036] Furthermore, the stabilizers in the calibration products and quality control products are magnesium chloride and sodium caseinate.

[0037] An application method of the above kit, the application method comprising the following steps:

[0038] After the calibration substance, reagent I and reagent II are mixed evenly, the pre-excitation solution and the excitation solution are added after incubation reaction and washing, and the number of photons generated is measured;

[0039] With the concentration of the calibrant as the horizontal axis and the luminescence value as the vertical axis, a standard curve was drawn to obtain a linear regression equation;

[0040] After the quality control product, reagent I and reagent II are mixed evenly, after incubation reaction and washing, pre-excitation solution and excitation solution are added, the number of photons generated is measured, and the luminescence value of the obtained quality control product is substituted into the linear regression equation to calculate the concentration of 11-dehydrothromboxane B2 in the quality control product. The measured result should be within the allowable range of the quality control product;

[0041] After the sample to be tested, reagent I and reagent II are mixed evenly, after incubation reaction and washing, pre-excitation solution and excitation solution are added, the number of photons generated is measured, and the luminescence value of the sample to be tested is substituted into the linear regression equation to calculate the concentration of 11-dehydrothromboxane B2 in the sample to be tested.

[0042] The beneficial effects of the 11-dehydrothromboxane B2-BSA conjugate, magnetic particle complex, kit and application thereof of the present invention are:

[0043] The kit prepared by the 11-dehydrothromboxane B2-BSA conjugate of the present invention has the advantages of strong specificity, high sensitivity, good repeatability, etc., and presents a certain linear range, which can meet a wide range of clinical diagnosis needs;

[0044] The reagent I in the kit of the present invention can provide a stable environment for the magnetic particle complex coated with 11-dehydrothromboxane B2-BSA conjugate by adding a phosphate buffer containing an anti-interference agent and a stabilizer, and can also inhibit the interference caused by excessive content of urine protein (including serine protease, metalloprotease, acid protease, etc.) in the sample to be tested (urine sample) due to partial renal filtration insufficiency, pH interference, bilirubin interference, etc.;

[0045] The reagent II in the kit of the present invention provides a stable environment for the acridinium ester conjugate labeled with the 11-dehydrothromboxane B2-BSA conjugate polyclonal antibody by adding a phosphate buffer containing a stabilizer; the 11-dehydrothromboxane B2-BSA conjugate polyclonal antibody of the present invention can specifically recognize 11-dehydrothromboxane B2, and promote the acridinium ester conjugate labeled with the 11-dehydrothromboxane B2-BSA conjugate polyclonal antibody to produce competitive binding with the magnetic particle complex coated with the 11-dehydrothromboxane B2-BSA conjugate in the reagent I or the 11-dehydrothromboxane B2 in the sample to be tested;

[0046] The calibrator and quality control product of the present invention include 11-dehydrothromboxane B2-BSA conjugate, which is used to compare with the sample to be tested and calculate the concentration of 11-dehydrothromboxane B2 in the sample to be tested;

[0047] The stabilizer used in the calibrator and quality control product of the present invention can maintain the structural stability of the 11-dehydrothromboxane B2-BSA conjugate for a long time;

[0048] The technical principle adopted by the present invention is as follows: when a certain concentration of 11-dehydrothromboxane B2 is contained in a sample to be tested, a magnetic particle complex coated with a 11-dehydrothromboxane B2-BSA conjugate in a reagent I competes with a 11-dehydrothromboxane B2-BSA conjugate polyclonal antibody labeled acridinium ester conjugate in a reagent II containing 11-dehydrothromboxane B2 at a certain concentration in the sample to be tested, after washing by a fully automatic chemiluminescence immunoassay instrument, a pre-excitation solution and an excitation solution are added to generate photon numbers, the concentration of 11-dehydrothromboxane B2 in the sample to be tested is negatively correlated with the photon number, and the concentration of 11-dehydrothromboxane B2 in the sample to be tested is calculated by using a standard curve drawn with a calibrator. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is the standard curve drawn using the calibrator in Example 1 of the present invention;

[0050] Figure 2 is a graph showing the results of an accelerated test of reagent II prepared using NSP-SA-NHS in Example 1 of the present invention;

[0051] Figure 3 is a graph showing the results of an accelerated test of reagent II prepared using NSP-DMAE-NHS in Example 20 of the present invention;

[0052] Figure 4 It is a graph of the correlation analysis results in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0053] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0054] The solution used in the present invention comprises:

[0055] Activation buffer: 10-50 mmol / L MES buffer, pH 5.5, preferably 50 mmol / L MES buffer.

[0056] Coupling buffer: 10-50 mmol / L borate buffer, pH 8.0, preferably 50 mmol / L borate buffer.

[0057] Activator: 10 mg / mL EDC solution, 10 mg / mL Sulfo-NHS;

[0058] Sealing agent: 0.5wt% sealing agent aqueous solution;

[0059] Labeling buffer: 10-50 mmol / L borate buffer, pH 8.0, preferably 50 mmol / L borate buffer.

[0060] Example 1 Preparation method of 11-dehydrothromboxane B2-BSA conjugate, magnetic particle complex and kit and application of the kit

[0061] This embodiment is a method for preparing a 11-dehydrothromboxane B2-BSA conjugate, a magnetic particle complex and a reagent I, comprising the following specific steps:

[0062] 1) Preparation of 11-dehydrothromboxane B2-BSA conjugate

[0063] 11) Preparation of modified compounds by modification of 11-dehydrothromboxane B2 with alkyl hydrocarbon ethers

[0064] A third-party company is commissioned to use 11-dehydrothromboxane B2 and alkyl hydrocarbon ether for esterification reaction (the skilled person in the art can adjust the conventional technology in the synthesis process according to common knowledge) to obtain a modified compound;

[0065] The structural formula of the alkyl hydrocarbon ether is:

[0066]

[0067] Wherein, x and y are each an integer greater than or equal to 1;

[0068] The structural formula of the alkyl hydrocarbon ether in this embodiment is:

[0069]

[0070] The structural formula of the modified compound obtained is:

[0071]

[0072] Wherein, x and y are each an integer greater than or equal to 1;

[0073] The structural formula of the modified compound prepared in this example is:

[0074]

[0075] 12) Dissolve 5 mg of the modified compound in 0.5 mL of DMSO to prepare a DMSO solution with a concentration of 10 mg / mL, then add the DMSO solution to 9.5 mL of activation buffer to dissolve, stir on a magnetic stirrer at 450-550 rpm (500 rpm in this embodiment) for 18-22 min (stir for 20 min in this embodiment), add 0.5 mL of activator EDC (i.e., 0.5 mL of EDC solution with a concentration of 10 mg / mL) and 0.5 mL of activator Surfo-NHS (i.e., 0.5 mL of Sulfo-NHS with a concentration of 10 mg / mL), continue stirring for more than 30 min (continue stirring for 30 min in this embodiment) to obtain an activated reaction solution;

[0076] 13) Dissolve 10 mg of BSA in 10 mL of coupling buffer, add the activated reaction solution to the obtained coupling buffer containing BSA, and continue stirring for more than 12 h (in this embodiment, continue stirring for 12 h); then add 2 μL of ethanolamine to terminate the reaction, continue stirring for 1 h, place the obtained reaction solution in a dialysis bag, and dialyze in 10 mM PBS buffer for more than 12 h (in this embodiment, dialyze for 12 h) to obtain 11-dehydrothromboxane B2-BSA conjugate.

[0077] The structural formula of the 11-dehydrothromboxane B2-BSA conjugate is:

[0078]

[0079] Wherein, x and y are each an integer greater than or equal to 1;

[0080] BSA is bovine serum albumin;

[0081] The structural formula of the 11-dehydrothromboxane B2-BSA conjugate in this example is:

[0082]

[0083] 2) Preparation of magnetic microparticle complex coated with 11-dehydrothromboxane B2-BSA conjugate

[0084] 21) 1 mL of superparamagnetic microspheres was added to 10 mL of activation buffer, and stirred on a magnetic stirrer at 450-550 rpm (500 rpm in this embodiment) for 18-22 min (stirred for 20 min in this embodiment), and then 0.5 mL of activator EDC (i.e., 0.5 mL of EDC solution with a concentration of 10 mg / mL) and 0.5 mL of activator Surfo-NHS (i.e., 0.5 mL of Sulfo-NHS with a concentration of 10 mg / mL) were added, and stirring was continued for more than 30 min (stirred for 30 min in this embodiment) to obtain an activated magnetic microparticle reaction solution;

[0085] 22) Add 2 mg of 11-dehydrothromboxane B2-BSA conjugate to 10 mL of coupling buffer, stir on a magnetic stirrer at 500-600 rpm (550 rpm in this embodiment) for more than 10 min (stir for 10 min in this embodiment), add the above-mentioned activated magnetic particle reaction solution, continue stirring for more than 3 h (stir for 3 h in this embodiment), then add 20 μL of 0.5 wt% sodium caseinate aqueous solution as a blocking agent, continue stirring for more than 2 h (stir for 2 h in this embodiment), and then use a magnetic separator to separate the solid magnetic particle complex and the reaction solution, and then wash twice with purified water to obtain the magnetic particle complex.

[0086] 3) Preparation of Reagent I

[0087] A storage solution is prepared by using a phosphate buffer with a concentration of 100 to 200 mmol / L (the concentration of the phosphate buffer in this embodiment is 100 mmol / L), an anti-interference agent, a stabilizer, sodium chloride and Proclin 300, wherein the storage solution contains 0.5 wt% of the stabilizer, 0.9 wt% of the sodium chloride and 0.2 wt% of Proclin 3000; the stabilizer is at least one of PEG200, PA1080, calf serum, sodium caseinate and fish skin gelatin

[0088] The magnetic particle complex was added with 20 mL of storage solution and stored as a magnetic particle complex solution.

[0089] The magnetic particle complex solution was prepared into a reagent solution with a mass concentration of 0.5 mg / mL using a storage solution according to the mass concentration, and the solution was placed at 45° C. for 24 hours to obtain reagent I.

[0090] 4) Preparation of Reagent II

[0091] 41) using 11-dehydrothromboxane B2-BSA conjugate to prepare 11-dehydrothromboxane B2-BSA conjugate polyclonal antibody;

[0092] First immunization: Inoculation of 11-dehydrothromboxane B2-BSA conjugate into host rabbits: Dilute 11-dehydrothromboxane B2-BSA conjugate to 1.0 mg / mL with 1 mL PBS buffer, add an equal volume of Freund's complete adjuvant, and after complete emulsification, the host rabbits are first immunized at a dose of 0.5 mg / rabbit;

[0093] Enhanced immunization: After an interval of 4 weeks, weigh 1.0 mg of 11-dehydrothromboxane B2-BSA conjugate and 1.0 mg of Freund's incomplete adjuvant, mix them evenly and fully emulsify them, and perform enhanced immunization at a dose of 0.5 mg / rabbit on the host rabbits that have completed the first immunization. The enhanced immunization was repeated 4 times, and the interval between each two enhanced immunizations was 4 weeks.

[0094] 42) Weigh 1 mg of acridinium ester (NSP-SA-NHS is used in this example) and dissolve it in 0.1 mL of DMSO to obtain an organic solution containing acridinium ester;

[0095] 1 mg of 11-dehydrothromboxane B2 polyclonal antibody was dissolved in 5 mL of labeling buffer, stirred on a magnetic stirrer at 450-550 rpm (500 rpm in this embodiment) for 18-22 min (stirred for 20 min in this embodiment), then 10 μL of an organic solution containing acridinium ester was added, and stirred on a magnetic stirrer at 500-600 rpm (550 rpm in this embodiment) in the dark for more than 2 h (stirred for 2 h in this embodiment), and then 2 μL of ethanolamine was added to terminate the reaction. After continuous stirring for 1 h, the reaction solution was placed in a dialysis bag and dialyzed in a PBS buffer solution with a concentration of 10 mmol / L for more than 12 h (dialyzed for 12 h in this embodiment) to obtain an acridinium ester conjugate labeled with 11-dehydrothromboxane B2-BSA conjugate polyclonal antibody.

[0096] 43) A storage solution is prepared by combining a phosphate buffer having a concentration of 100 to 200 mmol / L (the concentration of the phosphate buffer in this embodiment is 100 mmol / L), a stabilizer, sodium chloride and Proclin 300, wherein the storage solution contains a stabilizer selected from at least one of BSA, PA1080, calf serum, sodium caseinate and yellow sucrose.

[0097] Take the above acridinium ester conjugate and add 10 mL of the storage solution of the labeled conjugate to obtain reagent II.

[0098] 5) Preparation of Calibrators

[0099] A storage solution is prepared by using a phosphate buffer with a concentration of 100-200 mmol / L (the concentration of the phosphate buffer in this embodiment is 100 mmol / L), a stabilizer, sodium chloride and Proclin 300, wherein the storage solution contains a stabilizer of 1.0% BSA and 0.2% casein sodium. An appropriate amount of 11-dehydrothromboxane B2-BSA conjugate is dissolved in the above storage solution, and the calibration product is obtained after the value is determined.

[0100] 6) Preparation of quality control products

[0101] A storage solution is prepared by using a phosphate buffer with a concentration of 100-200 mmol / L (the concentration of the phosphate buffer in this embodiment is 100 mmol / L), a stabilizer, sodium chloride and Proclin 300, wherein the storage solution contains a stabilizer of 1.0% BSA and 0.2% casein sodium. An appropriate amount of 11-dehydrothromboxane B2-BSA conjugate is dissolved in the above storage solution, and the quality control product is obtained after the value is determined.

[0102] 7) Kit and its application

[0103] The kit for detecting 11-dehydrothromboxane B2 based on the acridinium ester chemiluminescence method in this embodiment includes the above-mentioned reagent I, reagent II, calibrator and quality control product, and the specific application method is as follows:

[0104] 71) placing the kit in a fully automatic chemiluminescence immunoassay analyzer, pipetting the calibrator, reagent I and reagent II, mixing them evenly, adding pre-excitation solution and excitation solution (pre-excitation agent and excitation solution can be used in the art) after incubation reaction and washing, and measuring the number of photons generated;

[0105] 72) Draw a standard curve with the concentration of the calibrant as the horizontal axis and the luminescence value as the vertical axis. Figure 1 .

[0106] 73) After the quality control product, reagent I and reagent II are mixed evenly, after incubation reaction and washing, the pre-excitation solution and the excitation solution are added, the number of photons generated is measured, and the luminescence value of the obtained quality control product is substituted into the linear regression equation to calculate the concentration of 11-dehydrothromboxane B2 in the quality control product. The measured result should be within the allowable range of the quality control product, indicating that the detection method is accurate and can be used to measure the concentration of 11-dehydrothromboxane B2 in the sample to be tested;

[0107] 74) After the sample to be tested, reagent I and reagent II are mixed evenly, after incubation reaction and washing, pre-excitation solution and excitation solution are added, the number of photons generated is measured, and the luminescence value of the sample to be tested is substituted into the linear regression equation to calculate the concentration of 11-dehydrothromboxane B2 in the sample to be tested.

[0108] Examples 2 to 10 Preparation Methods of Kits

[0109] Examples 2 to 10 are respectively methods for preparing a kit, and their steps are substantially the same as those of Example 1, except that the anti-interference agent in step 3) is different, as follows:

[0110] In Example 2, 30 μg / mL phenylmethylsulfonyl fluoride is used as an anti-interference agent, and 10.0-50.0 μg / mL phenylmethylsulfonyl fluoride can also be used as an anti-interference agent;

[0111] In Example 3, 0.2 μg / mL pepsin inhibitor was used as the anti-interference agent, and 0.1-1.0 μg / mL pepsin inhibitor may also be used as the anti-interference agent;

[0112] In Example 4, 0.25 μg / mL trypsin inhibitor was used as the anti-interference agent, and 0.1-0.5 μg / mL trypsin inhibitor may also be used as the anti-interference agent;

[0113] In Example 5, 0.5 μg / mL leupeptin was used as an anti-interference agent, and 0.2-1.0 μg / mL leupeptin could also be used as an anti-interference agent;

[0114] In Example 6, 10 mmol / mL of disodium ethylenediaminetetraacetate is used as an anti-interference agent, and 5.0 to 15.0 mmol / mL of disodium ethylenediaminetetraacetate can also be used as an anti-interference agent;

[0115] In Example 7, 30 μg / mL phenylmethylsulfonyl fluoride and 10 mmol / mL ethylenediaminetetraacetic acid disodium were mixed in a weight ratio of 1:1 as an anti-interference agent;

[0116] In Example 8, 0.2 μg / mL pepsin inhibitor and 10 mmol / mL EDTA disodium were mixed in a weight ratio of 1:1 as an anti-interference agent;

[0117] In Example 9, 0.25 μg / mL trypsin inhibitor and 10 mmol / mL ethylenediaminetetraacetic acid disodium were mixed in a weight ratio of 1:1 as an anti-interference agent;

[0118] In Example 10, 0.5 μg / mL leupeptin and 10 mmol / mL ethylenediaminetetraacetic acid disodium were mixed in a weight ratio of 1:1 as an anti-interference agent;

[0119] Among them, phenylmethylsulfonyl fluoride was purchased from Sigma, pepsin inhibitor, trypsin inhibitor, and leupeptin were purchased from Thermo Company, and disodium ethylenediaminetetraacetic acid was purchased from Sinopharm.

[0120] By using the kits in Examples 2 to 10 and the comparative reagent (aspirin assay kit produced by Corgenix, USA) to assay the test samples 1 to 5 respectively, the test results obtained are as follows:

[0121] Table 1 Effect of anti-interference agents in reagent I on test results (pg / mL)

[0122]

[0123] As can be seen from Table 1, the data in Example 7 are closest to the commercially available comparison reagent data, so the use of the anti-interference agent (Example 7) to which phenylmethylsulfonyl fluoride and disodium ethylenediaminetetraacetic acid are added can effectively improve the anti-interference ability of the reagent, which is closer to the test results of the comparison kit.

[0124] Examples 11 to 19 Preparation Methods of Kits

[0125] Examples 11 to 19 are respectively methods for preparing a kit, and their steps are substantially the same as those of Example 1, except that the stabilizer in step 3) is different, as follows:

[0126] In Example 11, 1.0 wt% PEG200 is used as a stabilizer, and 0.5-2.0 wt% PEG200 can also be used as a stabilizer;

[0127] In Example 12, 1.0 wt% PA1080 is used as a stabilizer, and 0.5-2.0 wt% PA1080 can also be used as a stabilizer;

[0128] In Example 13, 2.0 wt% calf serum is used as a stabilizer, and 1.0-5.0 wt% calf serum can also be used as a stabilizer;

[0129] In Example 14, 0.5 wt% sodium caseinate is used as a stabilizer, and 0.1-1.0 wt% sodium caseinate can also be used as a stabilizer;

[0130] In Example 15, 2.0 wt% fish skin gelatin is used as a stabilizer, and 1.0-3.0 wt% fish skin gelatin can also be used as a stabilizer;

[0131] In Example 16, 1.0 wt % PEG200 and 2.0 wt % fish skin gelatin were mixed in a weight ratio of 1:1 as a stabilizer;

[0132] In Example 17, 1.0 wt % PA1080 and 2.0 wt % fish skin gelatin were mixed in a weight ratio of 1:1 as a stabilizer;

[0133] In Example 18, 2.0 wt % calf serum and 2.0 wt % fish skin gelatin were mixed in a weight ratio of 1:1 as a stabilizer;

[0134] In Example 19, 0.5 wt % sodium caseinate and 2.0 wt % fish skin gelatin were mixed in a weight ratio of 1:1 as a stabilizer;

[0135] Among them, PEG200, sodium caseinate, and fish skin gelatin were purchased from Sigma, PA1080 was purchased from JSR, and calf serum was purchased from Beijing Yuanhengshengma Company.

[0136] Reagent I in Examples 11 to 19 was subjected to a 7-day 37°C thermal acceleration test using calibrators of different concentrations, and the data before acceleration was used as a comparison to analyze the effect of the stabilizer on the stability of the kit. The specific accelerated test results are shown in Table 2, where the comparison in Table 2 represents the luminescence value before acceleration;

[0137] Table 2 Effect of stabilizer in reagent I on test results (calibrator concentration: pg / mL, data corresponding to the example are luminescence values)

[0138]

[0139] The closer the result of the calibration sample is to the comparison value, the lower the attenuation of the luminescence value after acceleration and the higher the stability.

[0140] It can be seen from Table 2 that when the combination of PA1080 and fish skin gelatin is used as a stabilizer, the attenuation amplitude of the luminescence value after accelerated testing is lower than that of other stabilizers, so the stability is higher.

[0141] Example 20 Preparation method of the kit

[0142] Example 20 is a method for preparing a kit, and the steps are basically the same as those in Example 1, except that the acridinium ester in step 4) is different, as follows:

[0143] In Example 20, NSP-DMAE-NHS was used as the acridinium ester;

[0144] Among them, NSP-SA-NHS in Example 1 and NSP-DMAE-NHS in Example 20 were both purchased from Suzhou Yaco Company.

[0145] The same standard product was tested using the kits prepared in Example 1 and Example 20, respectively. The test results are shown in the following table:

[0146] Table 3 Effect of acridinium ester in reagent II on test results

[0147] NSP-SA-NHS NSP-DMAE-NHS Standard concentration RLUs RLUs 0 209936 259835 500 116907 159942 S / N 1.80 1.62

[0148] As can be seen from Table 3, the kit prepared using NSP-SA-NHS has a higher signal-to-noise ratio during the detection process, so it has a higher sensitivity.

[0149] The reagent II prepared in Example 1 and Example 20 was again subjected to thermal acceleration at 37°C for 7 days, and the stability at 4°C was used as a reference to observe the stability of the reagent II. The results are shown in Figures 2-3 , it can be seen Figure 2 The accelerated stability of reagent II prepared by NSP-SA-NHS is better than Figure 3 The stability of reagent II prepared with NSP-DMAE-NHS was accelerated, so reagent II prepared with NSP-SA-NHS had better stability.

[0150] Examples 21 to 29 Preparation of Kits

[0151] Examples 21 to 29 are respectively methods for preparing a kit, and their steps are substantially the same as those of Example 1, except that the stabilizer in step 4) is different, as follows:

[0152] In Example 21, 0.5 wt % BSA is used as a stabilizer, and 0.1-2.0 wt % BSA can also be used as a stabilizer;

[0153] In Example 22, 1.0 wt% PA1080 is used as a stabilizer, and 0.1-2.0 wt% PA1080 can also be used as a stabilizer;

[0154] In Example 23, 3.0 wt% calf serum is used as a stabilizer, and 0.5-5.0 wt% calf serum can also be used as a stabilizer;

[0155] In Example 24, 0.5 wt% sodium caseinate is used as a stabilizer, and 0.1-2.0 wt% sodium caseinate can also be used as a stabilizer;

[0156] In Example 25, 5.0 wt% yellow sucrose is used as a stabilizer, and 1.0-10.0 wt% yellow sucrose can also be used as a stabilizer;

[0157] In Example 26, 0.5 wt % BSA and 5.0 wt % yellow sucrose were mixed in a weight ratio of 1:1 as a stabilizer;

[0158] In Example 27, 1.0 wt% PA1080 and 5.0 wt% yellow sucrose were mixed in a weight ratio of 1:1 as a stabilizer;

[0159] In Example 28, 3.0 wt % calf serum and 5.0 wt % yellow sucrose were mixed in a weight ratio of 1:1 as a stabilizer;

[0160] In Example 29, 0.5 wt % sodium caseinate and 5.0 wt % yellow sucrose were mixed in a weight ratio of 1:1 as a stabilizer;

[0161] Among them, BSA and calf serum were purchased from Beijing Yuanhengshengma Company, PA1080 was purchased from JSR Company, and sodium caseinate and yellow sucrose were purchased from Sinopharm.

[0162] Reagent II prepared in Examples 21 to 29 was subjected to a 7-day 37°C thermal acceleration test using calibrators of different concentrations. The data before acceleration was used as a reference to analyze the effect of the stabilizer on the stability of the kit. The specific accelerated test results are shown in Table 4. The comparison in Table 4 represents the luminescence value before acceleration.

[0163] Table 4 Effect of stabilizer in reagent II on test results (calibrator concentration: pg / mL, data corresponding to the example are luminescence values)

[0164]

[0165] The closer the result of the calibration sample is to the comparison value, the lower the attenuation of the luminescence value after acceleration and the higher the stability.

[0166] It can be seen from Table 4 that when the combination of yellow sucrose and sodium caseinate is used as a stabilizer, the attenuation of the luminescence value after accelerated testing is lower than that of other stabilizers, so the stability is higher.

[0167] Embodiment 30

[0168] This example is a method for preparing a kit, and the steps are basically the same as those in Example 1, except that:

[0169] The anti-interference agent in step 3) is a mixture of 30 μg / mL phenylmethylsulfonyl fluoride and 10 mmol / mL disodium ethylenediaminetetraacetate in a weight ratio of 1:1 as an anti-interference agent;

[0170] The stabilizer in step 3) is a mixture of 1.0wt% PA1080 and 2.0wt% fish skin gelatin in a weight ratio of 1:1.

[0171] The stabilizer in step 4) is a mixture of 0.5 wt % sodium caseinate and 5.0 wt % yellow sucrose in a weight ratio of 1:1.

[0172] Clinical correlation analysis of test example 1

[0173] The kit prepared in Example 30 (acridinium ester chemiluminescence detection) and the commercially available 11-dehydrothromboxane B2 detection kit (aspirin determination kit (11-dehydrothromboxane B2) produced by Corgenix, USA, the detection method is relatively complicated) were used to detect the same clinical sample and compared, and correlation analysis was performed. The specific results are shown in Figure 4 , we can see the correlation coefficient R 2 It can reach 0.9948.

[0174] Test Example 2 Detection Sensitivity Test

[0175] The kit prepared in Example 30 (acridinium ester chemiluminescence detection) was compared with the commercially available 11-dehydrothromboxane B2 detection kit (detected using other methodological methods, which are relatively complex) for the same clinical sample detection, and the signal-to-noise ratio S / N was calculated. The higher the S / N, the higher the sensitivity. It can be concluded from Table 5 that the S / N of the patented kit is significantly higher than that of the comparison kit.

[0176] Table 5 Sensitivity comparison

[0177] Commercially available kits Example 1 Clinical samples Signal value Signal value 0 147221 210923 1382pg / mL 92817 73480 S / N 1.59 2.87

[0178] Test Example 3 Detection Specificity Test

[0179] The reagent of the present invention performed specificity analysis on the compound with the following structure. The compound with the following structure was prepared into gradient concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.1 ng / mL, 0.01 ng / mL, etc., and tested using the kit prepared in Example 30 of the present invention. No concentration value was detected, indicating that there was no cross reaction.

[0180] The structural formula of the specific compound is:

[0181]

[0182] On the other hand, the clinical correlation in Experimental Example 1 shows that the kit prepared by the present invention has good clinical correlation with the commercially available kit, further verifying the specificity of the patented reagent.

[0183] Test Example 4 Linear Analysis Test

[0184] The high concentration sample to be tested was gradiently diluted into 8 concentration points, and each point was tested using the kit in Example 30 of the present invention. Each point was tested 3 times and the average was taken. The average was fitted with the dilution factor, and the correlation coefficient r=0.9983, which met the requirements.

[0185] Table 6 Linear analysis

[0186] Dilution multiple Luminous value 0.007813 190864 0.015625 173429 0.03125 129467 0.0625 106245 0.125 75983 0.25 51290 0.5 33894 1 22835 r 0.9983

[0187] Therefore, the various performance indicators and clinical relevance of the kit prepared by the present invention meet the expected goals.

[0188] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A 11-dehydrothromboxane B2-BSA conjugate, characterized in that: The 11-dehydrothromboxane B2-BSA conjugate is prepared by modifying 11-dehydrothromboxane B2 with a straight-chain alkyl hydrocarbon ether and then coupling it with bovine serum albumin; The structural formula of the 11-dehydrothromboxane B2-BSA conjugate is: Among them, BSA is bovine serum albumin.

2. A magnetic particle complex coated with 11-dehydrothromboxane B2-BSA conjugate, characterized in that: The 11-dehydrothromboxane B2-BSA conjugate-coated magnetic particle complex is prepared by coating the 11-dehydrothromboxane B2-BSA conjugate described in claim 1 with magnetic particles.

3. A kit for detecting 11-dehydrothromboxane B2 based on acridinium ester chemiluminescence method, characterized in that: The kit includes a reagent I, a reagent II, a calibrator and a quality control product prepared by using the magnetic particle complex coated with the 11-dehydrothromboxane B2-BSA conjugate according to claim 2 and a phosphate buffer containing an anti-interference agent and a stabilizer; Reagent II is prepared by using an acridinium ester conjugate labeled with 11-dehydrothromboxane B2 polyclonal antibody and a phosphate buffer containing a stabilizer; The acridinium ester conjugate is prepared by reacting acridinium ester with a polyclonal antibody responsive to 11-dehydrothromboxane B2-BSA produced by immunizing an animal with the 11-dehydrothromboxane B2-BSA conjugate; the acridinium ester is NSP-SA-NHS or / and NSP-DMAE-NHS; The stabilizer in the reagent II is at least one of BSA, PA1080, calf serum, sodium caseinate and yellow sucrose.

4. The kit according to claim 3, characterized in that The concentration of the magnetic particle complex in the reagent I is 0.5-1.0 mg / mL.

5. The kit according to claim 3 or 4, characterized in that The anti-interference agent in the reagent I is at least one of phenylmethylsulfonyl fluoride, pepsin inhibitor, trypsin inhibitor, leupeptin and disodium ethylenediaminetetraacetate; The stabilizer in the reagent I is at least one of PEG200, PA1080, calf serum, sodium caseinate and fish skin gelatin; The molar concentration of the phosphate buffer is 100-200 mmol / L.

6. The kit according to claim 3 or 4, characterized in that The calibration product and quality control product are prepared by using 11-dehydrothromboxane B2-BSA conjugate and tris-hydroxyaminomethane buffer containing a stabilizer.

7. The kit according to claim 6, characterized in that The stabilizers in the calibration products and quality control products are magnesium chloride and sodium caseinate.

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