A chemiluminescence detection kit based on nucleic acid aptamer and a preparation method thereof

Abstract

The application belongs to the field of medical detection, and mainly relates to a chemiluminescence detection kit based on nucleic acid aptamer and a preparation method thereof. The chemiluminescence detection kit prepared by the application comprises reagents M and R. The reagent M comprises magnetic microparticle coupled recognition molecules, which can be called capture probes. The reagent R comprises chemiluminescence marker coupled recognition molecules, which can be called chemiluminescence probes. The recognition molecules are nucleic acid aptamers or antibodies, and the recognition molecules in the reagent M and the reagent R are not antibodies at the same time. The chemiluminescence detection kit combines the convenient detection and analysis of the magnetic microparticle chemiluminescence method and the high specificity and high affinity of the nucleic acid aptamer, and can timely and accurately detect various proteins. Different from the capture probes and the chemiluminescence probes constructed by protein antibodies on the market, the kit overcomes the problems of poor stability, difficult modification and high production cost of the protein antibodies, and will play an important role in the field of immunodetection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical detection, and mainly relates to a chemiluminescence detection kit based on nucleic acid aptamer and a preparation method thereof. BACKGROUND

[0002] Immune diagnosis is a series of detection methods for determining antigens, antibodies, immune cells and their secreted cytokines designed by applying immunology theory. In recent years, new immunological detection methods continue to iterate and innovate, among which the chemiluminescence immunoassay technology is the latest generation of immune detection technology after the radioimmunoassay technology, colloidal gold technology, immunofluorescence technology, enzyme-linked immunoassay technology and time-resolved fluorescence technology. In particular, the magnetic particle chemiluminescence technology is a detection technology combining magnetic separation technology, chemiluminescence technology and immune analysis technology, and has the characteristics of easy separation, high automation, high sensitivity and strong specificity, etc. It is one of the most mainstream immune diagnostic technologies in medical institutions at present.

[0003] In the magnetic particle chemiluminescence method, the antibody (or antigen) is first labeled to the solid carrier magnetic particle to construct a capture probe, and then a chemiluminescent label is labeled to the surface of the antibody (or antigen) to construct a chemiluminescent probe. The target antigen (or antibody) is added for immune reaction and magnetic separation and washing operation, then the chemiluminescence excitation solution is added, and finally the chemiluminescence measuring equipment is used to detect the generated luminescence signal. Since the concentration of the measured substance is linearly related to the luminescence intensity within a certain range, the quantitative detection of the target substance is realized.

[0004] The immune combination of antigen and antibody has high specificity, and is a commonly used recognition tool for chemiluminescence immunoassay technology. However, the production cost of antibody is high, the acquisition cycle is long, and the batch difference is large, which increases the production cost and batch stability of the immune detection reagent based on antibody. In addition, the chemical stability of antibody is poor, the modification and preparation process is complex, the binding number and modification site cannot be accurately controlled, which further limits the application of antibody in immune detection, mainly in the application in detection kit.

[0005] In view of the problems of antibodies, nucleic acid aptamer is more and more concerned. Nucleic acid aptamer is known as "chemist's antibody". Compared with antibodies, nucleic acid aptamer has unique advantages such as small size, low cost, easy chemical synthesis, precise modification, good programmability, high biocompatibility and wide target substances. By using the system evolution of ligands by exponential enrichment (SELEX), researchers can screen nucleic acid aptamer which specifically binds to the target from the library containing different nucleic acid sequences. Nucleic acid aptamer is a single-stranded oligonucleotide (DNA or RNA) with a length of about 20-100 bases, which can specifically bind to other targets such as proteins, metal ions, small molecules, polypeptides and even whole cells through folding into a unique tertiary structure, and is expected to replace antibodies in the field of chemical immunoassay.

[0006] At present, there is no magnetic microparticle chemiluminescence detection method based on nucleic acid aptamer and nucleic acid aptamer and antibody combination in the field of biomedical detection, and the development of a nucleic acid aptamer-based magnetic microparticle chemiluminescence technology is urgently needed in the field. SUMMARY

[0007] In view of the deficiencies of the prior art, the present application aims to provide a chemiluminescence detection kit based on nucleic acid aptamer and a preparation method thereof.

[0008] The present application provides a chemiluminescence detection kit based on nucleic acid aptamer, which comprises reagent M and reagent R.

[0009] Preferably, the reagent M comprises magnetic microparticle coupled recognition molecules.

[0010] Preferably, the reagent R comprises chemiluminescence marker coupled recognition molecules.

[0011] Preferably, the recognition molecules are nucleic acid aptamers or antibodies.

[0012] Preferably, the recognition molecules in the reagent M and the reagent R are not antibodies.

[0013] Preferably, the magnetic microparticles contain a modification group, and the group comprises one of streptavidin, carboxyl and amino.

[0014] Preferably, the chemiluminescence marker comprises one of acridinium ester, alkaline phosphatase, horseradish peroxidase, trispyridine ruthenium and other luminescent molecules or luminescent materials.

[0015] Preferably, the nucleotide sequence of the nucleic acid aptamer is one of the following:

[0016] The HBP nucleic acid aptamer is shown in SEQ ID NO. 1.

[0017] The nucleic acid aptamer of cTnI is shown as SEQ ID NO. 2.

[0018] The nucleic acid aptamer of NMP22 is shown as SEQ ID NO. 3.

[0019] The application further provides a preparation method of the chemiluminescence detection kit, comprising the following steps:

[0020] The magnetic particles and the recognition molecules are prepared into reagent M;

[0021] The chemiluminescence label and the recognition molecules are prepared into reagent R.

[0022] Preferably, the chemiluminescence detection kit comprises reagent M and reagent R.

[0023] Preferably, the recognition molecules are nucleic acid aptamers or antibodies.

[0024] Preferably, the recognition molecules in reagent M and reagent R can be antibodies at the same time.

[0025] Preferably, the nucleotide sequence of the nucleic acid aptamer is one of the following:

[0026] The nucleic acid aptamer of HBP is shown as SEQ ID NO. 1;

[0027] The nucleic acid aptamer of cTnI is shown as SEQ ID NO. 2;

[0028] The nucleic acid aptamer of NMP22 is shown as SEQ ID NO. 3.

[0029] In an embodiment of the application, the preparation method of the magnetic particle coupled nucleic acid aptamer is specifically as follows:

[0030] The magnetic particle solution and the nucleic acid aptamer solution are mixed at a certain volume ratio at room temperature for 0.5-1h; then the above solution is all magnetically separated, and the supernatant is discarded; then the magnetic particle buffer is added and mixed at room temperature for 0.5-1h; then the magnetic particle buffer is washed for 3-5 times to obtain the magnetic particle coupled nucleic acid aptamer, which is called reagent M; finally, the reagent M is diluted to 0.2-0.8mg / mL with the magnetic particle buffer.

[0031] Preferably, the concentration of the magnetic particle solution is 1-10mg / mL.

[0032] Preferably, the concentration of the nucleic acid aptamer solution is 1-100μM.

[0033] Preferably, the magnetic particle solution and the nucleic acid aptamer solution are mixed at a volume ratio of 500:1-10.

[0034] Preferably, the magnetic particles in the magnetic particle solution are modified with streptavidin.

[0035] Preferably, the concentration of the reagent M after dilution with the magnetic particle buffer is 0.2-0.8 mg / mL.

[0036] Preferably, the magnetic particle buffer comprises 0.1-1 wt% Tris, 1-2 wt% BSA, 0.01-0.1 wt% folic acid-chitosan-superoxide dismutase (FA-CS-SOD), and 0.1-0.5 wt% Tween 20.

[0037] In an embodiment of the present application, the preparation method of FA-CS-SOD is as follows:

[0038] Mix folic acid, EDC, NHS, and DMSO in a certain proportion, activate slowly under stirring at room temperature for 10-60 min to obtain an activation reaction solution; dissolve chitosan in 1-5% acetic acid solution to obtain a chitosan-acetic acid solution; slowly add the activation reaction solution into the chitosan-acetic acid solution, stir under light protection at room temperature for 10-24 h to obtain reaction solution 1; adjust the pH of reaction solution 1 with NaOH solution to obtain reaction solution 2; centrifuge at 2000-8000 rpm for 1-10 min to obtain a precipitate, wash the precipitate with NaHCO3 solution for 2-5 times; dissolve the precipitate by adding a certain amount of pure water, then dialyze with pure water, freeze dry the dialyzed solution to obtain folic acid-chitosan.

[0039] Mix superoxide dismutase, EDC, NHS, and phosphate buffer in a certain proportion, activate slowly under stirring at room temperature for 10-60 min to obtain an activation reaction solution 1. Dissolve folic acid-chitosan in phosphate buffer to obtain reaction solution 3, slowly add reaction solution 3 into the activation reaction solution 1, stir under light protection at room temperature for 3-8 h to obtain reaction solution 4; then transfer reaction solution 4 into a dialysis bag, dialyze with phosphate buffer, freeze dry the dialyzed solution to obtain FA-CS-SOD.

[0040] Preferably, the folic acid, EDC, NHS, and DMSO are mixed in a proportion of 1-10 mg: 1-5 mg: 1-5 mg: 10-20 mL.

[0041] Preferably, the amount of chitosan used is 0.1-1 wt% of the acetic acid solution.

[0042] Preferably, the pH of the acetic acid solution is 4.0-6.0.

[0043] Preferably, the mass ratio of folic acid to chitosan in the reaction solution is 1: 1-10.

[0044] Preferably, the concentration of the NaOH solution is 2-5 M.

[0045] Preferably, the pH of reaction solution 2 is 7.0-9.0.

[0046] Preferably, the pH of the NaHCO3 solution is 7.0-9.0.

[0047] Preferably, the ratio of the precipitate to pure water is 1-10 mg: 1-10 mL.

[0048] Preferably, the molecular weight cut-off of the dialysis bag is 3-30 KDa.

[0049] Preferably, the superoxide dismutase, EDC, NHS and phosphate buffer are mixed in a ratio of 1-10 mg: 5-15 mg; 1-5 mg: 1-10 mL.

[0050] Preferably, the concentration of the phosphate buffer is 2-5 M.

[0051] Preferably, the pH of the phosphate buffer is 6.0-8.0.

[0052] Preferably, the folic acid-chitosan and the phosphate buffer are mixed in a ratio of 1-5 mg: 1-5 mL.

[0053] Preferably, the superoxide dismutase in the activation reaction solution 1 and the folic acid-chitosan in the reaction solution 3 are mixed in a mass ratio of 1-5: 1-5.

[0054] The addition of FA-CS-SOD in the magnetic micro-particle buffer not only makes the magnetic micro-particle buffer have a certain antibacterial effect, but also ensures the stability of the magnetic micro-particle coupled nucleic acid aptamer and the magnetic micro-particle coupled antibody through the antioxidant property, and prevents the degradation of the antibody by protease to a certain extent.

[0055] In an embodiment of the present application, the preparation method of the magnetic micro-particle coupled antibody is as follows:

[0056] The magnetic micro-particles are washed 4-5 times in a morpholine ethanesulfonic acid buffer (MES-T) containing 1-5% Tween, and then washed 2-5 times in a morpholine ethanesulfonic acid buffer (MES); the magnetic micro-particles are resuspended in the MES to obtain a magnetic micro-particle solution 1, then the magnetic micro-particle solution 1, an EDC solution and an NHS solution are mixed in a certain volume ratio for activation reaction, and mixed uniformly at room temperature for 0.5-1 h; then the above solutions are all magnetically separated, and the supernatant is discarded; the magnetic micro-particles are resuspended in the MES to obtain a magnetic micro-particle solution 2; the magnetic micro-particle solution 2 and an antibody solution are mixed in a certain volume ratio at room temperature for 0.5-1 h; after the above solutions are washed 1-2 times with a magnetic micro-particle buffer, the magnetic micro-particle buffer is added for blocking at room temperature for 0.5-1 h; then the magnetic micro-particle buffer is used for washing 3-5 times to obtain the magnetic micro-particle coupled antibody, which is called reagent M; finally, the reagent M is diluted to 0.2-0.8 mg / mL with the magnetic micro-particle buffer.

[0057] Preferably, the concentration of the magnetic micro-particle solution is 10-50 mg / mL.

[0058] Preferably, the magnetic particles in the magnetic particle solution are modified with carboxyl groups.

[0059] Preferably, the concentration of the magnetic particle solution 1 is 10-50 mg / mL.

[0060] Preferably, the concentration of the EDC solution is 1-10 mg / mL.

[0061] Preferably, the concentration of the NHS solution is 1-10 mg / mL.

[0062] Preferably, the magnetic particle solution 1, the EDC solution and the NHS solution are activated at a volume ratio of 1-100: 1-10: 1-10, and then coupled with the antibody.

[0063] Preferably, the concentration of the magnetic particle solution 2 is 10-50 mg / mL.

[0064] Preferably, the concentration of the antibody solution is 1-10 mg / mL.

[0065] Preferably, the magnetic particle solution 2 and the antibody solution are mixed at a volume ratio of 10-50: 1-5.

[0066] Preferably, the concentration of the reagent M after being diluted with the magnetic particle buffer is 0.2-0.8 mg / mL.

[0067] Preferably, the magnetic particle buffer comprises 0.1-1 wt% of Tris, 1-2 wt% of BSA, 0.01-0.1 wt% of FA-CS-SOD, and 0.1-0.5 wt% of Tween 20.

[0068] In an embodiment of the present application, the preparation method of the chemiluminescent marker coupled aptamer is specifically as follows:

[0069] The chemiluminescent marker solution, the aptamer solution and the DPBS solution are mixed at a certain volume ratio at room temperature for 0.5-1 h; then the above solution is transferred into an ultrafiltration tube, centrifuged at 5000-10000 rpm for 10-30 min, and washed with DPBS for 2-3 times to obtain the chemiluminescent marker coupled aptamer, which is called reagent R; the reagent R is diluted 1000-5000 times with the alkaline phosphatase buffer.

[0070] Preferably, the concentration of the chemiluminescent marker solution is 0.1-1 mg / mL.

[0071] Preferably, the chemiluminescent marker in the chemiluminescent marker solution is modified with streptavidin.

[0072] Preferably, the concentration of the aptamer solution is 1-100 μM.

[0073] Preferably, the chemiluminescent marker solution, the nucleic acid aptamer solution and the DPBS are mixed in a volume ratio of 10-50:1-10:100-500.

[0074] Preferably, the reagent R is further diluted with an alkaline phosphatase buffer.

[0075] Preferably, the alkaline phosphatase buffer comprises 0.1-1wt% MOPS, 0.1-1wt% NaCl, 1-5wt% BSA, 0.1-0.2wt% ZrCl2, 0.01-0.1wt% MgCl2, 1-5wt% ethyl undecanoate raffinose and 0.1-0.5% Proclin 300.

[0076] Preferably, the addition of ethyl undecanoate raffinose in the alkaline phosphatase buffer can effectively protect the structure of proteins and nucleic acid aptamers, and can be used as a protective agent to maintain the stable state of the chemiluminescent marker conjugated antibody and the chemiluminescent marker conjugated nucleic acid aptamer in the experiment.

[0077] In an embodiment of the present application, the preparation method of the chemiluminescent marker conjugated antibody is specifically as follows:

[0078] The chemiluminescent marker solution, the antibody solution and the PBS solution are mixed in a certain volume ratio at room temperature and in the dark for 0.5-1h; then the same volume of lysine solution as the chemiluminescent marker solution is added to the above solution for further reaction for 0.5-1h to obtain a reaction solution 1; a certain amount of dextran gel G25 is pretreated by being placed in a desalting column, and then the reaction solution 1 is added to collect the flow-through solution, which is the chemiluminescent marker conjugated antibody; the chemiluminescent marker conjugated antibody is further diluted to 0.1-1mg / mL with PBS, and then the same volume of glycerol is added for further dilution, which is called reagent R; finally, the reagent R is diluted 1000-5000 times with acridinum ester buffer.

[0079] Preferably, the concentration of the chemiluminescent marker solution is 1-10mM.

[0080] Preferably, the concentration of the antibody solution is 1-10mg / mL.

[0081] Preferably, the chemiluminescent marker solution, the antibody solution and the PBS are mixed in a volume ratio of 1-10:10-50:100-200.

[0082] Preferably, the concentration of the lysine solution is 100-200mM.

[0083] Preferably, the amount of dextran gel G25 is 4-5 times the volume of the reaction solution 1.

[0084] Preferably, the pretreatment of the dextran gel G25 is washing with pure water for three times and then washing with PBS for three times.

[0085] Preferably, the amount of pure water and PBS used in the pretreatment of the dextran gel G25 is 2-5 times of the volume of the reaction solution.

[0086] Preferably, the reagent R is further diluted with acridinium ester buffer.

[0087] Preferably, the composition of the acridinium ester buffer is: 1-5 wt% NaHPO4·12H2O, 0.1-0.5 wt% NaH2PO4·2H2O, 0.1-1 wt% NaCl, 0.1-1 wt% BSA, 0.01-0.1 wt% MgCl2·6H2O, 0.1-0.5% Tween 20, 0.1-0.5% Proclin 300, and 1-5 wt% ethyl undecanoate raffinose.

[0088] Preferably, the addition of ethyl undecanoate raffinose in the acridinium ester buffer can effectively protect the structure of proteins and aptamers, and can be used as a protective agent to maintain the stable state of the chemiluminescent marker-coupled antibody and the chemiluminescent marker-coupled aptamer in the experiment.

[0089] The present application applies the above-mentioned chemiluminescence detection kit. The target protein to be detected is diluted to different concentrations and then loaded into the machine together with the reagents M and R. The volume ratio of the three is 1-10:2.5-25:5-50. After mixing, incubation and washing, the pre-activation solution and the activation solution are added, the relative light intensity is detected, and the content of the detected substance is calculated, thereby drawing the curve of the detected substance and the light intensity. The pre-activation solution and the activation solution are common reagents on the market.

[0090] The nucleic acid aptamer-based chemiluminescence detection kit prepared by the application comprises reagent M and reagent R. The reagent M comprises magnetic microparticle-coupled recognition molecules, and the reagent R comprises chemiluminescence marker-coupled recognition molecules, the recognition molecules are nucleic acid aptamers or antibodies, and the recognition molecules in the reagent M and the reagent R are not antibodies at the same time. Since the nucleic acid aptamer or the antibody is used as the recognition molecule, the chemiluminescence detection kit has the following beneficial effects: compared with the detection kit with only antibodies as the recognition molecules, the chemiluminescence detection kit has better stability, higher sensitivity, and can detect more substances, not limited to protein substances. In addition, the buffer of the prepared chemiluminescence detection kit is improved, FA-CS-SOD is added in the magnetic microparticle buffer, and ethyl undecanoate raffinose is added in the alkaline phosphatase buffer and the acridinium ester buffer, so that the reagent M and the reagent R are protected, the accuracy of the chemiluminescence detection kit is ensured, and the stability of the chemiluminescence detection kit is prolonged. BRIEF DESCRIPTION OF DRAWINGS

[0091] Figure 1 The change curve of the chemiluminescence value of the HBP quantitative determination kit for different types of HBP for detecting different concentrations of HBP recombinant antigens.

[0092] Figure 2 The standard curve of the chemiluminescence value of the HBP quantitative determination kit for different types of HBP for detecting different concentrations of HBP recombinant antigens.

[0093] Figure 3 The change curve of the chemiluminescence value of the cTnI quantitative determination kit for detecting different concentrations of cTnI recombinant antigens.

[0094] Figure 4 The standard curve of the chemiluminescence value of the cTnI quantitative determination kit for detecting different concentrations of cTnI recombinant antigens.

[0095] Figure 5 The change curve of the chemiluminescence value of the NMP22 quantitative determination kit for detecting different concentrations of NMP22 recombinant antigens.

[0096] Figure 6 The standard curve of the chemiluminescence value of the NMP22 quantitative determination kit for detecting different concentrations of NMP22 recombinant antigens. DETAILED DESCRIPTION

[0097] The application will be further described below in conjunction with the embodiments of the application. However, the scope of the application is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the application without departing from the spirit and scope of the application.

[0098] Example 1: Specific nucleotide sequences of each aptamer (all in 5'-3' direction)

[0099] HBP aptamer:

[0100] SEQ ID NO. 1: 5'-biotin-TTTCACGCATAACCACGATGTGTCACACGAGCTTTACTACCCCCGCATGGTTATGCGTG-3'

[0101] cTnI aptamer:

[0102] SEQ ID NO. 2: 5'-biotin-TTTACGCATAGGGTATGGGTGGGGGCGAGGGTCCATTCTATTGTGGCTACCTATGCGT-3'

[0103] NMP22 aptamer:

[0104] SEQ ID NO. 3: 5'-biotin-TTTCACGAGGGTTGGCGTTACGGGTTGGCTCATCGAAGATCGGGTGTG-3'

[0105] The above-mentioned aptamers are synthesized by Shengwo.

[0106] Example 2: Preparation method of magnetic microparticle buffer

[0107] The magnetic microparticle buffer is prepared by adding the materials in the following table into a beaker, stirring until the materials are completely dissolved, adjusting the pH to 7.4 and diluting to 100 mL.

[0108]

[0109] The preparation method of the folic acid-chitosan modified superoxide dismutase is as follows:

[0110] (1) Coupling of folic acid and chitosan

[0111] In a dark environment, 5 mg of FA, 2.6 mg of EDC and 2.0 mg of NHS were dissolved in 10 mL of DMSO by stirring, and activated at room temperature for 30 min to obtain an activated reaction solution. 20 mg of chitosan was added to 4 mL of 1% acetic acid solution (pH 5.0) to obtain a chitosan-acetic acid solution. The activated reaction solution was slowly added to the chitosan-acetic acid solution, and stirred at room temperature for 16 h in the dark to obtain reaction solution 1; the pH of reaction solution 1 was adjusted to 9.0 with a 3M NaOH solution to obtain reaction solution 2; then centrifuged (4000 rpm, 5 min) to obtain a yellow precipitate, which was washed with a NaHCO3 solution (pH 8.5) for 3 times, then dissolved in 10 mL of pure water, and dialyzed with pure water (the molecular weight cut-off of the dialysis bag was 12 KDa) for 3 days. The dialyzed solution was freeze-dried to obtain FA-CS.

[0112] (2) FA-CS modified superoxide dismutase

[0113] 10 mg of superoxide dismutase (SOD) was dissolved in 10 mL of 5M phosphate buffer (pH 6.0), and 10.2 mg of EDC and 3.4 mg of NHS were added in sequence, and activated at room temperature for 30 min by slow stirring to obtain activated reaction solution 1. Another 10 mg of FA-CS was dissolved in 10 mL of 5M phosphate buffer (pH 6.0) to obtain reaction solution 3; reaction solution 3 was slowly added to activated reaction solution 1, and stirred at room temperature in the dark for 6 h to obtain reaction solution 4. Then reaction solution 4 was transferred into a dialysis bag (molecular weight cut-off of 15 KDa), and dialyzed with 5M phosphate buffer (pH 6.0) for 3 days. The dialyzed solution was freeze-dried to obtain FA-CS-SOD. Chitosan (30-40 KDa, degree of deacetylation ≥95%) and folic acid were purchased from Shanghai Maikelin Biochemical Technology Co., Ltd. SOD was Cu / Zn-SOD from pig blood, purchased from Chengdu Kemai Biological Technology Co., Ltd.

[0114] Example 3: Preparation method of magnetic particle buffer

[0115] The difference between Example 3 and Example 2 is that the amount of FA-CS-SOD used in Example 3 is 0.01 g.

[0116] Example 4: Preparation method of magnetic particle buffer

[0117] The difference between Example 4 and Example 2 is that Example 4 does not contain FA-CS-SOD.

[0118] Example 5: Preparation method of acridine ester buffer

[0119] The acridine ester buffer was prepared by adding the materials in the table below into a beaker, stirring until the materials were completely dissolved, adjusting the pH to 7.4, and diluting to 1 L.

[0120]

[0121] Ethyl undecanoate raffinose was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.

[0122] Example 6: Preparation method of acridinium ester buffer

[0123] The difference between Example 6 and Example 5 is that the amount of ethyl undecanoate raffinose used in Example 6 is 10 g.

[0124] Example 7: Preparation method of acridinium ester buffer

[0125] The difference between Example 7 and Example 5 is that Example 7 does not contain ethyl undecanoate raffinose.

[0126] Example 8: Preparation method of alkaline phosphatase buffer

[0127] The alkaline phosphatase buffer was prepared by adding the materials in the following table into a beaker, stirring until the materials were completely dissolved, adjusting the pH to 7.4 and diluting to 2 L.

[0128]

[0129] Example 9: Preparation method of alkaline phosphatase buffer

[0130] The difference between Example 9 and Example 8 is that the amount of ethyl undecanoate raffinose used in Example 9 is 20 g.

[0131] Example 10: Preparation method of alkaline phosphatase buffer

[0132] The difference between Example 10 and Example 8 is that Example 10 does not contain ethyl undecanoate raffinose.

[0133] Example 11: Preparation of heparin binding protein (HBP) chemiluminescence detection kit

[0134] 1. Dilution of HBP aptamer:

[0135] The nucleotide sequence of the HBP aptamer is shown in SEQ ID NO. 1, which is concentrated to 100 μM with DPBS.

[0136] 2. Magnetic particle coupling HBP aptamer:

[0137] After the magnetic microparticle solution was shaken and mixed, 500 µL was taken out, 5.97 µL of HBP aptamer solution was added, and it was mixed for 30 min at room temperature on a three-dimensional rotator; the above solution was placed on a magnetic stand for 2 min to allow the magnetic microparticles to be completely separated, the supernatant was discarded, and then 500 µL of magnetic microparticle buffer was added and mixed for 30 min at room temperature on a three-dimensional rotator; then the magnetic microparticles were washed three times with 500 µL of magnetic microparticle buffer; the magnetic microparticle-coupled HBP aptamer was obtained, which was referred to as reagent M; finally, reagent M was diluted to 0.5 mg / mL with magnetic microparticle buffer. The magnetic microparticle solution was purchased from Boyue Biology, with a product number of M2800S3 and a concentration of 10 mg / mL; the magnetic microparticles in the magnetic microparticle solution were modified with streptavidin modification; the magnetic microparticle buffer was prepared by the preparation method of Example 4.

[0138] 3. Preparation of acridinium ester-coupled HBP antibody:

[0139] 3.34 µL of acridinium ester solution and 26.1 µL of HBP antibody solution were taken and added to 165 µL of PBS buffer, and reacted in a constant temperature shaking bath at room temperature for 30 min in the dark; then 3.34 µL of lysine solution was added and reacted for another 30 min, to obtain reaction solution 1; 900 µL of dextran gel G25 was taken and added to a desalting column, which was washed three times with 500 µL of purified water and then three times with 500 µL of PBS, and then the above reaction solution 1 was added to the desalting column for purification, and the flow-through solution was collected to obtain the acridinium ester-coupled HBP antibody; the concentration of the purified acridinium ester-coupled HBP antibody was calculated, and the acridinium ester-coupled HBP antibody was diluted to a concentration of 0.5 mg / mL with PBS, and then to a concentration of 0.25 mg / mL with glycerol for storage, which was referred to as reagent R. The concentration of the HBP antibody solution was 7.66 mg / mL; the concentration of the lysine solution was 100 mM; the acridinium ester solution was purchased from Jingjie Biology, with a concentration of 4 mM; the dextran gel G25 was purchased from Jingcheng Biology; the desalting column had a volume of 2 mL and a pore size of 5 µm.

[0140] 4. HBP chemiluminescence detection kit:

[0141] The reagent R was diluted to a concentration of 0.125 µg / mL with acridinium ester buffer and mixed for 10 min on a three-dimensional rotator; and the 0.5 mg / mL reagent M was mixed for 10 min on a three-dimensional rotator. The acridinium ester buffer was prepared by the preparation method of Example 5.

[0142] Example 12: Preparation of HBP chemiluminescence detection kit

[0143] The difference between Example 12 and Example 11 is that the magnetic microparticle buffer of Example 12 was prepared by the preparation method of Example 4; and the acridinium ester buffer was prepared by the preparation method of Example 6.

[0144] Example 13: Preparation of HBP chemiluminescent detection kit

[0145] Example 13 differs from Example 11 in that the magnetic particle buffer of Example 13 is prepared by the preparation method of Example 2; and the acridinium ester buffer is prepared by the preparation method of Example 5.

[0146] Example 14: Preparation of HBP chemiluminescent detection kit

[0147] Example 14 differs from Example 11 in that the magnetic particle buffer of Example 14 is prepared by the preparation method of Example 3; and the acridinium ester buffer is prepared by the preparation method of Example 5.

[0148] Comparative Example 1: Preparation of HBP chemiluminescent detection kit

[0149] Comparative Example 1 differs from Example 11 in that the acridinium ester buffer of Comparative Example 1 is prepared by the preparation method of Example 7.

[0150] The differences of Examples 11-14 and Comparative Example 1 are summarized in the following table.

[0151]

[0152] Test Example 1: Accuracy test of HBP chemiluminescent detection kit

[0153] Accuracy tests were performed on Examples 11-14 and Comparative Example 1.

[0154] 100 μL of high concentration HBP standard solution (300 ng / mL) was added to 900 μL of low concentration HBP standard solution (0 ng / mL), and after mixing, the mixture was tested. Each test was performed three times, and the average value and recovery rate were calculated. The recovery rate should be 95%-105%. The recovery formula is as follows:

[0155]

[0156] In the formula, R is the recovery rate, %; V is the volume of the high concentration standard solution added, mL; V0 is the volume of the low concentration standard solution, mL; C is the average value of the mixture test, ng / mL; C0 is the test value of the low concentration standard solution, ng / mL; and Cs is the known high concentration standard solution concentration, ng / mL.

[0157] The accuracy results are shown in Table 1. It can be seen that the accuracy of Examples 11, 12, 13 and 14 is better than that of Comparative Example 1. Compared with Example 11, Example 12 and Comparative Example 1, the addition of ethyl undecanoate raffinose improves the accuracy of the HBP chemiluminescence detection kit; compared with Example 11, Example 13 and Example 14, the further addition of FA-CS-SOD improves the accuracy of the HBP chemiluminescence detection kit to a certain extent; in addition, compared with Example 13, Example 14 and Comparative Example 1, the addition of FA-CS-SOD and ethyl undecanoate raffinose significantly improves the accuracy of the HBP chemiluminescence detection kit. Preferably, the accuracy of Example 13 is better.

[0158] Table 1: Accuracy data of HBP chemiluminescence detection kit

[0159]

[0160] Test Example 2: Stability experiment of HBP chemiluminescence detection kit

[0161] Stability experiments were performed on Examples 11-14 and Comparative Example 1.

[0162] The prepared HBP chemiluminescence detection kit was subjected to 37°C accelerated stability, and the initial (0 days) and 7-day kits were measured for luminescence value (RLU) of 300 ng / mL HBP protein, and the luminescence value drop rate (ROD) = (RLU 7天 / RLU 0天 -1) * 100% was calculated. A ROD value less than 10% was considered to be a pass standard.

[0163] The stability results are shown in Table 2. It can be seen that Examples 11, 12, 13 and 14 are all qualified, while Comparative Example 1 is not qualified, and the ROD is significantly high. Compared with Example 11, Example 12 and Comparative Example 1, the addition of ethyl undecanoate raffinose improves the stability of the HBP chemiluminescence detection kit; compared with Example 11, Example 13 and Example 14, the further addition of FA-CS-SOD improves the stability of the HBP chemiluminescence detection kit to a certain extent; in addition, compared with Example 13, Example 14 and Comparative Example 1, the addition of FA-CS-SOD and ethyl undecanoate raffinose significantly improves the stability of the HBP chemiluminescence detection kit. Preferably, the stability of Example 13 is better.

[0164] Table 2: Stability data of HBP chemiluminescence detection kit

[0165]

[0166] Test Example 3:

[0167] Test procedure of HBP chemiluminescence detection kit:

[0168] Load the HBP chemiluminescence detection kit of Example 13 onto the Shine i2910 system, and operate according to the corresponding Shine i2910 system operation manual to detect HBP.

[0169] Dilute the HBP protein to different concentrations, and load it onto the machine together with M and R reagents.

[0170] The Shine i2910 system will sequentially draw 10 μL of HBP protein solution of different concentrations, 25 μL of M reagent, and 50 μL of R reagent; after mixing, incubation, and washing according to the preset program, pre-priming liquid and priming liquid are added, the relative luminescence intensity is detected, and the content of the test substance is calculated, thereby drawing the curve of the test substance and the luminescence intensity. The above-mentioned pre-priming liquid and priming liquid are common reagents on the market.

[0171] It should be noted that the reagent M is mixed to re-suspend the magnetic microparticles that may have precipitated during transportation. The following operations can be taken:

[0172] 1) Turn the M reagent bottle over 30 times, and control the speed during turning to avoid generating bubbles;

[0173] 2) Visually inspect the reagent bottle to check whether the magnetic microparticles are suspended. If the magnetic microparticles still adhere to the bottle, continue to turn until the magnetic microparticles are completely suspended; if not, do not use it.

[0174] The reagent R should be gently turned over 8 times before use to mix.

[0175] Figure 1 Draw the standard curve of HBP recombinant antigen from the change curve of RLU of different concentrations of HBP recombinant antigen detected by different types of HBP quantitative determination kits, and select some points to draw the standard curve of HBP recombinant antigen. Figure 2 The HBP chemiluminescence detection kit prepared in Example 13 is based on aptamer as capture probe, while the other kit is based on antibody as capture probe, combined with Figure 1 and Figure 2 From the above, the HBP chemiluminescence detection kit prepared in Example 13 has a higher luminescence signal, indicating better detection sensitivity; a wider linear detection range, which can be better detected within the HBP recombinant antigen concentration range of 0-400 ng / mL, and the linear correlation coefficient is R 2 =0.998.

[0176] Example 15: Preparation of hypersensitive cardiac troponin I (cTnI) chemiluminescence detection kit

[0177] 1. Magnetic particle coupled cTnI antibody:

[0178] The magnetic particle solution was shaken and 100 μL was taken out. After adding 100 μL of 1% Tween-containing morpholine ethanesulfonic acid buffer (MES-T) and mixing, the above solution was placed on a magnetic stand for 2 min to completely separate the magnetic particles, the supernatant was discarded, and the magnetic particles were washed once with 500 μL of MES-T and twice with 500 μL of morpholine ethanesulfonic acid buffer (MES). The magnetic particles were resuspended in 400 μL of MES to obtain magnetic particle solution 1. Then 50 μL of EDC solution and 50 μL of NHS solution were added to the magnetic particle solution 1 for activation reaction, and mixed with a three-dimensional rotator at room temperature for 30 min. The magnetic particles were separated, and the supernatant was discarded. The magnetic particles were resuspended in 500 μL of MES to obtain magnetic particle solution 2. 20 μL of cTnI antibody solution was added to the magnetic particle solution 2, and mixed with a three-dimensional rotator at room temperature for 30 min. The magnetic particles were separated, the supernatant was discarded, and washed once with MES. 500 μL of magnetic particle buffer was added and incubated at 37°C for 2 h. The magnetic particles were washed three times with 500 μL of magnetic particle buffer to obtain the magnetic particle coupled cTnI antibody, which was referred to as reagent M. Finally, reagent M was diluted to 0.5 mg / mL with magnetic particle buffer. The cTnI antibody solution was 5 mg / mL. The magnetic particle solution was purchased from Boyue Biology, with the product number M2800C3 and a concentration of 50 mg / mL. The magnetic particles in the magnetic particle solution were modified with carboxyl groups. The EDC solution had a concentration of 10 mg / mL and was prepared fresh. The NHS solution had a concentration of 10 mg / mL and was prepared fresh. The magnetic particle buffer was prepared according to the preparation method of Example 4.

[0179] 2. Dilution of cTnI aptamer:

[0180] The nucleotide sequence of the cTnI aptamer is shown in SEQ ID NO. 2, which was diluted to 100 μM with DPBS.

[0181] 3. Preparation of alkaline phosphatase coupled cTnI aptamer:

[0182] 20 μL of alkaline phosphatase solution and 2 μL of cTnI aptamer solution were added to 128 μL of DPBS, and incubated at room temperature for 30 min. The above solution was subjected to ultrafiltration treatment with an ultrafiltration tube (8000 rpm, 10 min). The sample was washed twice with 450 μL of DPBS (8000 rpm, 10 min), and collected by inverting the centrifuge tube (1000 rpm, 1 min) to obtain the alkaline phosphatase coupled cTnI aptamer, which was referred to as reagent R. The alkaline phosphatase solution was purchased from Yixing Biology, with a concentration of 0.5 mg / mL. The alkaline phosphatase in the alkaline phosphatase solution was modified with streptavidin. The ultrafiltration tube was Millipore, 30 KDa.

[0183] 4. cTnI chemiluminescence detection kit:

[0184] Dilute reagent R 2000 times with alkaline phosphatase buffer and mix with a three-dimensional rotator for 10 min; dilute 0.5 mg / mL reagent M 2000 times with alkaline phosphatase buffer and mix with a three-dimensional rotator for 10 min. The alkaline phosphatase buffer is prepared by the preparation method of Example 8.

[0185] Example 16: Preparation of a cTnI chemiluminescence detection kit

[0186] Example 16 differs from Example 15 in that the magnetic particle buffer of Example 16 is prepared by the preparation method of Example 4; the alkaline phosphoric acid buffer is prepared by the preparation method of Example 9.

[0187] Example 17: Preparation of a cTnI chemiluminescence detection kit

[0188] Example 17 differs from Example 15 in that the magnetic particle buffer of Example 17 is prepared by the preparation method of Example 2; the alkaline phosphoric acid buffer is prepared by the preparation method of Example 8.

[0189] Example 18: Preparation of a cTnI chemiluminescence detection kit

[0190] Example 18 differs from Example 15 in that the magnetic particle buffer of Example 18 is prepared by the preparation method of Example 3; the alkaline phosphoric acid buffer is prepared by the preparation method of Example 8.

[0191] Comparative Example 2: Preparation of a cTnI chemiluminescence detection kit

[0192] Comparative Example 2 differs from Example 15 in that the alkaline phosphoric acid buffer of Comparative Example 2 is prepared by the preparation method of Example 10.

[0193] The differences between Examples 15-18 and Comparative Example 2 are summarized in the following table.

[0194]

[0195] Test Example 4: Accuracy experiment of cTnI chemiluminescence detection kit

[0196] Accuracy experiments were performed on Examples 15-18 and Comparative Example 2.

[0197] 1. Take 100 μL high concentration cTnI standard solution (100 ng / mL) into 900 μL low concentration cTnI standard solution (0 ng / mL), mix well to be mixed solution, test together with low concentration cTnI standard solution, test 3 times each, calculate the average value and recovery rate. The recovery rate should be 95%-105%. The recovery formula is as follows:

[0198]

[0199] In the formula: R is the recovery rate, %; V is the volume of high concentration standard solution added, mL; V0 is the volume of low concentration standard solution, mL; C is the average value of mixed solution test, ng / mL; C0 is the test value of low concentration standard solution, ng / mL; Cs is the known high concentration standard solution concentration, ng / mL.

[0200] The accuracy results are shown in Table 3, it can be seen that the accuracy of examples 15, 16, 17 and 18 is better than that of comparative example 2. Compared with example 15, example 16 and comparative example 2, the addition of ethyl undecanoate raffinose improves the accuracy of HBP chemiluminescence detection kit; compared with example 15, example 17 and example 18, after further adding FA-CS-SOD, the accuracy of cTnI chemiluminescence detection kit is improved to a certain extent; in addition, compared with example 17, example 18 and comparative example 2, after adding FA-CS-SOD and ethyl undecanoate raffinose, the accuracy of cTnI chemiluminescence detection kit is significantly improved. Among them, preferably, the accuracy of example 17 is better.

[0201] Table 3 cTnI chemiluminescence detection kit accuracy data

[0202]

[0203] Test example 5: cTnI chemiluminescence detection kit stability experiment

[0204] Stability experiments were carried out on examples 15-18 and comparative example 2.

[0205] The prepared cTnI chemiluminescence detection kit was subjected to 37℃ accelerated stability, and the initial (0 day) and 7 day kits were measured, the luminescence value (RLU) of 100 ng / mL cTnI protein was detected, and the luminescence value drop rate (ROD) = (RLU 7天 / RLU 0天 -1) * 100% was calculated, and the ROD value less than 10% was considered as the qualified standard.

[0206] The stability results are shown in Table 4. It can be seen that Examples 15, 16, 17 and 18 are qualified, while Comparative Example 2 is not qualified, and the ROD is significantly high. Compared with Example 15, Example 16 and Comparative Example 2, the addition of ethyl undecanoate raffinose improves the stability of the cTnI chemiluminescence detection kit; compared with Example 15, Example 17 and Example 18, the further addition of FA-CS-SOD further improves the stability of the cTnI chemiluminescence detection kit to a certain extent; in addition, compared with Example 17, Example 18 and Comparative Example 2, the addition of FA-CS-SOD and ethyl undecanoate raffinose significantly improves the stability of the cTnI detection kit. Preferably, the stability of Example 17 is better.

[0207] Table 4 cTnI chemiluminescence detection kit stability data

[0208]

[0209] Test Example 6:

[0210] cTnI chemiluminescence detection kit test procedure:

[0211] The cTnI chemiluminescence detection kit of Example 17 was loaded onto the incare i2900 system, and the operation was performed according to the corresponding incare i2900 system operation manual, and cTnI was detected.

[0212] The cTnI protein was diluted to different concentrations, and was loaded onto the machine together with M and R reagents.

[0213] The incare i2900 system will sequentially draw 10 μL of cTnI protein solution of different concentrations, 25 μL of M reagent, and 50 μL of R reagent; after mixing, incubating and washing according to the preset program, pre-priming liquid and priming liquid are added, the relative luminescence intensity is detected, and the content of the measured substance is calculated, thereby drawing the curve of the measured substance and the luminescence intensity. The above-mentioned pre-priming liquid and priming liquid are common reagents on the market.

[0214] It should be noted that the reagent M is mixed to re-suspend the magnetic microparticles that may precipitate during transportation. The following operations can be taken:

[0215] 1) Turn the M reagent bottle 30 times, and control the speed during turning to avoid generating bubbles;

[0216] 2) Visually inspect the reagent bottle to check whether the magnetic microparticles are suspended. If the magnetic microparticles still adhere to the bottle, continue to turn until the magnetic microparticles are completely suspended; if not, do not use it.

[0217] The reagent R should be gently turned 8 times before use to mix.

[0218] Figure 3 To determine the change curve of RLU of different concentrations of cTnI recombinant antigen, some points are selected from it to make Figure 4 The standard curve of cTnI recombinant antigen, in this test example, it can be seen that when the concentration of cTnI recombinant antigen is in the range of 0-100 ng / mL, it can be better detected, and the linear correlation coefficient is R 2 =0.992. In addition, it can be seen that the chemiluminescence signal in this test example is strong, indicating that the cTnI chemiluminescence detection kit has good sensitivity.

[0219] Example 19: Preparation of a urine nuclear matrix protein 22 (NMP22) chemiluminescence detection kit

[0220] 1. Dilution of NMP22 aptamer:

[0221] The nucleotide sequence of NMP22 aptamer is shown as SEQ ID NO. 3, which is concentrated to 100 μM with DPBS.

[0222] 2. Magnetic microparticle coupling NMP22 aptamer:

[0223] The magnetic microparticle solution was shaken and mixed, and 500 µL was taken out, 6.92 µL of NMP22 aptamer solution was added, and it was mixed for 30 min at room temperature with a three-dimensional rotator; the above solution was placed on a magnetic stand for 2 minutes to magnetically separate all the magnetic microparticles, and the supernatant was discarded; then 500 µL of magnetic microparticle buffer was added, and it was mixed for 30 min at room temperature with a three-dimensional rotator; then the magnetic microparticles were washed with 500 µL of magnetic microparticle buffer for three times, to obtain magnetic microparticle coupling NMP22 aptamer, which was called reagent M; finally, reagent M was diluted to 0.5 mg / mL with magnetic microparticle buffer. The magnetic microparticle solution was purchased from Boyue Biological, with the product number M2800S3 and a concentration of 10 mg / mL. The magnetic microparticles in the magnetic microparticle solution were modified with streptavidin. The magnetic microparticle buffer was prepared by the preparation method of Example 4.

[0224] 3. Alkaline phosphatase coupling NMP22 aptamer:

[0225] Add 20 μL alkaline phosphatase solution and 2 μL NMP22 aptamer solution into 128 μL DPBS, incubate at room temperature for 30 min. Ultrafiltration treatment is performed on the above solution (8000 rpm, 10 min); wash twice with 450 μL DPBS (8000 rpm, 10 min), collect the sample by inverting the centrifuge tube (1000 rpm, 1 min) to obtain alkaline phosphatase coupled NMP22 aptamer, which is referred to as reagent R. The alkaline phosphatase solution is purchased from Yixing Biological Technology Co., Ltd., and the concentration is 0.5 mg / mL. The alkaline phosphatase in the alkaline phosphatase solution is modified with streptavidin. The filter tube is Millipore, 30 KDa.

[0226] 4. NMP22 chemiluminescence detection kit:

[0227] Dilute reagent R 2000 times with alkaline phosphoric acid buffer and mix well with a three-dimensional rotator for 10 min; dilute 0.5 mg / mL reagent M 2000 times with alkaline phosphoric acid buffer and mix well with a three-dimensional rotator for 10 min. The alkaline phosphoric acid buffer is prepared by the preparation method of Example 8.

[0228] Example 20: Preparation of NMP22 chemiluminescence detection kit

[0229] The difference between Example 20 and Example 19 is that the magnetic particle buffer of Example 20 is prepared by the preparation method of Example 4; the alkaline phosphoric acid buffer is prepared by the preparation method of Example 9.

[0230] Example 21: Preparation of NMP22 chemiluminescence detection kit

[0231] The difference between Example 21 and Example 19 is that the magnetic particle buffer of Example 21 is prepared by the preparation method of Example 2; the alkaline phosphoric acid buffer is prepared by the preparation method of Example 8.

[0232] Example 22: Preparation of NMP22 chemiluminescence detection kit

[0233] The difference between Example 22 and Example 19 is that the magnetic particle buffer of Example 22 is prepared by the preparation method of Example 3; the alkaline phosphoric acid buffer is prepared by the preparation method of Example 8.

[0234] Comparative Example 3: Preparation of NMP22 chemiluminescence detection kit

[0235] The difference between Comparative Example 3 and Example 19 is that the alkaline phosphoric acid buffer of Comparative Example 3 is prepared by the preparation method of Example 10.

[0236] The differences of the above Examples 19-22 and Comparative Example 3 are summarized in the following table.

[0237]

[0238] Test Example 7: Accuracy experiment of NMP22 chemiluminescence detection kit

[0239] Accuracy experiments were performed on Examples 19-22 and Comparative Example 3.

[0240] 100 μL of high-concentration NMP22 standard solution (400 ng / mL) was added to 900 μL of low-concentration NMP22 standard solution (0 ng / mL), mixed, and used as a mixed solution, which was tested together with the low-concentration NMP22 standard solution. Each test was performed three times, and the average value and recovery rate were calculated. The recovery rate should be 95-105%. The recovery formula is as follows:

[0241]

[0242] In the formula, R is the recovery rate, %; V is the volume of the added high-concentration standard solution, mL; V0 is the volume of the low-concentration standard solution, mL; C is the average value of the mixed solution test, ng / mL; C0 is the test value of the low-concentration standard solution, ng / mL; and Cs is the known high-concentration standard solution concentration, ng / mL.

[0243] The accuracy results are shown in Table 5. It can be seen that the accuracy of each group of Examples is better than that of Comparative Example 3. Compared with Example 19, Example 20, and Comparative Example 3, the addition of ethyl undecanoate raffinose improves the accuracy of the NMP22 chemiluminescence detection kit. Compared with Example 19, further addition of FA-CS-SOD in Example 21 and Example 22 improves the accuracy of the NMP22 chemiluminescence detection kit to a certain extent. In addition, compared with Example 21, Example 22, and Comparative Example 3, the addition of FA-CS-SOD and ethyl undecanoate raffinose significantly improves the accuracy of the NMP22 chemiluminescence detection kit. Preferably, the accuracy of Example 21 is better.

[0244] Table 5: Accuracy data of NMP22 chemiluminescence detection kit

[0245]

[0246] Test Example 8: Stability experiment of NMP22 chemiluminescence detection kit

[0247] Stability experiments were performed on Examples 19-22 and Comparative Example 3.

[0248] The prepared NMP22 chemiluminescence detection kit was subjected to 37°C accelerated stability, and the kit was measured at the initial (0 days) and 7 days. The luminescence value (RLU) of 400 ng / mL NMP22 protein was detected, and the luminescence value drop rate (ROD) = (RLU7天 RLU 0天 -1) * 100%, and the ROD value less than 10% is considered as the qualified standard.

[0249] The stability results are shown in Table 6. It can be seen that the stability of each group of examples is qualified, while Comparative Example 3 is not qualified, and the ROD is significantly high. Compared with Example 19, Example 20 and Comparative Example 3, the addition of ethyl undecanoate raffinose improves the stability of the NMP22 chemiluminescence detection kit; compared with Example 19, Example 21 and Example 22, after further adding FA-CS-SOD, the stability of the NMP22 chemiluminescence detection kit is improved to a certain extent; in addition, compared with Example 21, Example 22 and Comparative Example 3, after adding FA-CS-SOD and ethyl undecanoate raffinose, the stability of the NMP22 chemiluminescence detection kit is significantly improved. Preferably, the accuracy of Example 21 is better.

[0250] Table 6 NMP22 chemiluminescence detection kit stability data

[0251]

[0252] Test Example 9:

[0253] NMP22 chemiluminescence detection kit test procedure:

[0254] The NMP22 chemiluminescence detection kit of Example 21 was loaded onto the Shine i2910 system, and the corresponding Shine i2910 system operation manual was followed to detect HBP.

[0255] The NMP22 protein was diluted to different concentrations, and was loaded onto the machine together with M and R reagents.

[0256] The Shine i2910 system will sequentially suck 10 μL of HBP protein solution with different concentrations, 25 μL of M reagent, and 50 μL of R reagent; after mixing, incubating and washing according to the preset program, pre-priming liquid and priming liquid are added, the relative luminous intensity is detected, and the content of the measured substance is calculated, so as to draw the curve of the measured substance and the luminous intensity. The above-mentioned pre-priming liquid and priming liquid are common reagents on the market.

[0257] It should be noted that the reagent M is mixed to re-suspend the magnetic microparticles that may precipitate during transportation. The following operations can be taken:

[0258] 1) Turn the M reagent bottle over 30 times, and control the speed during turning to avoid generating bubbles;

[0259] 2) Visually inspect the reagent bottle to check whether the magnetic particles are suspended. If the magnetic particles are still attached to the bottle, continue to flip the bottle until the magnetic particles are completely suspended. If they are not suspended, do not use.

[0260] Before using reagent R, gently turn the R reagent bottle 8 times to mix it evenly.

[0261] Figure 5 In order to determine the RLU change curve of different concentrations of NMP22 recombinant antigen, some points were selected to make Figure 6 The standard curve of NMP22 recombinant antigen. In this test case, NMP22 recombinant antigen has a wide linear range and can be well detected in the range of 200-500 ng / mL. The linear correlation coefficient is R 2 =0.994. In addition, it can be seen that the chemiluminescent signal in this test example is strong, indicating that the NMP22 chemiluminescent detection kit has good sensitivity.

[0262] By obtaining different nucleic acid aptamers, the chemiluminescence detection kit prepared in the above examples can be used to detect multiple proteins, including HBP, cTnI and NMP22.

[0263] The above embodiments and / or implementation methods are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

Claims

1. A nucleic acid aptamer-based chemiluminescent detection kit, the chemiluminescent detection kit comprising: Reagent M and reagent R, the reagent M includes magnetic particle coupling recognition molecule, the reagent R includes chemiluminescence label coupling recognition molecule, the recognition molecule is nucleic acid aptamer or antibody, the recognition molecule in reagent M and reagent R is not simultaneously antibody, the nucleotide sequence of nucleic acid aptamer includes one of the following: The HBP nucleic acid aptamer is shown as SEQ ID NO.1; The cTnI nucleic acid aptamer is shown as SEQ ID NO.2; The NMP22 nucleic acid aptamer is shown as SEQ ID NO.3; The magnetic particle contains a modified group, and the group includes one of streptavidin, carboxyl and amino;The chemiluminescence label includes acridan ester; The chemiluminescence detection kit further includes a buffer containing the acridan ester, and the acridan ester buffer contains ethyl undecanoate raffinose;Wherein, the kit further includes a magnetic particle buffer containing the magnetic particle, and the magnetic particle buffer includes folic acid-chitosan-super oxide dismutase.

2. The chemiluminescent detection kit according to claim 1, characterized by The components of the acridan ester buffer are 1-5wt% NaHPO4·12H2O, 0.1-0.5wt% NaH2PO4·2H2O, 0.1-1wt% NaCl, 0.1-1wt% BSA, 0.01-0.1wt% MgCl2·6H2O, 0.1-0.5% Tween 20, 0.1-0.5% Proclin 300, and 1-5wt% ethyl undecanoate raffinose.

3. The chemiluminescence detection kit according to claim 1, wherein the components of the magnetic particle buffer are tris, BSA, teween20 and the folic acid-chitosan-super oxide dismutase, and the mass ratio is 0.6:1:0.1:0.

1.

4. The use of the buffer containing acridinium ester of undecanoic acid ethyl cellobioside and the magnetic microparticle buffer containing folic acid-chitosan-super oxide dismutase for preparing a kit for detecting nucleic acid aptamer by chemiluminescence method to improve stability and accuracy, wherein, The kit includes: reagent M and reagent R, the reagent M includes magnetic particle coupling recognition molecule, the reagent R includes chemiluminescence label coupling recognition molecule, the recognition molecule is nucleic acid aptamer or antibody, the recognition molecule in reagent M and reagent R is not simultaneously antibody, the nucleotide sequence of nucleic acid aptamer includes one of the following: The HBP nucleic acid aptamer is shown as SEQ ID NO.1; The cTnI nucleic acid aptamer is shown as SEQ ID NO.2; The NMP22 nucleic acid aptamer is shown as SEQ ID NO.3; The magnetic particle contains a modified group, and the group includes one of streptavidin, carboxyl and amino;The chemiluminescence label includes acridan ester; The chemiluminescence detection kit further includes a buffer containing the acridan ester, and the acridan ester buffer contains ethyl undecanoate raffinose;Wherein, the kit further includes a magnetic particle buffer containing the magnetic particle, and the magnetic particle buffer includes folic acid-chitosan-super oxide dismutase.

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