Myocardial N69 protein protective solution and myocardial N69 protein detection kit

Abstract

The present application discloses a myocardial N69 protein protective solution and a myocardial N69 protein detection kit. The present application designs a myocardial N69 protein protective solution, the ratio of which includes: 0.12% to 0.48% by mass of Tris-HCl buffer, 1% to 5% by mass of BSA or 0.1% to 0.5% by mass of caseinate sodium, 0.2% to 2% by mass of disodium EDTA, 5% to 20% by mass of trehalose, 0.02% to 0.06% by volume of a surfactant, and 0.08% to 0.12% by volume of a preservative, ProClin300; it also includes a protease inhibitor and a reducing agent. The present application also designs a myocardial N69 protein detection kit. The present application can effectively and long-term preserve N69 protein calibrants and can quickly and accurately detect N69 protein.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a myocardial N69 protein protective solution and a myocardial N69 protein detection kit. Background Art

[0002] With my country's aging population, increasing urbanization, and increasingly prominent sub-health issues, the incidence of cardiovascular disease (CVD) in the country continues to rise. According to the "China Cardiovascular Health and Disease Report 2020," the prevalence of CVD in China continues to rise. In 2018, the total number of cardiovascular and cerebrovascular disease discharges from Chinese hospitals reached 23.1613 million, accounting for 12.80% of all discharges during the same period. Of these, cardiovascular disease accounted for 6.31% and cerebrovascular disease for 6.48%. Among CVD discharges, ischemic heart disease accounted for the highest proportion (36.95%). From 1980 to 2018, the average annual growth rate of CVD discharges in China was 9.73%, faster than the average annual growth rate of discharges for all diseases during the same period (6.34%). The top three diseases with the highest average annual growth rates were cerebral infarction (12.03%), ischemic heart disease (11.22%), and acute myocardial infarction (10.94%). The economic burden of cardiovascular disease on residents and society is increasing day by day and has become a major public health issue.

[0003] Currently, the main methods for detecting myocardial ischemia include electrocardiogram (ECG), coronary CT angiography (CTA), and cardiac marker testing. ECG is a commonly used method for diagnosing myocardial ischemia, offering high sensitivity and specificity, and is convenient and rapid. However, for transient ischemic episodes, ECG changes are only noticeable during the ischemic episode. Once the ischemic episode resolves, the ECG returns to normal. ECG changes are not significant in patients with milder ischemia. Coronary CTA is primarily used to diagnose coronary artery stenosis. It involves injecting a contrast agent into a peripheral vein, followed by spiral CT scanning and computer-processed reconstruction to obtain images of the coronary arteries. It can accurately determine whether coronary artery sclerosis, calcification, stenosis, or occlusion exists, with an accuracy rate of up to 90%. However, there are still some differences compared to coronary angiography, and a small number of patients experience allergic reactions to the contrast agent. Coronary angiography, the gold standard for diagnosing coronary artery disease, requires arterial puncture in the hand or leg, which carries the risk of vascular injury and other complications, making it an invasive test. Coronary angiography requires the use of iodine contrast agents. Even if an iodine allergy skin test has been performed before the examination, a small number of patients may experience allergic reactions or serious complications such as arrhythmia, acute myocardial infarction, or even heart rupture due to the disease, which may be life-threatening.

[0004] Compared with the aforementioned methods, myocardial biomarker testing, due to its noninvasive, rapid, highly specific, and short diagnostic window, is widely used for the early diagnosis of myocardial ischemia or myocardial injury. Creatine kinase isoenzyme (CKMB), once the gold standard for diagnosing myocardial infarction, has been gradually replaced by troponin (cTn), which has become the globally recognized gold standard for diagnosing myocardial injury. In the early stages of myocardial cell injury, cTn is rapidly released from the cytoplasm, with serum / plasma levels rising within 4-6 hours and peaking 8-14 hours after the onset of acute myocardial infarction. However, cTn cannot meet the clinical need for detecting myocardial injury caused by myocardial ischemia in the early stages (within 0-3 hours).

[0005] Recent studies have found that during myocardial ischemia-reperfusion, calpain 1 mediates the enzymatic cleavage of cTnT at position 69 on the N-terminus, producing the myocardial N69 protein. This may represent an acute mechanism for regulating muscle contractility under stress. N69, with a molecular weight of approximately 8 kDa, is approximately one-quarter the size of the intact cTnT molecule (34 kDa). In the early stages of myocardial ischemia-induced myocardial injury (before myocardial necrosis), it can enter the bloodstream before cTnT or the cTn-ITC complex, making it crucial for the early diagnosis of myocardial injury caused by myocardial ischemia. However, due to the difficulty in long-term storage of N69 protein calibrators and the lack of clinical research, there are currently no N69 protein detection products. Summary of the Invention

[0006] In order to solve at least one of the above technical problems, a detection product that can effectively and long-term preserve N69 protein calibrators and can quickly and accurately detect N69 protein is developed. The present application provides a myocardial N69 protein protective solution and a myocardial N69 protein detection kit.

[0007] On the one hand, the present application provides a myocardial N69 protein protective solution, the ratio of which includes: 0.12%~0.48% by mass of Tris-HCl buffer, 1%~5% by mass of BSA or 0.1%~0.5% by mass of caseinate sodium, 0.2%~2% by mass of disodium EDTA, 5%~20% by mass of trehalose, 0.02%~0.06% by volume of surfactant, and 0.08%~0.12% by volume of preservative; it also includes protease inhibitors and reducing agents.

[0008] Optionally, the pH of the Tris-HCl buffer is not lower than 7.2.

[0009] Optionally, the protease inhibitor is 0.05% to 0.3% by mass of benzamidine and / or 0.5% to 2% by mass of aprotinin.

[0010] Optionally, the reducing agent is 0.5% to 3% by mass of vitamin C and 0.2% to 0.8% by mass of tris(2-formylethyl)phosphine hydrochloride.

[0011] Optionally, the surfactant is Tween 20 and the preservative is ProClin 300.

[0012] Optionally, the preferred ratio of the myocardial N69 protein protective solution includes: 0.24% mass fraction of Tris-HCl buffer with pH 7.4, 2% mass fraction of BSA, 0.4% mass fraction of disodium EDTA, 0.15% mass fraction of benzamidine, 1% mass fraction of aprotinin, 10% mass fraction of trehalose, 1.5% mass fraction of vitamin C, 0.4% mass fraction of tris(2-formylethyl)phosphine hydrochloride, 0.04% volume fraction of Tween 20, and 0.1% volume fraction of ProClin300.

[0013] On the other hand, the present application provides a myocardial N69 protein detection kit, comprising a reagent strip and a calibrator; the reagent strip comprises at least one sample well, 11 reagent wells and one light-proof detection cup, which are arranged in a straight line along the reagent strip; the sample well is used to load the sample, the reagent well located on one side of the sample well is loaded with a working wash solution, the three reagent wells located on one side of the light-proof detection cup are respectively loaded with an enzyme-labeled antibody reagent, a magnetic bead reagent and a chemiluminescent substrate solution, and the light-proof detection cup is used to detect the chemiluminescent signal; the calibrator is configured with an N69 protein standard and the above-mentioned myocardial N69 protein protective solution.

[0014] Optionally, the working wash solution includes 200 mM Tris-HCl buffer at pH 8.0, 0.9% mass fraction of NaCl, 0.5% volume fraction of Tween 20, and 0.1% volume fraction of Proclin 300.

[0015] Optionally, the enzyme-labeled antibody reagent enzyme label diluent is a proportional dilution of the ALP-labeled N69 antibody, and the enzyme label diluent includes 200 mM Tris-HCl buffer at pH 8.0, 0.9% mass fraction of NaCl, 3% mass fraction of BSA, 10% volume fraction of a stabilizer, and 0.1% volume fraction of Proclin300.

[0016] Optionally, the magnetic bead working solution reagent is an N69 antibody coupled to magnetic beads diluted proportionally with a magnetic bead diluent, wherein the magnetic bead diluent includes 200 mM Tris-HCl buffer at pH 8.0, 0.9% mass fraction of NaCl, 30% volume fraction of glycerol, 10% mass fraction of gelatin, 5% mass fraction of trehalose, and 0.1% volume fraction of Proclin300.

[0017] In summary, the present invention includes at least one of the following beneficial technical effects:

[0018] 1. The myocardial N69 protein protective solution provided by the present invention adopts an appropriate ratio to provide long-term preservation and protection for N69 protein, and can effectively and long-term preserve myocardial N69 protein calibrators.

[0019] 2. The present invention uses the myocardial N69 protein protective solution designed in this application to prepare a calibrator that can be preserved for a long time, and then designs a myocardial N69 protein detection kit, which effectively fills the market gap and can provide early and accurate diagnosis of myocardial ischemia.

[0020] 3. The present invention has a simple configuration, easy-to-obtain components, relatively low cost, and high detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the structure of the test strip of the present invention. Implementation Method

[0022] The present application is further described in detail below with reference to the accompanying drawings and examples.

[0023] It should be noted that if no specific conditions are specified in the following examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the instruments, materials, reagents, etc. used in the following examples can be obtained through commercial channels.

[0024] Antibodies and antigens used in magnetic-linked reagents and enzyme-labeled antibodies were commercially available, and magnetic particles were purchased from Thermo Fisher Scientific. Healthy and myocardial ischemia samples were obtained from the hospital.

[0025] The present application designs a myocardial N69 protein protective solution, which includes: 0.12%~0.48% by mass of Tris-HCl buffer, 1%~5% by mass of BSA or 0.1%~0.5% by mass of caseinate sodium, 0.2%~2% by mass of disodium EDTA, 5%~20% by mass of trehalose, 0.02%~0.06% by volume of a surfactant, and 0.08%~0.12% by volume of a preservative; it also includes a protease inhibitor and a reducing agent.

[0026] Optionally, the protease inhibitor is 0.05% to 0.3% by mass of benzamidine and / or 0.5% to 2% by mass of aprotinin.

[0027] Optionally, the reducing agent is 0.5% to 3% by mass of vitamin C and 0.2% to 0.8% by mass of tris(2-formylethyl)phosphine hydrochloride.

[0028] Optionally, the surfactant is Tween 20 and the preservative is ProClin 300.

[0029] The present invention uses EDTA to chelate metal ions in the solution to prevent N69 protein degradation by proteases with metal ions as active centers. Protease inhibitors are also used to prevent N69 protein degradation by proteases. BSA or sodium caseinate acts as a protective protein to indirectly prevent N69 protein degradation by proteases. The present invention also adds a reducing agent to prevent chemical and photochemical oxidation of methionine in N69 protein to form methionine sulfoxide, thereby resolving the issue of N69 protein degradation leading to inaccurate detection of N69 protein by reagents.

[0030] The following is an example of a protective solution for myocardial N69 protein: Example

[0031] The myocardial N69 protein protective solution of this example comprises: 0.12% (w / v) Tris-HCl buffer at a pH of 7.2, containing a final concentration of 1% (w / v) BSA, 0.2% (w / v) disodium EDTA, 0.05% (w / v) benzamidine, 0.5% (w / v) aprotinin, 5% (w / v) trehalose, 0.5% (w / v) vitamin C, 0.2% (w / v) tris(2-formylethyl)phosphine hydrochloride, 0.02% (v / v) Tween 20, and 0.1% (v / v) ProClin 300 (all w / v values ​​above refer to g / mL, and v / v values ​​refer to mL / mL). Example

[0032] The myocardial N69 protein protective solution of this example has the following ratio: 0.48% (w / v) Tris-HCl buffer with a pH of 7.2, containing a final concentration of 5% (w / v) BSA, 2% (w / v) disodium EDTA, 0.3% (w / v) benzamidine, 2% (w / v) aprotinin, 20% (w / v) trehalose, 3% (w / v) vitamin C, 0.8% (w / v) tris(2-formylethyl)phosphine hydrochloride, 0.06% (v / v) Tween 20, and 0.1% (v / v) ProClin 300 (all w / v values ​​above refer to g / mL, and v / v values ​​refer to mL / mL). Example

[0033] The myocardial N69 protein protective solution of this example comprises: 0.12% (w / v) Tris-HCl buffer at a pH of 7.2, containing a final concentration of 0.1% (w / v) sodium caseinate, 0.2% (w / v) disodium EDTA, 0.05% (w / v) benzamidine, 0.5% (w / v) aprotinin, 5% (w / v) trehalose, 0.5% (w / v) vitamin C, 0.2% (w / v) tris(2-formylethyl)phosphine hydrochloride, 0.02% (v / v) Tween 20, and 0.1% (v / v) ProClin 300 (all w / v values ​​above refer to g / mL, and v / v values ​​refer to mL / mL). Example

[0034] The myocardial N69 protein protective solution of this example has the following ratio: 0.48% (w / v) Tris-HCl buffer with a pH of 7.2, containing a final concentration of 0.5% (w / v) sodium caseinate, 2% (w / v) disodium EDTA, 0.3% (w / v) benzamidine, 2% (w / v) aprotinin, 20% (w / v) trehalose, 3% (w / v) vitamin C, 0.8% (w / v) tris(2-formylethyl)phosphine hydrochloride, 0.06% (v / v) Tween 20, and 0.1% (v / v) ProClin 300 (all w / v values ​​above refer to g / mL, and v / v values ​​refer to mL / mL). Example

[0035] The myocardial N69 protein protective solution of this example comprises: 0.24% (w / v) Tris-HCl buffer at a pH of 7.4, containing a final concentration of 2% (w / v) BSA, 0.4% (w / v) disodium EDTA, 0.15% (w / v) benzamidine, 1% (w / v) aprotinin, 10% (w / v) trehalose, 1.5% (w / v) vitamin C, 0.4% (w / v) tris(2-formylethyl)phosphine hydrochloride, 0.04% (v / v) Tween 20, and 0.1% (v / v) ProClin 300 (all w / v values ​​above refer to g / mL, and v / v values ​​refer to mL / mL). Example

[0036] The myocardial N69 protein protective solution of this example comprises: 0.36% (w / v) Tris-HCl buffer at a pH of 7.8, containing a final concentration of 4% (w / v) BSA, 1.6% (w / v) disodium EDTA, 0.15% (w / v) benzamidine, 1% (w / v) aprotinin, 10% (w / v) trehalose, 2% (w / v) vitamin C, 0.6% (w / v) tris(2-formylethyl)phosphine hydrochloride, 0.04% (v / v) Tween 20, and 0.1% (v / v) ProClin 300 (all w / v values ​​above refer to g / mL, and v / v values ​​refer to mL / mL). Example

[0037] The myocardial N69 protein protective solution of this example comprises: 0.36% (w / v) Tris-HCl buffer at a pH of 7.0, containing a final concentration of 0.3% (w / v) sodium caseinate, 1% (w / v) disodium EDTA, 0.15% (w / v) benzamidine, 1% (w / v) aprotinin, 15% (w / v) trehalose, 1% (w / v) vitamin C, 0.6% (w / v) tris(2-formylethyl)phosphine hydrochloride, 0.04% (v / v) Tween 20, and 0.1% (v / v) ProClin 300 (all w / v values ​​above refer to g / mL, and v / v values ​​refer to mL / mL). Example

[0038] The myocardial N69 protein protective solution of this example has the following ratio: 0.24% (w / v) Tris-HCl buffer with a pH of 7.2, containing a final concentration of 2% (w / v) BSA, 0.4% (w / v) disodium EDTA, 0.15% (w / v) benzamidine, 1% (w / v) aprotinin, 10% (w / v) trehalose, 2% (w / v) astaxanthin, 0.6% (w / v) sodium bisulfite, 0.04% (v / v) Tween 20, and 0.1% (v / v) ProClin 300 (all w / v values ​​above refer to g / mL, and v / v values ​​refer to mL / mL). Example

[0039] The myocardial N69 protein protective solution of this example has the following ratio: 0.24% (w / v) Tris-HCl buffer with a pH of 7, containing a final concentration of 2% (w / v) BSA, 0.4% (w / v) disodium EDTA, 0.15% (w / v) 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 1% (w / v) nafamostat, 10% (w / v) trehalose, 1.5% (w / v) vitamin C, 0.4% (w / v) tris(2-formylethyl)phosphine hydrochloride, 0.04% (v / v) Tween 20, and 0.1% (v / v) ProClin 300 (all w / v values ​​above refer to g / mL, and v / v values ​​refer to mL / mL).

[0040] The present application also designs a myocardial N69 protein detection kit, including a reagent strip and a calibrator; the reagent strip has at least one sample well, 11 reagent wells and one light-proof detection cup, which are arranged in a straight line along the reagent strip; the sample well is used to load the sample, the reagent well located on one side of the sample well is loaded with a working wash solution, the three reagent wells located on one side of the light-proof detection cup are respectively loaded with an enzyme-labeled antibody reagent, a magnetic bead reagent and a chemiluminescent substrate solution, and the light-proof detection cup is used to detect the chemiluminescent signal; the calibrator is configured using an N69 protein standard and the above-mentioned myocardial N69 protein protective solution.

[0041] Optionally, the working wash solution includes 200 mM Tris-HCl buffer at pH 8.0, 0.9% mass fraction of NaCl, 0.5% volume fraction of Tween 20, and 0.1% volume fraction of Proclin 300.

[0042] Optionally, the enzyme-labeled antibody reagent enzyme label diluent is a proportional dilution of the ALP-labeled N69 antibody, and the enzyme label diluent includes 200 mM Tris-HCl buffer at pH 8.0, 0.9% mass fraction of NaCl, 3% mass fraction of BSA, 10% volume fraction of a stabilizer, and 0.1% volume fraction of Proclin300.

[0043] Optionally, the magnetic bead working solution reagent is an N69 antibody coupled to magnetic beads diluted proportionally with a magnetic bead diluent, wherein the magnetic bead diluent includes 200 mM Tris-HCl buffer at pH 8.0, 0.9% mass fraction of NaCl, 30% volume fraction of glycerol, 10% mass fraction of gelatin, 5% mass fraction of trehalose, and 0.1% volume fraction of Proclin300.

[0044] The chemiluminescent substrate solution is prepared according to the method described in the invention patent publication number CN107449771B.

[0045] The following is an example of a kit for detecting myocardial N69 protein in real time Example

[0046] The kit of this embodiment consists of a reagent strip and a calibrator; the reagent strip includes at least one sample well (1#), 11 reagent wells (2#-12#), and one light-proof detection cup; the sample well (1#) is loaded with 50 μL of the test sample; reagent wells 2#-6# are each loaded with 400 μL of the working wash solution; reagent wells 9#, 10#, and 11# are respectively loaded with 60 μL of the enzyme-labeled antibody reagent, 50 μL of the magnetic bead reagent, and 110 μL of the chemiluminescent substrate solution; and the light-proof detection cup (13#) is used to detect the chemiluminescent signal.

[0047] The working wash solution was 200 mM Tris-HCl buffer at pH 8.0, containing a final concentration of 0.9% (w / v) NaCl, 0.5% (v / v) TW-20, and 0.1% (v / v) Proclin300 (all w / v values ​​above refer to g / mL);

[0048] The enzyme-labeled antibody reagent is ALP-labeled N69 antibody diluted in enzyme label diluent at a volume ratio of 1:500; the enzyme label diluent is 200 mM Tris-HCl buffer, pH 8.0, containing a final concentration of 0.9% (w / v) NaCl, 3% (w / v) BSA, 10% (v / v) stabilizer, and 0.1% (v / v) Proclin 300 (all w / v values ​​above refer to g / mL, and v / v values ​​refer to mL / mL);

[0049] The magnetic bead reagent consisted of N69 antibody coupled to magnetic beads diluted in magnetic bead diluent at a volume ratio of 20:1. The magnetic bead diluent consisted of 200 mM Tris-HCl buffer, pH 8.0, containing a final concentration of 0.9% (w / v) NaCl, 30% (v / v) glycerol, 10% (w / v) gelatin, 5% (w / v) trehalose, and 0.1% (v / v) Proclin 300 (all w / v values ​​refer to g / mL, and v / v values ​​refer to mL / mL).

[0050] The chemiluminescent substrate solution is 0.2 mol / L Tris-HCl buffer at pH 9.0, containing 0.5 g / L 9-(4-chlorophenylthiobenzoyloxymethylene)-10-methyl-9,10-dihydroacridinium disodium salt, 0.3 mg / L 5-(myristoylamino)fluorescein, 0.5 g / L aryl acylhydrazone-9,10-acridine derivative, 1.0 mM MgCl2, 1.0 mM ZnCl2, 5 mg / L hexadecyltrimethylammonium chloride, 0.5 g / L Proclin 300, and 0.5 wt% Tween 20.

[0051] The calibrator is N69 protein diluted with a calibrator diluent at different ratios. The calibrator diluent is the myocardial N69 protein protective solution described in Example 1.

[0052] When using the detection kit of this embodiment for detection, first, the calibrator is added to the sample well (1#), and the reagent strip is placed in the detection instrument. The chemiluminescence intensity value is detected, and a calibration curve is prepared with the different concentrations of N69 protein in the calibrator as the horizontal axis and the chemiluminescence intensity value as the vertical axis; then, the sample is added to the sample well (1#), and the reagent strip is placed in the detection instrument. The chemiluminescence intensity value is detected, and the concentration of N69 protein in the sample is calculated based on the linear relationship.

[0053] Examples 11 to 18

[0054] The difference from Example 10 is that the calibrator diluents are the myocardial N69 protein protection solutions in Examples 2 to 9 respectively; the rest is the same as Example 10.

[0055] Comparative Example 1

[0056] The difference from Example 10 is that the calibrator diluent adopts a conventional diluent, whose composition is: 0.24% (w / v) Tris-HCl buffer with a pH of 7.2, containing a final concentration of 10% (v / v) newborn calf serum and 0.1% (v / v) ProClin300 (all w / v refers to g / mL, and v / v refers to mL / mL); otherwise, it is the same as Example 10.

[0057] Performance testing

[0058] (1) Comparison of the stability of the N69 protein assay kits prepared in Example 10 and Comparative Example 1.

[0059] The N69 protein assay kits prepared in Example 10 and Comparative Example 1 were placed under conditions of 2-8°C, 37°C for 3 days, and 37°C for 6 days (accelerated destruction test, simulating storage conditions at 2-8°C for 1 year), respectively. After removal, the kits were recalibrated and the luminescence values ​​of the calibrators were compared. The results are shown in Tables 1 and 2.

[0060] Table 1 Detection of calibrators after accelerated destruction of two N69 test kits

[0061]

[0062] Table 2 Deviation of luminescence values ​​of calibrators detected by two N69 test kits after accelerated destruction

[0063]

[0064] It can be seen from Tables 1 and 2 that the luminescence value of the calibrator was detected under the conditions of 37 degrees for 3 days and 6 days (accelerated destruction test, simulating storage conditions of 2 to 8 degrees for 1 year) of the N69 test kit of Example 10 of the present application. The relative deviation between the detected luminescence value and the luminescence value of the test kit stored at 2 to 8 degrees was less than 15%; while the luminescence value of the calibrator was detected under the conditions of 37 degrees for 3 days and 6 days (accelerated destruction test, simulating storage conditions of 2 to 8 degrees for 1 year) of the N69 test kit prepared in Comparative Example 1. The relative deviation between the detected luminescence value and the luminescence value of the test kit stored at 2 to 8 degrees was -33% to -96%.

[0065] Through the comparison of the above data, it can be fully demonstrated that the detection stability of the N69 assay kit prepared in Example 10 of the present application is far superior to the detection stability of the kit prepared in Comparative Example 1, that is, the N69 protein protective solution prepared in Example 10 of the present application better solves the problem that the N69 protein calibrator is difficult to preserve for a long time.

[0066] (2) Stability of N69 protein calibrants prepared using the N69 protein protective solutions prepared in Examples 1 to 5.

[0067] Using the N69 protein protection solution prepared in Examples 1 to 5 as the diluent, the N69 protein was gradiently diluted to prepare its calibrator. The luminescence value of the calibrator was detected under conditions of 2 to 8 degrees, 37 degrees for 3 days, and 37 degrees for 6 days (accelerated destruction test, simulating storage conditions at 2 to 8 degrees for 1 year). The specific results are shown in Tables 3 and 4.

[0068] Table 3-1 Stability of calibrants prepared using five N69 protein protective solutions

[0069]

[0070] Table 3-2 Stability of calibrants prepared using five N69 protein protective solutions

[0071]

[0072] Table 4-1 Luminescence value deviation of calibrants prepared using five N69 protein protective solutions

[0073]

[0074] Table 4-2 Luminescence value deviation of calibrants prepared from five N69 protein protective solutions

[0075]

[0076] As shown in Tables 3 and 4, the N69 protein protection solutions prepared in Examples 1 to 5 of the present application have a good protective effect on the N69 protein. The relative deviation between the luminescence value of the calibrator detected at 37°C for 3 days and 6 days (accelerated destruction test, simulating storage conditions of 2 to 8°C for 1 year) and the luminescence value detected by the test kit stored at 2 to 8°C is less than 10%, which effectively solves the problem of the long-term storage of N69 protein calibrators. It can also be seen from Tables 3 and 4 that the N69 protein protection solution prepared in Example 5 of the present application has the best protective effect on the N69 protein.

[0077] (3) Comparison of the stability of N69 protein calibrants prepared using the N69 protein protection solutions prepared in Examples 6 to 9.

[0078] Using the N69 protein protection solution prepared in Examples 6 to 9 as the diluent, the N69 protein was gradiently diluted to prepare its calibrator. The luminescence value deviation of the calibrator was detected under conditions of 2 to 8 degrees, 37 degrees for 3 days, and 37 degrees for 6 days (accelerated destruction test, simulating storage conditions at 2 to 8 degrees for 1 year). The specific results are shown in Table 5.

[0079] Table 5-1 Luminescence value deviation of calibrants prepared with four N69 protein protection solutions

[0080]

[0081] Table 5-2 Luminescence value deviation of calibrants prepared with four N69 protein protection solutions

[0082]

[0083] As can be seen from Table 5, the N69 protein protection solution prepared in Examples 6 to 9 of the present application also has a good protective effect on the N69 protein. The relative deviation of the luminescence value of the calibrator detected under the conditions of 37 degrees for 3 days and 6 days (accelerated destruction test, simulating the storage conditions of 2 to 8 degrees for 1 year) and the luminescence value detected by the test kit stored at 2 to 8 degrees is less than 10%, which can better solve the problem of long-term storage of N69 protein calibrators.

[0084] Table 5 also shows that the N69 protein protection solution prepared in Example 7 of the present application uses a Tris-HCl buffer having a pH of 7.0, and its protective effect on the N69 protein is slightly lower than that of the N69 protein protection solutions prepared in Examples 1 to 6. Therefore, it is preferable that the pH of the Tris-HCl buffer in the N69 protein protection solution of the present application be controlled at or above 7.2.

[0085] Table 5 also shows that the N69 protein protection solution prepared in Example 8 of the present application, after using astaxanthin and sodium bisulfite as reducing agents, has a significantly worse protection effect on N69 protein than the N69 protein protection solutions prepared in Examples 1 to 7; the N69 protein protection solution prepared in Example 9 of the present application, after using 4(-2-aminoethyl)benzenesulfonyl fluoride hydrochloride and nafamostat as protease inhibitors, has a significantly worse protection effect on N69 protein than the N69 protein protection solutions prepared in Examples 1 to 7. It can be seen that although astaxanthin and sodium bisulfite have excellent antioxidant properties, their antioxidant properties are comparable to those of vitamin C and tris(2-formylethyl)phosphine hydrochloride, for the present application, the selection of vitamin C and tris(2-formylethyl)phosphine hydrochloride as reducing agents has a more prominent effect on the protection of N69 protein. In addition, although 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and nafamostat are both excellent serine protease inhibitors, their inhibitory effects on serine proteases are better than those of benzamidine and aprotinin, especially nafamostat, which is an artificially developed serine protease inhibitor with extremely excellent performance; however, for the present application, benzamidine and aprotinin are selected as protease inhibitors, which have a more prominent effect on the protection of N69 protein.

[0086] (4) Comparison of the N69 protein assay kit prepared in Example 10 with commercially available myocardial markers in detecting clinical myocardial ischemia samples.

[0087] The emergency samples were tested using the N69 protein assay kit prepared in Example 10 and a commercially available troponin T assay kit (chemiluminescence method). Subsequently, 6 normal samples and 6 cases of myocardial ischemia were confirmed. The test results are shown in Table 6 below.

[0088] Table 6-1 Comparison of the N69 protein assay kit and the commercially available myocardial marker cTnT in the detection of clinical myocardial ischemia samples

[0089]

[0090] Table 6-2 Comparison of N69 protein assay kit and commercially available myocardial marker cTnT in the detection of clinical myocardial ischemia samples

[0091]

[0092] As shown in Table 6, the two detection methods had good consistency in the test results for normal samples. For myocardial ischemia samples, due to the smaller molecular weight of N69, it may enter the blood earlier in the early stage of myocardial ischemia. All 6 myocardial ischemia samples were detected, while cTnT detection only detected 5 out of 6 myocardial ischemia samples, which may be due to the fact that cTnT enters the blood later than N69.

[0093] (5) Detection of samples 7 to 12 using the N69 protein assay kit prepared in Examples 11 to 18.

[0094] The above 6 myocardial ischemia samples were also tested using the N69 protein assay kit prepared in Examples 11 to 18. The test results are shown in Table 7 below.

[0095] Table 7 N69 protein assay kit for clinical myocardial ischemia samples

[0096]

[0097] As shown in Table 7, the detection results of the above 6 myocardial ischemia samples using the detection kits of Examples 11 to 18 of the present application are consistent with the results of Example 10 of the present application. It can be seen that the detection kit designed in the present application can accurately detect myocardial ischemia samples.

[0098] The above are all preferred embodiments of the present application, and are not intended to limit the scope of protection of the present application. Therefore, any equivalent changes made based on the structure, shape, and principle of the present application should be included in the scope of protection of the present application.

Claims

1. A myocardial N69 protein detection kit, characterized in that: The apparatus comprises a reagent strip and a calibrator; the reagent strip comprises at least one sample well, 11 reagent wells and one light-proof detection cup, which are arranged in a straight line along the reagent strip; the sample well is used to load the sample, the reagent well located on one side of the sample well is loaded with a working wash solution, the three reagent wells located on one side of the light-proof detection cup are loaded with an enzyme-labeled antibody reagent, a magnetic bead reagent and a chemiluminescent substrate solution respectively, and the light-proof detection cup is used to detect chemiluminescent signals; the calibrator is prepared by using an N69 protein standard and a myocardial N69 protein protective solution, and the ratio of the myocardial N69 protein protective solution is: 0.12% to 0.48% by mass of Tris -HCl buffer, 1%~5% by mass of BSA or 0.1%~0.5% by mass of caseinate sodium, 0.2%~2% by mass of disodium EDTA, 5%~20% by mass of trehalose, 0.02%~0.06% by volume of Tween 20, 0.08%~0.12% by volume of ProClin300, protease inhibitors and reducing agents; the protease inhibitors are 0.05%~0.3% by mass of benzamidine and 0.5%~2% by mass of aprotinin, and the reducing agent is 0.5%~3% by mass of vitamin C and 0.2%~0.8% by mass of tris(2-formylethyl)phosphine hydrochloride.

2. A myocardial N69 protein detection kit according to claim 1, characterized in that: The pH of the Tris-HCl buffer is not lower than 7.

2.

3. A myocardial N69 protein detection kit according to claim 1, characterized in that: The myocardial N69 protein protective solution comprises: 0.24% by mass of pH 7.4 Tris-HCl buffer, 2% by mass of BSA, 0.4% by mass of disodium EDTA, 0.15% by mass of benzamidine, 1% by mass of aprotinin, 10% by mass of trehalose, 1.5% by mass of vitamin C, 0.4% by mass of tris(2-formylethyl)phosphine hydrochloride, 0.04% by volume of Tween 20, and 0.1% by volume of ProClin 300.

4. A myocardial N69 protein detection kit according to claim 1, characterized in that: The working wash solution includes 200 mM Tris-HCl buffer with a pH of 8.0, 0.9% mass fraction of NaCl, 0.5% volume fraction of Tween 20, and 0.1% volume fraction of Proclin 300.

5. A myocardial N69 protein detection kit according to claim 1, characterized in that: The enzyme-labeled antibody reagent is an ALP-labeled N69 antibody diluted proportionally with an enzyme-labeled diluent, wherein the enzyme-labeled diluent includes 200 mM Tris-HCl buffer at pH 8.0, 0.9% mass fraction of NaCl, 3% mass fraction of BSA, 10% volume fraction of a stabilizer, and 0.1% volume fraction of Proclin 300.

6. A myocardial N69 protein detection kit according to claim 1, characterized in that: The magnetic bead reagent is an N69 antibody coupled to magnetic beads diluted in proportion with a magnetic bead diluent, wherein the magnetic bead diluent includes 200 mM Tris-HCl buffer at pH 8.0, 0.9% mass fraction of NaCl, 30% volume fraction of glycerol, 10% mass fraction of gelatin, 5% mass fraction of trehalose, and 0.1% volume fraction of Proclin 300.

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