A creatine kinase isoenzyme detection kit

Abstract

The invention discloses a creatine kinase isoenzyme detection kit, belonging to the field of biological detection technology. The creatine kinase isoenzyme detection kit includes reagent R1 and reagent R2; wherein the components of reagent R1 are: buffer, coagulant, chelating agent, blocking agent, surfactant, stabilizer and preservative; the components of reagent R2 are: buffer, latex particles coated with creatine kinase isoenzyme antibodies, blocking agent, stabilizer and preservative. The creatine kinase isoenzyme detection kit of the invention overcomes the defects of the existing creatine kinase isoenzyme detection kit, has the advantages of strong anti-interference, high sensitivity, high specificity, high accuracy, wide linear range and good stability, and provides a safe, fast, accurate and pollution-free detection means for the detection of creatine kinase isoenzymes.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a creatine kinase isoenzyme detection kit. Background Art

[0002] Creatine kinase (CK) is a dimeric enzyme that has four different forms: mitochondrial isozyme, cytoplasmic isozyme CK-MM (muscle type), creatine kinase brain isozyme CK-BB (brain type), and creatine kinase isozyme (CK-MB), among which creatine kinase isozyme is mainly located in the myocardium.

[0003] Creatine kinase isoenzymes have extremely high sensitivity and specificity in diagnosing transmural myocardial infarction. Under normal conditions, their expression level is low. Creatine kinase isoenzymes are excreted from damaged cells 4-8 hours after onset and begin to increase rapidly in the blood, reaching the highest level after 12-24 hours and returning to normal within 24-48 hours. If creatine kinase isoenzymes remain at a high level after acute myocardial infarction, it indicates that myocardial necrosis is still ongoing; if they rise again after returning to normal, it indicates that the original infarction site has expanded or a new infarction site has appeared. Therefore, quantitative detection of creatine kinase isoenzyme levels in the blood is of great value for the early diagnosis of acute myocardial infarction (AMI).

[0004] At present, the methods for determining creatine kinase isoenzymes include electrophoresis, immunosuppression, radioimmunoassay, immunoenzyme labeling, gold labeling, ion exchange column chromatography, etc. The most commonly used methods are electrophoresis and immunosuppression, but electrophoresis cannot be applied to fully automatic biochemical analyzers, and the instruments used are expensive and cannot be popularized; immunosuppression has the advantages of rapidity, time saving and high sensitivity, but there are many influencing factors. Other commonly used detection methods include immunoturbidimetry and enzyme-linked immunosorbent assay. The enzyme-linked immunosorbent assay has the disadvantages of cumbersome operation, low sensitivity, narrow linear range and long detection cycle, and cannot meet the requirements of rapid quantitative simple detection in large hospitals. With the popularization of fully automatic biochemical analyzers, immunoturbidimetry has developed a variety of rapid immunoturbidimetric detection technologies.

[0005] Latex enhanced immunoturbidimetry is a dynamic detection method for antigen-antibody binding. By coating the antibody corresponding to the target antigen to be detected on latex microspheres, the volume of the antigen-antibody conjugate is increased. In the detection light path, the intensity changes of the transmitted light and scattered light are more significant, thereby improving the sensitivity of the detection.

[0006] Although there are creatine kinase isoenzyme detection kits based on latex-enhanced immunoturbidimetry on the market, these kits have problems such as narrow detection linear range, low sensitivity, poor anti-interference ability and unstable reagent storage.

[0007] Based on the above, the art needs to research and develop a creatine kinase isoenzyme detection kit with strong anti-interference, high sensitivity, high specificity, high accuracy, wide detection range and good stability, so that creatine kinase isoenzymes can be widely promoted and applied in the clinical diagnosis of acute myocardial infarction. Summary of the invention

[0008] In view of the above problems, the present invention provides a creatine kinase isoenzyme detection kit with strong anti-interference ability, high sensitivity, high specificity, high accuracy, wide linear range and good stability.

[0009] The present invention is achieved through the following technical solutions:

[0010] A creatine kinase isoenzyme detection kit, comprising reagent R1 and reagent R2;

[0011] The components and contents of the reagent R1 are: buffer 10-50 mM, coagulant 2-5 g / L, chelating agent 1-2 g / L, blocking agent 0.5-2 g / L, surfactant 1-5 g / L, stabilizer 0.5-1 g / L, preservative 1-2 g / L;

[0012] The components and contents of the reagent R2 are: buffer solution 10-50 mM, latex particles coated with creatine kinase isozyme antibodies 0.05-0.4% w / v, blocking agent 1-10 g / L, stabilizer 5-20 g / L, and preservative 1-2 g / L.

[0013] Furthermore, the surfactant in the reagent R1 is compounded by TritonX-100 and styrene polyoxyethylene ether in a mass ratio of 2-5:1.

[0014] Furthermore, the blocking agent in the reagent R2 is prepared by compounding triethanolamine, glycine and casein in a mass ratio of 0.5-2:1:1-4.

[0015] Furthermore, the blocking agent in the reagent R1 is mouse IgG; and the chelating agent is EGTA.

[0016] Furthermore, the coagulant in the reagent R1 is any one of PEG-4000, PEG-6000, PEG-8000, and dextran.

[0017] Furthermore, the buffers in the reagents R1 and R2 are each independently selected from one of phosphate buffer, glycine buffer, Tris buffer, HEPES buffer, and MES buffer.

[0018] Furthermore, the stabilizers in the reagent R1 and the reagent R2 are each independently selected from one of bovine serum albumin, trehalose, chitosan and glycerol.

[0019] Furthermore, the preservatives in the reagents R1 and R2 are each independently selected from one of ProClin300, ProClin950, phenol, p-hydroxybenzoic acid, and ethyl p-hydroxybenzaldehyde.

[0020] Furthermore, the creatine kinase isozyme antibody in the reagent R2 is a mouse monoclonal antibody; the latex particles are polystyrene latex microspheres with a diameter of 250-400 nm, and the surface of the microspheres is modified with functional groups, and the modified functional groups are selected from carboxyl, amino or hydroxyl groups.

[0021] Furthermore, the creatine kinase isozyme antibody in the reagent R2 is coated onto the surface of the latex microspheres by chemical crosslinking, the chemical crosslinking agent used is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, maleimide, carboimide, mercaptonicotinamide, and isocyanate, the crosslinking buffer is selected from HEPES buffer, MOPS buffer, MES buffer, and MOPSO buffer, and the pH is between 6.0 and 8.0.

[0022] Further, the method for preparing the above-mentioned creatine kinase isoenzyme detection kit comprises the following method:

[0023] (1) Preparation of reagent R1: Take each component of reagent R1, add it to purified water, mix it evenly, adjust the pH value to 6.5-8.0, filter it through a filter membrane, and make up the volume to obtain reagent R1;

[0024] (2) Preparation of reagent R2:

[0025] ① Activation of microspheres: Add polystyrene latex microspheres with a diameter of 250-400 nm and surface modification with carboxyl, amino or hydroxyl groups to a buffer solution with a pH of 6.0-8.0, and add an activator, react at 25-30°C for 30-40 minutes, remove excess activator from the supernatant after centrifugation, take the precipitate, and resuspend it in a buffer solution to obtain activated polystyrene latex microspheres;

[0026] ② Antibody cross-linking: Add creatine kinase isoenzyme antibodies to activated polystyrene latex microspheres and react at 25-30°C for 1.5-2h to cross-link the antibodies on the surface of the latex microspheres;

[0027] ③ Microsphere blocking: Add blocking agent to the latex microsphere reaction solution after cross-linking with antibodies, block for 1-2 hours, and block the area on the surface of the latex microsphere that is not adhered by antibodies;

[0028] ④Impurity removal: The mixture of blocked latex microspheres is centrifuged to remove excess cross-linking agent, cross-linking byproducts, antibodies and blocking agent. The bottom precipitate is the latex particles coated with creatine kinase isoenzyme antibodies;

[0029] ⑤Mixing and preservation: Take the buffer solution, stabilizer and preservative in reagent R2, add them to purified water, mix well, add latex particles coated with creatine kinase isoenzyme antibodies, stir well, mix well, and obtain reagent R2 after constant volume.

[0030] The creatine kinase isozyme detection kit of the present invention adopts latex enhanced immunoturbidimetry to quantitatively detect the content of creatine kinase isozyme in blood. The principle is that creatine kinase isozyme antigen in the sample to be tested reacts with creatine kinase isozyme antibody coated on latex particles to form antigen-antibody-latex particle complex and agglutinate, resulting in increased turbidity, thereby changing the light diffusion or light transmission performance of the reaction solution. A standard curve is drawn using a standard product, and the content of the substance to be tested can be determined according to the change in the sample absorbance value.

[0031] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0032] 1. The creatine kinase isoenzyme detection kit of the present invention overcomes the defects of the existing creatine kinase isoenzyme detection kit, has the advantages of strong anti-interference, high sensitivity, high specificity, high accuracy, wide linear range and good stability, and provides a safe, fast, accurate and pollution-free detection method for the detection of creatine kinase isoenzymes, thereby providing a powerful experimental diagnosis basis for the clinical diagnosis of acute myocardial infarction.

[0033] 2. The surfactant in the reagent R1 of the present application is a compound of TritonX-100 and styrene polyoxyethylene ether, which can well promote the uniform dispersion of various components in the reagent system and various substances in the test sample, thereby improving the precision of the test, ensuring the reproducibility of the test results, making the test results stable, and reducing the influence of sample turbidity on the test results, thereby improving the anti-interference ability of the reagent.

[0034] 3. The blocking agent in the reagent R2 of the present invention is compounded with triethanolamine, glycine and casein, which can bind to the free -COOH sites in the latex particles to avoid the adverse effects of the free carboxyl sites on the detection results, and effectively improve the sensitivity of the detection and the stability of the kit.

[0035] 4. The present invention adds blocking agent mouse IgG to reagent R1, which can effectively reduce nonspecific adsorption during the antigen-antibody reaction process, avoid interference of nonspecific reaction on the test results, significantly improve the sensitivity of sample detection, and make the kit have a lower detection limit. The present invention also adds chelating agent EGTA to reagent R1, which effectively increases the upper limit of the detection range of the kit. Compared with the existing method, the linear range of the kit of the present invention is wider, and the detection range of the kit of the present invention is 3.0-180.0ng / mL.

[0036] 5. The surface functional groups of the polystyrene latex microspheres in the reagent R2 of the present invention are carboxyl, amino or hydroxyl groups, and the surface functional groups can combine with the amino groups on the surface of the antibody to form a covalent coupling structure, so that the creatine kinase isoenzyme antibody is firmly bound to the surface of the latex microspheres, ensuring that the reagent R2 has good stability and effectively extending the shelf life of the reagent.

[0037] 6. The particle size of the polystyrene latex particles used in the present invention is 250-400 nm. The larger particle size can increase the turbidity of the reaction between creatine kinase isoenzymes and creatine kinase isoenzyme antibodies in the blood, thereby increasing the sensitivity of the reaction during the detection process and effectively shortening the reaction time. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The creatine kinase isoenzyme detection kit prepared in Example 1 was compared with the Beijing Jiuqiang kit.

[0039] Figure 2 The creatine kinase isoenzyme detection kit prepared in comparative example 1 was compared with the Beijing Jiuqiang kit.

[0040] Figure 3 The stability trend diagram of the reagents of Example 1 and Comparative Example 2 stored at 2-8°C for 14 months. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below by way of examples. These examples are only used to illustrate the present invention and do not limit the protection scope of the present invention.

[0042] Example 1

[0043] The creatine kinase isoenzyme detection kit provided in this embodiment includes reagent R1 and reagent R2, and the compositions of the reagent R1 and reagent R2 are as follows:

[0044] Reagent R1:

[0045]

[0046]

[0047] In the table: the mass ratio of TritonX-100 and styrene polyoxyethylene ether is 3.2:1.

[0048] Preparation of reagent R1: According to the components and contents of reagent R1 in the above table, take the components of reagent R1, add them into purified water, mix them evenly, adjust the pH value to 7.0, filter through a filter membrane, and make up the volume to obtain reagent R1.

[0049] Reagent R2:

[0050]

[0051] In the table: the mass ratio of triethanolamine, glycine and casein is 0.5:1:2.5.

[0052] Preparation of reagent R2:

[0053] ① Activation of microspheres: Add polystyrene latex microspheres with a diameter of 300 nm and surface modified with carboxyl groups to a MOPS buffer with a pH of 7.2, and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, react at 25°C for 30 minutes, remove excess activator in the supernatant after centrifugation, take the precipitate, and resuspend it in MOPS buffer to obtain activated polystyrene latex microspheres;

[0054] ② Antibody cross-linking: Add creatine kinase isoenzyme antibodies to activated polystyrene latex microspheres and react at 25°C for 1.5 hours to cross-link the antibodies on the surface of the latex microspheres;

[0055] ③ Microsphere blocking: triethanolamine, glycine and casein were added to the latex microsphere reaction solution after cross-linking with antibodies, and the blocking was carried out for 1.5 hours to block the area on the surface of the latex microsphere that was not adhered by the antibodies;

[0056] ④ Impurity removal: The mixture of the blocked latex microspheres is centrifuged to remove excess cross-linked byproducts, creatine kinase isoenzyme antibodies, triethanolamine, glycine and casein. The bottom precipitate is the latex particles coated with creatine kinase isoenzyme antibodies;

[0057] ⑤Mixing and preservation: Take phosphate buffer, chitosan and ProClin950, add to purified water, mix evenly, add latex particles coated with creatine kinase isoenzyme antibodies, stir thoroughly, mix evenly, and obtain reagent R2 after constant volume.

[0058] Example 2

[0059] The creatine kinase isoenzyme detection kit provided in this embodiment includes reagent R1 and reagent R2, and the compositions of the reagent R1 and reagent R2 are as follows:

[0060] Reagent R1:

[0061]

[0062] In the table: the mass ratio of TritonX-100 and styrene polyoxyethylene ether is 2.5:1.

[0063] Preparation of reagent R1: According to the components and contents of reagent R1 in the above table, take the components of reagent R1, add them into purified water, mix them evenly, adjust the pH value to 6.5, filter through a filter membrane, and make up the volume to obtain reagent R1.

[0064] Reagent R2:

[0065]

[0066] In the table: the mass ratio of triethanolamine, glycine and casein is 1:1:3.

[0067] Preparation of reagent R2:

[0068] ① Activation of microspheres: Add polystyrene latex microspheres with a diameter of 400 nm and amino groups on the surface to a HEPES buffer at pH 6.0, and add mercapto nicotinamide, react at 30°C for 35 minutes, remove excess activator in the supernatant after centrifugation, take the precipitate, and resuspend it in HEPES buffer to obtain activated polystyrene latex microspheres;

[0069] ② Antibody cross-linking: Add creatine kinase isoenzyme antibodies to activated polystyrene latex microspheres and react at 30°C for 2 hours to cross-link the antibodies on the surface of the latex microspheres;

[0070] ③ Microsphere blocking: Add triethanolamine, glycine and casein to the latex microsphere reaction solution after cross-linking with antibodies, block for 1 hour, and block the area on the surface of the latex microsphere that is not adhered by antibodies;

[0071] ④ Impurity removal: The mixture of the blocked latex microspheres is centrifuged to remove excess cross-linked byproducts, creatine kinase isoenzyme antibodies, triethanolamine, glycine and casein. The bottom precipitate is the latex particles coated with creatine kinase isoenzyme antibodies;

[0072] ⑤Mixing and preservation: Take glycine buffer, chitosan and phenol, add to purified water, mix evenly, add latex particles coated with creatine kinase isoenzyme antibodies, stir thoroughly, mix evenly, and obtain reagent R2 after constant volume.

[0073] Example 3

[0074] The creatine kinase isoenzyme detection kit provided in this embodiment includes reagent R1 and reagent R2, and the compositions of the reagent R1 and reagent R2 are as follows:

[0075] Reagent R1:

[0076]

[0077] In the table: the mass ratio of TritonX-100 and styrene polyoxyethylene ether is 4.5:1.

[0078] Preparation of reagent R1: According to the components and contents of reagent R1 in the above table, take the components of reagent R1, add them into purified water, mix them evenly, adjust the pH value to 8.0, filter through a filter membrane, and make up the volume to obtain reagent R1.

[0079] Reagent R2:

[0080]

[0081]

[0082] In the table: the mass ratio of triethanolamine, glycine and casein is 2:1:4.

[0083] Preparation of reagent R2:

[0084] ① Activation of microspheres: Add polystyrene latex microspheres with a diameter of 250 nm and surface modified with hydroxyl groups to MOPSO buffer at pH 7.5, and add isocyanate, react at 25°C for 40 minutes, remove excess activator in the supernatant after centrifugation, take the precipitate, and resuspend it in MOPSO buffer to obtain activated polystyrene latex microspheres;

[0085] ② Antibody cross-linking: Add creatine kinase isoenzyme antibodies to activated polystyrene latex microspheres and react at 25°C for 2 hours to cross-link the antibodies on the surface of the latex microspheres;

[0086] ③ Microsphere blocking: Add triethanolamine, glycine and casein to the latex microsphere reaction solution after cross-linking with antibodies, block for 1 hour, and block the area on the surface of the latex microsphere that is not adhered by antibodies;

[0087] ④ Impurity removal: The mixture of the blocked latex microspheres is centrifuged to remove excess cross-linked byproducts, creatine kinase isoenzyme antibodies, triethanolamine, glycine and casein. The bottom precipitate is the latex particles coated with creatine kinase isoenzyme antibodies;

[0088] ⑤Mixing and storage: Take Tris buffer, bovine serum albumin and p-hydroxybenzoic acid, add to purified water, mix evenly, add latex particles coated with creatine kinase isoenzyme antibodies, stir thoroughly, mix evenly, and obtain reagent R2 after constant volume.

[0089] Example 4

[0090] The creatine kinase isoenzyme detection kit provided in this embodiment includes reagent R1 and reagent R2, and the compositions of the reagent R1 and reagent R2 are as follows:

[0091] Reagent R1:

[0092]

[0093] In the table: the mass ratio of TritonX-100 and styrene polyoxyethylene ether is 5:1.

[0094] Preparation of reagent R1: According to the components and contents of reagent R1 in the above table, take the components of reagent R1, add them into purified water, mix them evenly, adjust the pH value to 6.5, filter through a filter membrane, and make up the volume to obtain reagent R1.

[0095] Reagent R2:

[0096]

[0097] In the table: the mass ratio of triethanolamine, glycine and casein is 1:1:3.5.

[0098] Preparation of reagent R2:

[0099] ① Microsphere activation: Add polystyrene latex microspheres with a diameter of 400 nm and surface modified with carboxyl groups to MES buffer at pH 6.0, and add carboimide, react at 30°C for 40 minutes, remove excess activator in the supernatant after centrifugation, take the precipitate, and resuspend it in MES buffer to obtain activated polystyrene latex microspheres;

[0100] ② Antibody cross-linking: Add creatine kinase isoenzyme antibodies to activated polystyrene latex microspheres and react at 30°C for 2 hours to cross-link the antibodies on the surface of the latex microspheres;

[0101] ③ Microsphere blocking: Add triethanolamine, glycine and casein to the latex microsphere reaction solution after cross-linking with antibodies, block for 1 hour, and block the area on the surface of the latex microsphere that is not adhered by antibodies;

[0102] ④ Impurity removal: The mixture of the blocked latex microspheres is centrifuged to remove excess cross-linked byproducts, creatine kinase isoenzyme antibodies, triethanolamine, glycine and casein. The bottom precipitate is the latex particles coated with creatine kinase isoenzyme antibodies;

[0103] ⑤Mixing and preservation: Take MES buffer, glycerol and ethyl p-hydroxybenzaldehyde, add to purified water, mix evenly, add latex particles coated with creatine kinase isoenzyme antibodies, stir thoroughly, mix evenly, and obtain reagent R2 after constant volume.

[0104] Example 5

[0105] The creatine kinase isoenzyme detection kit provided in this embodiment includes reagent R1 and reagent R2, and the compositions of the reagent R1 and reagent R2 are as follows:

[0106] Reagent R1:

[0107]

[0108]

[0109] In the table: the mass ratio of TritonX-100 and styrene polyoxyethylene ether is 2:1.

[0110] Preparation of reagent R1: According to the components and contents of reagent R1 in the above table, take the components of reagent R1, add them into purified water, mix them evenly, adjust the pH value to 7.0, filter through a filter membrane, and make up the volume to obtain reagent R1.

[0111] Reagent R2:

[0112]

[0113] In the table: the mass ratio of triethanolamine, glycine and casein is 2:1:1.

[0114] Preparation of reagent R2:

[0115] ① Activation of microspheres: Add polystyrene latex microspheres with a diameter of 300 nm and surface modified with carboxyl groups to MES buffer at pH 8.0, and add maleimide, react at 25°C for 40 minutes, remove excess activator in the supernatant after centrifugation, take the precipitate, and resuspend it in MES buffer to obtain activated polystyrene latex microspheres;

[0116] ② Antibody cross-linking: Add creatine kinase isoenzyme antibodies to activated polystyrene latex microspheres and react at 25°C for 2 hours to cross-link the antibodies on the surface of the latex microspheres;

[0117] ③ Microsphere blocking: triethanolamine, glycine and casein were added to the latex microsphere reaction solution after cross-linking with antibodies, and the blocking was carried out for 2 hours to block the area on the surface of the latex microsphere that was not adhered by the antibodies;

[0118] ④ Impurity removal: The mixture of the blocked latex microspheres is centrifuged to remove excess cross-linked byproducts, creatine kinase isoenzyme antibodies, triethanolamine, glycine and casein. The bottom precipitate is the latex particles coated with creatine kinase isoenzyme antibodies;

[0119] ⑤Mixing and storage: Take HEPES buffer, bovine serum albumin and ProClin300, add them to purified water, mix well, add latex particles coated with creatine kinase isoenzyme antibodies, stir well, mix well, and obtain reagent R2 after constant volume.

[0120] Comparative Example 1

[0121] The difference between Comparative Example 1 and Example 1 is that the surfactant in the reagent R1 of Comparative Example 1 is only Triton X-100, and the rest is the same as that of Example 1.

[0122] Comparative Example 2

[0123] The difference between Comparative Example 2 and Example 1 is that the blocking agent in the reagent R2 of Comparative Example 2 is only casein, and the rest is the same as that of Example 1.

[0124] Comparative Example 3

[0125] The difference between Comparative Example 3 and Example 1 is that the reagent R1 of Comparative Example 3 does not contain blocking agent mouse IgG, and the rest is the same as that of Example 1.

[0126] Performance Evaluation

[0127] Test Example 1 Linear range test of creatine kinase isoenzyme detection kit

[0128] 1. Test samples

[0129] (1) Test sample: the creatine kinase isozyme detection kit prepared in Example 1-5;

[0130] (2) Control sample: the creatine kinase isozyme detection kit prepared in Comparative Examples 1-3;

[0131] 2. Test methods

[0132] The high-concentration sample and the low-concentration sample are diluted in multiples. The preparation scheme is as follows. The concentration and volume units of each sample are unified. When aspirating liquid, a pipette with good precision and accuracy should be selected; when preparing, the sample should be completely mixed and evaporation or other situations that may cause the sample to deteriorate should be avoided.

[0133] Sample No. 1 2 3 4 5 6 Low concentration serum 3.2ng / ml 1.00 0.80 0.60 0.40 0.20 0.00 High concentration serum 180ng / ml 0.00 0.20 0.40 0.60 0.80 1.00

[0134] The creatine kinase isoenzyme detection kits prepared in Examples 1-5 and Comparative Examples 1-3 were used to detect the creatine kinase isoenzyme, and the standard working curves of the detection kits were drawn.

[0135] The detection method is a two-point endpoint method, including the following steps: add 150 μL of reagent R1 to 10 μL of the sample to be detected (calibration tube uses calibration material as sample, blank uses distilled water as sample), mix thoroughly, keep constant temperature at 37°C for 5 minutes, then add 50 μL of reagent R2, mix, keep constant temperature at 37°C for 40 seconds, measure the absorbance Al of each tube at a wavelength of 660nm, read the absorbance A2 after 260 seconds, and the reaction absorbance OA=A2-A1. According to the concentration of multi-point calibration material and the corresponding absorbance change value OA, a multi-point nonlinear calibration mode is used to determine the working curve, and the concentration value corresponding to the sample absorbance change on the working curve is the measured concentration.

[0136] 3. Test results

[0137] The analysis results of the creatine kinase isozyme detection kits of Examples 1-5 and Comparative Examples 1-3 are as follows: (Technical indicators: within [3, 180] ng / mL, the linear correlation coefficient r ≥ 0.990. Within the range of [3, 20] ng / mL, the absolute linear deviation should not exceed ± 3 ng / mL, within the range of (20, 180] ng / mL, the relative linear deviation should not exceed ± 15%)

[0138]

[0139]

[0140]

[0141] Result analysis: Through the experimental data in the above table, it can be seen that the linear range indicators of Examples 1-5 all meet the technical indicator requirements, while Comparative Examples 1 and Comparative Examples 2 do not meet the requirements at low concentrations, indicating that the surfactant in reagent R1 and the blocking agent in reagent R2 have an impact on the dispersibility and background value of the reagent latex, and the linear range data of the comparative example 3 test is very messy, indicating that there is no blocking agent mouse IgG in reagent R1, which will cause strong interference.

[0142] Test Example 2 Minimum Detection Limit Test of Creatine Kinase Isoenzyme Detection Kit

[0143] 1. Test samples

[0144] (1) Test sample: the creatine kinase isozyme detection kit prepared in Example 1;

[0145] (2) Control sample: the creatine kinase isozyme detection kit prepared in Comparative Example 1;

[0146] 2. Test methods

[0147] Distilled water was used as a blank sample, and the creatine kinase isoenzyme detection kits prepared in Example 1 and Comparative Example 1 were used to detect it for 10 consecutive times. The mean and standard deviation of the results were calculated for 10 times, and the blank mean plus two times the standard deviation was taken as the minimum detection limit (+2SD).

[0148] 3. Test results

[0149] (Technical indicators: Use blank samples to test the kit, the blank limit of the reagent should be ≤2ng / mL)

[0150] Test results of the creatine kinase isozyme detection kit of Example 1

[0151]

[0152] Test results of the creatine kinase isoenzyme detection kit of comparative example 1

[0153]

[0154] Result analysis: The minimum detection limit results of Example 1 and Comparative Example 1 show that the minimum detection limit of Example 1 reaches 0.43, while the background value of the reagent in Comparative Example 1 is very high, resulting in unqualified minimum detection limit.

[0155] Test Example 3 Accuracy Test of Creatine Kinase Isoenzyme Detection Kit

[0156] 1. Test samples

[0157] (1) Test sample: the creatine kinase isozyme detection kit prepared in Example 1;

[0158] (2) Control sample: the creatine kinase isozyme detection kit prepared in Comparative Example 1;

[0159] 2. Test methods

[0160] The creatine kinase isoenzyme detection kits prepared in Example 1 and Comparative Example 1 and the Beijing Jiuqiang Biological Creatine Kinase Isoenzyme Detection Kit, which has been on the market, were used to perform clinical tests on 40 creatine kinase isoenzyme samples with different contents, and correlation analysis was performed on the measured values.

[0161] 3. Test results

[0162] The determination results of the creatine kinase isozyme detection kit of Example 1 and Comparative Example 1 are as follows: Figure 1 and Figure 2 shown.

[0163] Analysis of results: It can be seen from the results that in the serum clinical comparison with Beijing Jiuqiang's marketed products, Example 1 has a better correlation than Comparative Example 1 (Example 1 correlation r=0.99, Comparative Example 1 correlation r=0.92). At the same time, it can be seen that there are a lot of interferences in Comparative Example 1 when measuring serum, and the deviation is large at low concentration levels.

[0164] Test Example 4 Stability Test of Creatine Kinase Isoenzyme Detection Kit

[0165] 1. Test samples

[0166] (1) Test sample: the creatine kinase isozyme detection kit prepared in Example 1;

[0167] (2) Control sample: the creatine kinase isoenzyme detection kit prepared in Comparative Example 2;

[0168] 2. Test methods

[0169] Under storage conditions of 2-8°C, the creatine kinase isoenzyme detection kits prepared in Example 1 and Comparative Example 2 were used to detect creatine kinase isoenzyme samples with a high concentration of 120 ng / mL and a low concentration of 50 ng / mL after 0 month, 3 months, 6 months, 9 months, 12 months and 14 months, respectively. Each sample was tested 10 times and the average value was taken.

[0170] 3. Test results

[0171]

[0172]

[0173]

[0174] Analysis of results: From the variance analysis of the stability data of Example 1, it can be obtained that the reagent of Example 1 was stored at 2-8°C for 14 months, the high concentration p=0.868, the low concentration p=0.252, and the high and low concentration p>0.05, so the trend is not significant, that is, the reagent of Example 1 was stored at 2-8°C for 14 months, and the change trend of its test results was not significant, and it was in a stable state.

[0175]

[0176]

[0177]

[0178] Result analysis: From the variance analysis of the stability data of Comparative Example 2, it can be obtained that the reagent test value of Comparative Example 2 was significantly decreased after being stored at 2-8℃ for 14 months, and the p of high and low concentrations was less than 0.05, that is, the reagent of Comparative Example 2 was stored at 2-8℃ for 14 months, and its test result had a significant trend of change, and was in an extremely unstable state.

[0179] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A creatine kinase isoenzyme detection kit, characterized in that: The kit comprises reagent R1 and reagent R2; The components and contents of the reagent R1 are: buffer 10-50mM, coagulant 2-5g / L, chelating agent 1-2g / L, blocking agent 0.5-2g / L, surfactant 1-5g / L, stabilizer 0.5-1g / L, preservative 1-2g / L; the surfactant is prepared by compounding TritonX-100 and styrene polyoxyethylene ether at a mass ratio of 2-5:1; the blocking agent is mouse IgG; the chelating agent is EGTA; The components and contents of the reagent R2 are: buffer solution 10-50 mM, latex particles coated with creatine kinase isoenzyme antibodies 0.05-0.4% w / v, blocking agent 1-10 g / L, stabilizer 5-20 g / L, preservative 1-2 g / L; the blocking agent is compounded with triethanolamine, glycine and casein in a mass ratio of 0.5-2:1:1-4.

2. The creatine kinase isozyme detection kit according to claim 1, characterized in that: The coagulant in the reagent R1 is any one of PEG-4000, PEG-6000, PEG-8000 and dextran.

3. The creatine kinase isozyme detection kit according to claim 1, characterized in that: The buffers in the reagents R1 and R2 are each independently selected from one of phosphate buffer, glycine buffer, Tris buffer, HEPES buffer and MES buffer.

4. The creatine kinase isozyme detection kit according to claim 1, characterized in that: The stabilizers in the reagents R1 and R2 are each independently selected from one of bovine serum albumin, trehalose, chitosan and glycerol.

5. The creatine kinase isozyme detection kit according to claim 1, characterized in that: The preservatives in the reagents R1 and R2 are each independently selected from one of ProClin300, ProClin950, phenol, p-hydroxybenzoic acid, and ethyl p-hydroxybenzaldehyde.

6. The creatine kinase isozyme detection kit according to claim 1, characterized in that: The creatine kinase isozyme antibody in the reagent R2 is a mouse monoclonal antibody; the latex particles are polystyrene latex microspheres with a diameter of 250-400nm, and the surface of the microspheres is modified with functional groups, and the modified functional groups are selected from carboxyl, amino or hydroxyl groups.

7. The creatine kinase isozyme detection kit according to claim 1, characterized in that: The creatine kinase isozyme antibody in the reagent R2 is coated onto the surface of the latex microspheres by chemical crosslinking, the chemical crosslinking agent used is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, maleimide, carboimide, mercaptonicotinamide, and isocyanate, the crosslinking buffer is selected from HEPES buffer, MOPS buffer, MES buffer, and MOPSO buffer, and the pH is between 6.0 and 8.0.

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