Creatine kinase isoenzyme CK-MB detection kit

Abstract

The invention discloses a creatine kinase isoenzyme CK-MB detection kit, and belongs to the technical field of biochemical detection. The creatine kinase isoenzyme CK-MB detection kit disclosed by the invention comprises a reagent R1 and a reagent R2, the reagent R1 is prepared from the following components: 30 to 35 mmol / L of first buffer solution, 12 to 25 mL / L of compound latex sensitizer, 5.5 to 12 mL / L of compound surfactant and 0.65 g / L of preservative; the reagent R2 is prepared from the following components: 120 to 130 mmol / L of a second buffer solution, 12 to 32 mL / L of a microsphere solution coated with a CK-MB antibody, 12 to 25 mL / L of a compound latex stabilizer, 5.5 to 12 mL / L of a compound surfactant and 0.65 g / L of a preservative. The detection kit provided by the invention has the advantages of high sensitivity, good stability, wide linear range and the like, and can effectively detect the content of creatine kinase isoenzyme CK-MB.

Description

Technical Field

[0001] This invention relates to the field of biochemical detection technology, specifically to a creatine kinase isoenzyme CK-MB detection kit. Background Technology

[0002] Creatine kinase is a dimer composed of a brain-type subunit (B) and a muscle-type subunit (M). Normal human tissues typically contain three isoenzymes, listed in order of electrophoretic speed: CK-BB (CK1), CK-MB (CK2), and CK-MM (CK3). CK-MB is mainly found in myocardial tissue, accounting for 0-4% of serum CK. CK and CK-MB are sensitive indicators of myocardial tissue damage, with CK-MB being particularly specific. Elevated CK levels during myocardial injury are primarily due to elevated CK-MB, resulting in a simultaneous increase in both. When myocardial tissue damage is severe, CK-MB is released into the bloodstream, making serum CK-MB an important criterion for diagnosing acute myocardial infarction. Elevated serum CK-MB is commonly seen in acute myocardial infarction, skeletal muscle injury, trauma, and strenuous exercise. Elevated serum CK-MB can also be observed in patients with malignant fever and some of their family members. Patients with rhabdomyosarcoma may have significantly elevated serum CK-MB. In addition, cardiac surgery that damages the myocardium may cause a transient increase in CK-MB, which usually returns to normal within 24 hours after surgery.

[0003] Currently, common methods for detecting creatine kinase isoenzyme (CK-MB) include immunosuppression (activity assay) and electrophoresis. Immunosuppression has poor specificity and is easily affected by high bilirubin and hemoglobin; while electrophoresis has disadvantages such as complex operation, low sensitivity, and unsuitability for automated analysis.

[0004] Latex-enhanced immunoturbidimetry is a dynamic assay for antigen-antibody binding. In a specific dilution system, antigen and antibody bind; a change in turbidity occurs before and after binding. This turbidity change is detected by a fully automated biochemical analyzer, and a linear curve is plotted using standards to determine the concentration of the analyte in the sample. While there are reports on the detection of creatine kinase isoenzyme CK-MB based on latex-enhanced immunoturbidimetry, existing kits generally suffer from narrow linear ranges, low sensitivity, and poor stability. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a creatine kinase isoenzyme CK-MB detection kit. The detection kit of this invention has advantages such as high sensitivity, good stability, and a wide linear range, and can effectively detect the content of creatine kinase isoenzyme CK-MB.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A creatine kinase isoenzyme CK-MB detection kit, comprising reagent R1 and reagent R2;

[0008] The reagent R1 consists of: 30-35 mmol / L of the first buffer solution, 12-25 mL / L of the compound latex sensitizer, 5.5-12 mL / L of the compound surfactant, and 0.65 g / L of the preservative.

[0009] The reagent R2 consists of: 120-130 mmol / L second buffer, 12-32 mL / L microsphere solution coated with CK-MB antibody, 12-25 mL / L compound latex stabilizer, 5.5-12 mL / L compound surfactant, and 0.65 g / L preservative.

[0010] Furthermore, the first buffer is a POPSO-Tricine buffer.

[0011] In a preferred embodiment of the present invention, the POPSO-Tricine buffer is prepared by the following method:

[0012] Take 11.60g piperazine-N,N-bis(2-hydroxypropanesulfonic acid) (POPSO), dissolve it in 800mL of deionized water, then add 5.73g of N-tris(hydroxymethyl)methylglycine (Tricine), stir to dissolve, adjust the pH to 8.06 with an appropriate amount of sodium hydroxide or hydrochloric acid, and finally make up the volume to 1L with deionized water.

[0013] Furthermore, the compound latex sensitizer is composed of choline chloride, polyethylene glycol 8000, polyethylene glycol 6000, lithium chloride and sodium chloride in a weight ratio of (8-12):(10-14):(8-12):(4-6):(10-14).

[0014] In a preferred embodiment of the present invention, the compound latex sensitizer is prepared by the following method:

[0015] Add 10g of choline chloride to 200mL of water and stir at 25℃ for 0.5h to dissolve. Then add 12g of polyethylene glycol 8000 (PEG8000), 10g of polyethylene glycol 6000, 5g of lithium chloride, and 12g of sodium chloride. Stir at 30℃ for 1h until completely dissolved and set aside.

[0016] Furthermore, the compound surfactant is composed of a mixture of trimethylolpropane trioleate, alcohol ether hexyl sulfosuccinic acid mixed diester salt and phosphate betaine amphoteric surfactant.

[0017] In a preferred embodiment of the present invention, the compound surfactant is prepared by the following method:

[0018] Add 5g of trimethylolpropane trioleate to 300mL of deionized water and stir at 35℃ for 1.5h. Then add 2.2g of mixed diester salt of alcohol ether hexyl sulfosuccinic acid and 3.6g of phosphate betaine amphoteric surfactant, stir at 30℃ for 3h, adjust the pH to 7.50 with HCl or NaOH, and cool to 25℃ before use.

[0019] Furthermore, the second buffer is a CAPS-EPPS buffer.

[0020] In a preferred embodiment of the present invention, the CAPS-EPPS buffer solution is prepared by the following method:

[0021] Weigh 27.89g of CAPS (3-cyclohexylaminopropanesulfonic acid), dissolve it completely in 800mL of deionized water, then add 31.79g of EPPS (N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid), stir to dissolve, adjust the pH to 7.13 with hydrochloric acid, and finally bring the volume to 1L with deionized water.

[0022] Furthermore, the microsphere solution coated with CK-MB antibody is prepared by conjugating carboxyl latex microspheres with three different particle sizes of 80-100nm, 150-170nm, and 280-300nm with mouse anti-human CK-MB monoclonal antibody.

[0023] In a preferred embodiment of the present invention, the microsphere solution coated with CK-MB antibody is prepared by the following method:

[0024] Take 4 mL of 85 nm carboxylate microspheres, 3 mL of 166 nm carboxylate microspheres, and 0.3 mL of 285 nm carboxylate microspheres. Add 18 mL of 126 mmol / L CAPS-EPPS buffer (pH 7.13, 25℃), stir to mix, and vortex at 30℃ for 20 min (200 rpm). Then add 0.8-1.6 mg of EDC, and vortex at 35℃ for 50 min (120 rpm). Next, add 32-68 mg of mouse anti-human CK-MB monoclonal antibody, adjust the vortex speed to 160 rpm, and vortex at 30℃ for 1 hour. Then, add 2.5 mL of masking agent (composed of 15 g / L BSA and 10 g / L ethylenediamine), adjust the vortex speed to 200 rpm, and vortex at 30℃ for 3 hours. The final solution is the microsphere solution coated with CK-MB antibody.

[0025] Furthermore, the compound latex stabilizer is composed of sedum heptanose, polyethylene glycol 20000, sorbitol, arbutin, potassium chloride and lithium chloride in a weight ratio of (4-8):(8-12):(50-70):(6-10):(8-12):(4-6).

[0026] In a preferred embodiment of the present invention, the compound latex stabilizer is prepared by the following method:

[0027] Add 6g of sedaridin heptanose to 150mL of water and stir at 25℃ for 0.5h to dissolve. Then add 10g of polyethylene glycol 20000 (PEG20000), 20g of sorbose, 8g of azosulfose, 10g of potassium chloride, and 5g of lithium chloride. Stir at 30℃ for 1h. Then add an appropriate amount of purified water to make up to 200mL and stir at 30℃ for 0.5h until completely dissolved.

[0028] Furthermore, the preservative is selected from Nuosha PC-150 antibacterial agent.

[0029] The detection principle of the creatine kinase isoenzyme CK-MB detection kit of the present invention is as follows: mouse anti-human CK-MB monoclonal antibody is coated on latex particles, and the specific antigen in diluted human serum is bound to the antibody by incubation. After incubation, the change in absorbance is proportional to the concentration of the specific antigen in the detection sample.

[0030] The method of using the creatine kinase isoenzyme CK-MB detection kit of the present invention is as follows: the determination is performed using an automated biochemical analyzer with an endpoint method, the main detection wavelength is 700nm, and the ratio of reagent R1 to reagent R2 is 4:1.

[0031] The beneficial effects of this invention are:

[0032] (1) This invention uses carboxyl latex microspheres of different particle sizes in combination. By optimizing the coupling process, scientifically proportioning and adjusting the coupling buffer system, the reaction sensitivity and linear range of the reagent are greatly enhanced, and the repeatability and anti-interference ability of the reagent are stronger.

[0033] (2) The compound surfactant solution of the present invention can promote and maintain antibody stability, prevent system turbidity, and significantly enhance the stability and anti-interference ability of the reagent.

[0034] (3) The compound latex sensitizer of the present invention can effectively enhance the analytical sensitivity of the reagent.

[0035] (4) The compound latex stabilizer of the present invention can promote and maintain antibody stability through scientific formulation, and significantly enhance the stability of the reagent.

[0036] (5) This reagent is easy and quick to use, suitable for automated analysis, and is a more stable, sensitive, and interference-resistant creatine kinase isoenzyme (CK-MB) reagent. The reagent has good accuracy and stability, strong anti-interference ability, and a wide linear range. It is convenient to use and can fully meet clinical needs. Attached Figure Description

[0037] Figure 1 The stability test results of the creatine kinase isoenzyme CK-MB detection kit of the present invention. Detailed Implementation

[0038] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0039] As mentioned earlier, latex-enhanced immunoturbidimetry involves selecting latex microspheres of appropriate particle size and material, and using physical or chemical forces to stably bind antibodies to the microspheres. After the antibody reacts with a specific analyte, it will rapidly aggregate, leading to a change in the turbidity of the reaction system. This change is proportional to the degree of particle aggregation. By comparing the result with a calibrator that has undergone the same treatment at a specific wavelength, the content of the analyte in the specific analyte can be obtained.

[0040] The stability of latex-enhanced immunoturbidimetric reagents is extremely important for detection accuracy. This invention significantly improves the sensitivity, accuracy, and stability of creatine kinase isoenzyme CK-MB detection by optimizing the latex-enhanced immunoturbidimetric detection system, and also broadens the linear range of detection. Specifically:

[0041] The creatine kinase isoenzyme CK-MB detection kit of the present invention includes reagent R1 and reagent R2, wherein:

[0042] Reagent R1 provides a suitable environment for antigen-antibody reactions while reducing non-specific reactions and interference. Through extensive experimental screening, this invention selected POPSO-Tricine buffer as the buffer component in reagent R1, which effectively maintains the stability of the reaction system. This invention also adds a "compound latex sensitizer" to reagent R1, which can prevent interference from impurity ions, promote effective binding of antigen and antibody, and improve the accuracy and sensitivity of the detection.

[0043] Reagent R2 provides a specific antibody that reacts with the antigen in the sample to form an immune complex. This invention selects three different sizes of carboxylated latex microspheres and conjugates them with mouse anti-human CK-MB monoclonal antibody, comprehensively balancing the antibody binding efficiency, reaction rate, and extent. Furthermore, this invention adds a "compound latex stabilizer" to reagent R2, which effectively protects and stabilizes the cross-linked antibody on the surface of the carboxylated latex microspheres, allowing the latex particles to remain suspended in solution for a long time without easily settling.

[0044] In this invention, reagents R1 and R2 are both selected as "compound surfactants" obtained by compounding trimethylolpropane trioleate, alcohol ether hexyl sulfosuccinic acid mixed diester salt and phosphate betaine amphoteric surfactants, which can reduce the surface tension of the reaction system and increase the reaction kinetics.

[0045] When conducting the test, reagents R1 and R2 are mixed at a volume ratio of 4:1. The "compound latex sensitizer" and "compound latex stabilizer" can synergistically improve the sensitivity and accuracy of the test and improve the stability of the test, achieving a synergistic effect of 1+1>2.

[0046] In summary, the creatine kinase isoenzyme CK-MB detection kit of the present invention significantly improves the detection effect of creatine kinase isoenzyme CK-MB through the optimization of components, thus proposing the present invention.

[0047] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0048] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions.

[0049] Example 1: Preparation and detection of creatine kinase isoenzyme CK-MB assay kit:

[0050] 1. Composition of the creatine kinase isoenzyme CK-MB detection kit:

[0051] The creatine kinase isoenzyme CK-MB detection kit of this embodiment includes reagent R1 and reagent R2; wherein:

[0052] The composition of reagent R1 is as follows:

[0053]

[0054] The composition of reagent R2 is as follows:

[0055]

[0056] The POPSO-Tricine buffer (pH 8.06, 25°C) in reagent R1 was prepared by the following method:

[0057] Weigh 11.60 g of POPSO (piperazine-N,N-bis(2-hydroxypropanesulfonic acid)), dissolve it completely in 800 mL of deionized water, then add 5.73 g of Tricine (N-tris(hydroxymethyl)methylglycine), stir to dissolve, adjust the pH to 8.06 with an appropriate amount of sodium hydroxide or hydrochloric acid, and finally make up the volume to 1 L with deionized water.

[0058] The compound latex sensitizer is prepared by the following method:

[0059] Add 10g of choline chloride to 200mL of water and stir at 25℃ for 0.5h to dissolve. Then add 12g of polyethylene glycol 8000 (PEG8000), 10g of polyethylene glycol 6000, 5g of lithium chloride, and 12g of sodium chloride. Stir at 30℃ for 1h until completely dissolved and set aside.

[0060] The compound surfactant used in reagents R1 and R2 is the same and is prepared by the following method:

[0061] Add 5g of trimethylolpropane trioleate to 300mL of deionized water and stir at 35℃ for 1.5h. Then add 2.2g of alcohol ether hexyl sulfosuccinic acid mixed diester salt and 3.6g of phosphate betaine amphoteric surfactant, stir at 30℃ for 3h, adjust the pH to 7.50 with HCl or NaOH, and cool to 25℃ to prepare the compound surfactant.

[0062] CAPS-EPPS buffer solution is prepared by the following method:

[0063] Weigh 27.89g of CAPS (3-cyclohexylaminopropanesulfonic acid), dissolve it completely in 800mL of deionized water, then add 31.79g of EPPS (N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid), stir to dissolve, adjust the pH to 7.13 with hydrochloric acid, and finally bring the volume to 1L with deionized water.

[0064] The microsphere solution coated with CK-MB antibody was prepared by the following method:

[0065] Take 4 mL of commercially available carboxylated latex microspheres with an average particle size of 85 nm, 3 mL of carboxylated latex microspheres with an average particle size of 166 nm, and 0.3 mL of carboxylated latex microspheres with an average particle size of 285 nm. Add 18 mL of 126 mmol / L CAPS-EPPS buffer (pH 7.13, 25℃), stir to mix, and vortex at 30℃ for 20 min (200 rpm). Then add 0.8-1.6 mg EDC and vortex at 35℃ for 50 min (120 rpm). Next, add 60 mg of commercially available mouse anti-human CK-MB monoclonal antibody, adjust the vortex speed to 160 rpm, maintain the temperature at 30℃, and vortex for 1 hour. Finally, add 2.5 mL of masking agent (component: 15 g / L). Add BSA and 10 g / L ethylenediamine, adjust the oscillation rate to 200 r / min, the temperature to 30℃, and oscillate for 3 hours; the final solution is the microsphere solution coated with CK-MB antibody.

[0066] The compound latex stabilizer is prepared by the following method:

[0067] Add 6g of sedaridin heptanose to 150mL of water and stir at 25℃ for 0.5h to dissolve. Then add 10g of polyethylene glycol 20000 (PEG20000), 20g of sorbose, 8g of azosulfose, 10g of potassium chloride, and 5g of lithium chloride. Stir at 30℃ for 1h. Then add an appropriate amount of purified water to make up to 200mL and stir at 30℃ for 0.5h until completely dissolved.

[0068] The preservative used in both reagents R1 and R2 is Nuosha PC-150 antibacterial agent.

[0069] 2. Testing and Usage Method:

[0070] The creatine kinase isoenzyme (CK-MB) assay kit described in this embodiment is used with a fully automated biochemical analyzer featuring dual reagent functionality, such as the Hitachi 7180 fully automated analyzer, and the endpoint method is employed for determination. R1 and R2 are placed in their corresponding reagent positions at a 4:1 ratio. Distilled water, standards, and samples are placed in their corresponding positions on the sample tray, as shown in Table 1.

[0071] Table 1: Reagent and Detection Methods

[0072]

[0073]

[0074] Calculation: Creatine kinase isoenzyme (CK-MB) content (ng / mL) = (ΔA determination ÷ ΔA standard) × C standard.

[0075] Example 2: Preparation and detection of creatine kinase isoenzyme CK-MB assay kit:

[0076] 1. Composition of the creatine kinase isoenzyme CK-MB detection kit:

[0077] The creatine kinase isoenzyme CK-MB detection kit of this embodiment includes reagent R1 and reagent R2; wherein:

[0078] The composition of reagent R1 is as follows:

[0079]

[0080] The composition of reagent R2 is as follows:

[0081]

[0082] The preparation methods for POPSO-Tricine buffer, compound latex sensitizer, compound surfactant, CAPS-EPPS buffer, microsphere solution coated with CK-MB antibody, and compound latex stabilizer are the same as in Example 1; the preservatives used in reagents R1 and R2 are both Nossa PC-150 antibacterial agent.

[0083] 2. Testing and Usage Method:

[0084] The detection method of the kit is the same as in Example 1.

[0085] Example 3: Preparation and detection of creatine kinase isoenzyme CK-MB assay kit:

[0086] 1. Composition of the creatine kinase isoenzyme CK-MB detection kit:

[0087] The creatine kinase isoenzyme CK-MB detection kit of this embodiment includes reagent R1 and reagent R2; wherein:

[0088] The composition of reagent R1 is as follows:

[0089]

[0090] The composition of reagent R2 is as follows:

[0091]

[0092] The preparation methods for POPSO-Tricine buffer, compound latex sensitizer, compound surfactant, CAPS-EPPS buffer, microsphere solution coated with CK-MB antibody, and compound latex stabilizer are the same as in Example 1. The preservative used in reagents R1 and R2 is NutraSat PC-150 antibacterial agent.

[0093] 2. Testing and Usage Method:

[0094] The detection method of the kit is the same as in Example 1.

[0095] Comparative Example 1:

[0096] The creatine kinase isoenzyme CK-MB assay kit includes reagent R1 and reagent R2; wherein:

[0097] The composition of reagent R1 is as follows:

[0098] POPSO-Tricine buffer (pH 8.06, 25℃) 32 mmol / L

[0099] Compound surfactant 5.5 mL / L,

[0100] Preservative 0.65g / L;

[0101] The composition of reagent R2 is as follows:

[0102]

[0103] The difference between this comparative example and Example 1 is that "compound latex sensitizer" is omitted in reagent R1.

[0104] Comparative Example 2:

[0105] The creatine kinase isoenzyme CK-MB assay kit includes reagent R1 and reagent R2; wherein:

[0106] The composition of reagent R1 is as follows:

[0107]

[0108]

[0109] The composition of reagent R2 is as follows:

[0110]

[0111] The difference between this comparative example and Example 1 is that "compound latex stabilizer" is omitted in reagent R2.

[0112] Comparative Example 3:

[0113] The creatine kinase isoenzyme CK-MB assay kit includes reagent R1 and reagent R2; wherein:

[0114] The composition of reagent R1 is as follows:

[0115] POPSO-Tricine buffer (pH 8.06, 25℃) 32 mmol / L

[0116] Compound surfactant 5.5 mL / L,

[0117] Preservative 0.65g / L;

[0118] The composition of reagent R2 is as follows:

[0119]

[0120] The difference between this comparative example and Example 1 is that "compound latex sensitizer" is omitted in reagent R1 and "compound latex stabilizer" is omitted in reagent R2.

[0121] Experiment 1: Accuracy Comparison Experiment

[0122] One copy each of the traceable high-value quality control (target value 20.04 ng / mL) and low-value quality control (target value 4.25 ng / mL) of creatine kinase isoenzyme (CK-MB) were prepared using the formulations of Examples 1-3 and Comparative Examples 1-3, respectively, to obtain creatine kinase isoenzyme (CK-MB) detection kits. Control tests were performed under the same detection conditions, with each test repeated 6 times. The average value was calculated and compared with the target value of the quality control. The results are shown in Tables 2 and 3.

[0123] Table 2: Accuracy Test Data of High-Value Quality Control Materials

[0124] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Maximum value 20.05 20.15 20.09 22.35 21.36 23.13 Minimum value 19.95 19.98 19.94 20.85 20.34 19.81 average value 20.04 20.06 20.01 21.64 21.12 22.55

[0125] Table 3: Accuracy Test Data for Low-Value Control Materials

[0126] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Maximum value 4.26 4.27 4.25 3.95 4.03 3.84 Minimum value 4.18 4.11 4.15 3.53 3.70 3.49 average value 4.24 4.21 4.23 3.68 3.90 3.59

[0127] As can be seen from the test results in Tables 2 and 3, compared with Comparative Examples 1-3, the test values ​​of the reagents prepared in Examples 1, 2 and 3 are closer to the target values, and the difference between the average value and the target value is within 0.03.

[0128] This invention uses the "compliance" between the average detected value and the target value to evaluate the detection effect. The "compliance" is calculated as follows:

[0129]

[0130] A higher degree of conformity indicates better accuracy in the detection; a conformity of 100% means that the average value of the detection is completely consistent with the target value.

[0131] The results showed that for high-value quality control materials, the compliance rate of the test results of the kit in Example 1 was 100%, the compliance rate of Comparative Example 1 was 92.0%, the compliance rate of Comparative Example 2 was 94.6%, and the compliance rate of Comparative Example 3 was 87.5%.

[0132] For low-value quality control materials, the compliance rate of the test results of the kit in Example 1 was 99.8%, the compliance rate of Comparative Example 1 was 86.6%, the compliance rate of Comparative Example 2 was 91.8%, and the compliance rate of Comparative Example 3 was 84.5%.

[0133] Therefore, the detection kit of Example 1 of the present invention has higher accuracy, which shows that by adding compound latex sensitizer and compound latex stabilizer, the present invention can promote and maintain antibody stability, prevent system turbidity, and significantly enhance the anti-interference ability of the reagent; through scientific formulation, the reaction system is optimized, which greatly improves the accuracy of detection.

[0134] Experiment 2: Comparison of Sensitivity

[0135] Seven concentrations of creatine kinase isoenzyme (CK-MB) samples were obtained by diluting traceable calibrators from low to high concentrations. Reagents were prepared using the formulations of Examples 1-3 and Comparative Examples 1-3, respectively, to obtain creatine kinase isoenzyme (CK-MB) detection reagents for control testing. The detection results were compared with the theoretical concentrations. The results are shown in Table 4.

[0136] Table 4: Sensitivity Comparison Test Data Table

[0137] Theoretical concentration Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 0.05 0.05 0.04 0.06 - - - 1 1.01 0.99 1.02 0.87 0.92 0.82 10 10.01 10.11 9.98 9.32 9.44 9.03 50 50.02 50.06 50.11 51.56 50.67 52.21 100 100.06 100.13 100.17 100.42 99.67 101.38 200 200.03 200.13 200.23 192.29 195.08 190.78 450 450.15 450.78 449.98 376.38 385.48 370.51

[0138] Note: "-" in the table indicates that it was not detected.

[0139] As shown in Table 4, comparative examples 1-3 failed to detect samples with concentrations as low as 0.05 ng / mL; however, the reagents formulated in Examples 1, 2, and 3 could still accurately detect the samples. Furthermore, compared to comparative examples 1-3, the reagents formulated in Examples 1, 2, and 3 showed higher accuracy in detecting low-value samples (0.08-10 ng / mL) close to the lower limit of linearity. Additionally, when the sample concentration was at the upper limit of linearity (450 ng / mL), the comparative examples showed detection values ​​between 370.51 and 385.48 ng / mL, which were significantly lower than expected. However, the reagents formulated in Examples 1, 2, and 3 could still accurately detect the samples. This indicates that the combination of latex sensitizers and latex stabilizers in Examples 1, 2, and 3 resulted in test kits with higher analytical sensitivity, accuracy, and a wider linear range.

[0140] Experiment 3: Stability Comparison Test

[0141] The reagents from Examples 1-3 and Comparative Examples 1-3 were uniformly dispensed into 17 groups, with each group containing 20 mL for R1 and 5 mL for R2. The reagents were stored in a refrigerator at 2-8°C. On the same day each month, one group of reagents was taken out to test the creatine kinase isoenzyme (CK-MB) quality control (target value 20.04 ng / mL).

[0142] Test results as follows Figure 1 As shown, after 17 months of storage, the detection values ​​of Comparative Examples 1-3 were 15.13-18.03, differing from the target value by 2.01-4.91, and the detection values ​​showed a clear decreasing trend with prolonged storage time. In contrast, the detection values ​​of the reagents formulated in Examples 1, 2, and 3 were 19.59-20.54, differing from the target value by 0.45-0.50. This indicates that the reagents formulated in Examples 1, 2, and 3, and the reagents of Examples 1-3, are more stable than the comparative examples under storage conditions of 2-8℃. This demonstrates that the present invention, through optimizing the preparation method of microsphere solutions coated with mouse anti-human CK-MB monoclonal antibodies, adding compound latex sensitizers and compound latex stabilizers, and scientifically proportioning them, optimizes the reaction system and greatly improves the stability of the reagents.

[0143] In summary, through verification, the reagent provided by this invention has high sensitivity, wide linear range, and high accuracy compared to the control group, and is a more stable and better creatine kinase isoenzyme (CK-MB) detection kit.

[0144] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A creatine kinase isoenzyme CK-MB detection kit, characterized in that, Including reagent R1 and reagent R2; The reagent R1 consists of: 30-35 mmol / L of the first buffer solution, 12-25 mL / L of the compound latex sensitizer, 5.5-12 mL / L of the compound surfactant, and 0.65 g / L of the preservative. The reagent R2 consists of: 120-130 mmol / L second buffer, 12-32 mL / L microsphere solution coated with CK-MB antibody, 12-25 mL / L compound latex stabilizer, 5.5-12 mL / L compound surfactant, and 0.65 g / L preservative.

2. The creatine kinase isoenzyme CK-MB detection kit according to claim 1, characterized in that, The first buffer solution is POPSO-Tricine buffer.

3. The creatine kinase isoenzyme CK-MB detection kit according to claim 2, characterized in that, POPSO-Tricine buffer was prepared by the following method: Take 11.60g piperazine-N,N-bis(2-hydroxypropanesulfonic acid), dissolve it in 800mL of deionized water, then add 5.73g of N-tris(hydroxymethyl)methylglycine, stir to dissolve, adjust the pH to 8.06, and finally make up to 1L with deionized water.

4. The creatine kinase isoenzyme CK-MB detection kit according to claim 1, characterized in that, The compound latex sensitizer is composed of choline chloride, polyethylene glycol 8000, polyethylene glycol 6000, lithium chloride and sodium chloride in a weight ratio of (8-12):(10-14):(8-12):(4-6):(10-14).

5. The creatine kinase isoenzyme CK-MB detection kit according to claim 1, characterized in that, The compound surfactant is composed of a mixture of trimethylolpropane trioleate, alcohol ether hexyl sulfosuccinic acid mixed diester salt and phosphate betaine amphoteric surfactant.

6. The creatine kinase isoenzyme CK-MB detection kit according to claim 1, characterized in that, The second buffer is CAPS-EPPS buffer.

7. The creatine kinase isoenzyme CK-MB detection kit according to claim 1, characterized in that, The microsphere solution coated with CK-MB antibody was prepared by conjugating carboxyl latex microspheres with three different particle sizes (80-100 nm, 150-170 nm, and 280-300 nm) with mouse anti-human CK-MB monoclonal antibody.

8. The creatine kinase isoenzyme CK-MB detection kit according to claim 7, characterized in that, The microsphere solution coated with CK-MB antibody was prepared by the following method: Take 4 mL of 85 nm carboxylate microspheres, 3 mL of 166 nm carboxylate microspheres, and 0.3 mL of 285 nm carboxylate microspheres. Add 18 mL of 126 mmol / L CAPS-EPPS buffer, stir to mix, and vortex at 30 °C for 20 min. Then add 0.8-1.6 mg of EDC and vortex at 35 °C for 50 min. Then add 32-68 mg of mouse anti-human CK-MB monoclonal antibody, adjust the vortex rate to 160 r / min, and vortex at 30 °C for 1 hour. Then add 2.5 mL of masking agent, adjust the vortex rate to 200 r / min, and vortex at 30 °C for 3 hours.

9. The creatine kinase isoenzyme CK-MB detection kit according to claim 1, characterized in that, The compound latex stabilizer is composed of sedum heptanose, polyethylene glycol 20000, sorbitol, arbutin, potassium chloride and lithium chloride in a weight ratio of (4-8):(8-12):(50-70):(6-10):(8-12):(4-6).

10. The creatine kinase isoenzyme CK-MB detection kit according to claim 7, characterized in that, The preservative is selected from Nuosha PC-150 antibacterial agent.

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