Formula for stably preserving myocardial enzyme CK-MB and application thereof

Abstract

The invention relates to the field of immunodetection, in particular to a formula for stably preserving CK-MB and application thereof.The formula comprises 10-500 mM of a buffer solution, 100-500 mM of inorganic salt, 10-50% of glycerin, 1-5 mM of a chelating agent, 1-10 mM of a reducing agent, 5-50 mM of an antioxidant, 10-100 mM of amino acid, 10-200 g / L of sugar, 10-200 g / L of polyhydric alcohol, 10-100 g / L of inert protein and 0.5-2 g / L of a preservation.By means of the formula of the preserving fluid, a prepared liquid CKMB calibration product or quality control product can be better in stability; and the detection accuracy and repeatability are not influenced.

Description

Technical Field

[0001] The invention relates to the field of immune detection, in particular to a formula for stably storing CK-MB and an application thereof. Background Art

[0002] Creatine kinase isoenzymes, also known as serum creatine kinase isoenzymes, are an enzyme component with four isoenzyme forms: muscle type (MM), brain type (BB), hybrid type (MB) and mitochondrial type (MiMi). The MM type is mainly present in various muscle cells, the BB type is mainly present in brain cells, the MB type is mainly present in cardiomyocytes, and the MiMi type is mainly present in cardiac and skeletal muscle mitochondria. The muscle creatine kinase molecule is a dimer composed of two identical subunits. According to the primary structure of rabbit, human, chicken, and mouse creatine kinase that has been determined, the M-type subunit is composed of 387 amino acid residues, with a molecular weight of about 43kDa, 8 sulfhydryl groups in the molecule, but no disulfide bonds. The CK in the serum of normal adults is mainly composed of CK-MM isoenzymes, with only trace amounts of CK-MB. Within the detection limit of most CK tests, the CK-BB isoenzyme is usually not present in serum. The detection of a large amount of CK-MB in serum usually indicates acute myocardial infarction (AMI). CKBB is a brain isoenzyme, while CKMM is present in all muscles. CKMB mainly occurs in the myocardium, accounting for about 20% of the total myocardial CK, and is of great significance for the diagnosis of AMI. There are two subtypes of CKMB, namely CKMB1 and CKMB2. CKMB2 is a tissue isomer. When released into the plasma, the terminal lysine of the m subunit is cut off by carboxypeptidase, resulting in serum CKMB1 subtype.

[0003] CK-MB For a long time, the increase of creatine kinase MB (CKMB) in serum has been used as a marker for acute myocardial infarction (AMI). CKMB is usually divided into two isoforms, MB1 and MB2. In myocardial cells, CKMB mainly exists in the form of MB2. Once myocardial cells are damaged, MB2 will be released, causing the CKMB level in serum to rise rapidly in a short period of time. The rise time is usually within 6 hours of onset, and it reaches a peak at around 24 hours, and gradually decreases after 72 hours until it returns to normal levels. This shows that CK-MB can reflect myocardial damage at an early stage.

[0004] There are generally two methods of CK-MB detection in clinical practice, which respectively detect the CK-Mb enzyme activity in the "five items of myocardial enzymes" and the CK-Mb enzyme quality in the "three items of myocardial infarction". One is the enzyme activity and the other is the enzyme content (quality).

[0005] In the detection of CK-MB, it is inevitable to use CKMB antigen for antibody preparation, calibration products, quality control products and other purposes, which leads to the need for the preparation and preservation of CKMB protein antigen. However, CKMB protein is easily inactivated. In order to maintain the stability of the protein, most of them are stored in freeze-dried form on the market. The production of freeze-dried products requires equipment investment and the process is cumbersome. Freeze-dried calibrators and quality control products need to be re-dissolved before use. After re-dissolution, it is difficult to maintain long-term stability at 2-8°C, and the concentration of the calibrator may change after re-dissolution, and there is a risk of batch-to-batch and bottle-to-bottle differences. Therefore, liquid stable CKMB calibrators and quality control products are urgently needed in clinical practice. Summary of the invention

[0006] The invention provides a formula for stably preserving CK-MB, which comprises 10-500 mM buffer, 100-500 mM inorganic salt, 10-50% glycerol, 1-5 mM chelating agent, 1-10 mM reducing agent, 5-50 mM antioxidant, 10-100 mM amino acid, 10-200 g / L sugar, 10-200 g / L polyol, 10-100 g / L inert protein and 0.5-2 g / L preservative.

[0007] Preferably, the buffer is any one of phosphate buffer, tris(hydroxymethylaminomethane) buffer, 4-hydroxyethylpiperazineethanesulfonic acid buffer, glycine buffer and borate buffer; the pH of the buffer is 7.0-8.5, and the concentration of the buffer is 10-200 mM.

[0008] Furthermore, the inorganic salt is any one or more of sodium chloride, magnesium chloride, potassium chloride, sodium sulfite, magnesium sulfate, and magnesium acetate, and the concentration of the inorganic salt is 100 to 500 mM.

[0009] Furthermore, the chelating agent is any one or more of EDTA, EGTA, and ammonium citrate, with a concentration of 1 to 5 mM.

[0010] Furthermore, the reducing agent is any one or more of DTT, mercaptoethanol, TCEP, and THPP, and the concentration is 1 to 10 mM.

[0011] Furthermore, the antioxidant is any one or more of vitamin C, sodium succinate, and reduced glutathione, and the concentration is 5 to 50 mM.

[0012] Furthermore, the stabilizer is any one or more of sugars, polyols, small molecule amino acids, and large molecule inert proteins.

[0013] Furthermore, the sugar is any one or more of sucrose, trehalose, mannose, and glucose, with a concentration of 10 to 200 g / L; the polyol is sorbitol or mannitol, with a concentration of 10 to 200 g / L; the small molecule amino acid is any one or more of glycine, proline, cysteine, and arginine, with a concentration of 10 to 100 mM; and the macromolecular inert protein is any one or more of bovine serum albumin, casein, and fish skin gelatin, with a concentration of 10 to 100 g / L.

[0014] Furthermore, the preservative is any one or more of sodium azide, Proclin 300 and gentamicin, with a concentration of 0.5 to 2 g / L.

[0015] The present invention also provides an application of the CKMB preservation solution for preparing stable liquid CKMB calibrants and quality control products.

[0016] Compared with the prior art, the preservation formula of the present invention has the following remarkable effects:

[0017] (1) The combined use of reducing agent + antioxidant + chelating agent + amino acid + sugar + polyol + inert protein + preservative is different and improved from the existing combined use of 3 to 4 types of reagents.

[0018] (2) After multiple comparisons and repeated adjustments, the inventors have obtained a preservation solution formula that can make the prepared liquid CKMB calibrator or quality control product more stable, without the need for freeze-drying or ultra-low temperature storage at -80°C, and can be stable at 4°C for at least 1 year;

[0019] (3) The CKMB preservation solution of the present invention does not affect the detection accuracy and repeatability. DETAILED DESCRIPTION

[0020] The present invention is described in detail below by way of examples, but the protection scope of the present invention is not limited to the following examples.

[0021] The embodiment of the present invention provides a formula for stably preserving CK-MB, which is composed of a basic component + a reducing agent + an antioxidant + a chelating agent + an amino acid + a sugar + a polyol + an inert protein + a preservative.

[0022] Wherein: the basic components include buffer, inorganic salt and glycerol, the buffer provides a stable pH range; the inorganic salt provides a certain concentration of salt ions, which can promote the dissolution of protein in water; glycerol can reduce the polarity of the buffer, thereby preventing the hydrophobic aggregation of protein. Sugars, polyols and inert proteins can promote protein dissolution, prevent water loss on the surface of the protein, and maintain the stability of the spatial conformation of the protein; preservatives can prevent the protein from being degraded by bacteria. The buffer, inorganic salt, stabilizer and preservative in the above materials are the basic formulas for most protein stability, but because CKMB is a catalytic enzyme, it mainly exists in a reduced form, with 8 sulfhydryl groups in the molecule, including two cysteines in the active site, but no intramolecular disulfide bonds, the structure is extremely unstable, and the addition of a reducing agent is the most critical, and the combination of an antioxidant can prevent the reducing agent from being oxidized, thereby protecting the sulfhydryl groups in the CKMB molecule. Further addition of chelating agents and amino acids plays a role in protecting the active center of the CKMB enzyme and improving the stability and activity of the enzyme.

[0023] The following is explained in conjunction with specific embodiments:

[0024] Example 1

[0025] Take a 1L clean beaker, add 500mL of purified water, add the following components in sequence, mix thoroughly and test the pH to 7.4±0.1, make up to 1L, stir and mix, and filter through a 0.45μm filter membrane.

[0026]

[0027]

[0028] Example 2

[0029] Take a 1L clean beaker, add 500mL of purified water, add the following components in sequence, mix thoroughly, adjust the pH to 8.0±0.1 with HCl, make up to 1L, stir and mix, and filter through a 0.45μm filter membrane.

[0030] materials Mass / L Tris 15g Sodium chloride 20g glycerin 100g THPP 0.5g Sodium succinate 2g EGTA 2g Glycine 5g sucrose 50g Sorbitol 10g Sodium Caseinate 10g PC300 1ml

[0031] Example 3

[0032] Take a 1L clean beaker, add 500mL of purified water, add the following components in sequence, mix thoroughly, adjust the pH to 7.5±0.1 with NaOH, make up to 1L, stir and mix, and filter through a 0.45μm filter membrane.

[0033]

[0034]

[0035] Example 4

[0036] Take a 1L clean beaker, add 500mL of purified water, add the following components in sequence, mix thoroughly, adjust the pH to 7.5±0.1 with NaOH, make up to 1L, stir and mix, and filter through a 0.45μm filter membrane.

[0037] materials Mass / L Hepes7.4 12.1g Sodium chloride 2.5g glycerin 250g DTT 0.8g Reduced Glutathione 2g EDTA 2ml Proline 5g Trehalose 50g Mannitol 10g BSA 10g PC300 1ml

[0038] Comparative Example 1:

[0039] The method of Example 1 was used to prepare the product, except that no antioxidant was added, and the other components were the same as those in Example 1.

[0040] Comparative Example 2:

[0041] Prepared according to the method of Example 2, except that amino acids and sugars are not included, and the other components are the same as those added in Example 2

[0042] Comparative Example 3

[0043] Prepared according to the method of Example 3, except that polyol and inert protein are not contained, and other components are the same as those added in Example 3

[0044] The storage solutions prepared in the above Examples 1 to 4 and Comparative Examples 1 to 3 were used to dilute the CKMB antigen to a concentration of 5 ng / mL, 50 ng / mL, and 300 ng / mL, respectively, mixed, and stored at 2-8° C. The CKMB antigen is a recombinant antigen produced by Yibaixin Biotechnology Co., Ltd.

[0045] The stability of the 3 CKMB concentrations at room temperature and the real-time stability test at 2-8°C were performed. The specific experimental methods are as follows:

[0046] Room temperature open bottle stability: Take samples prepared with the preservation solutions of Examples 1-4 and Comparative Examples 1-3, a total of 7 groups, place them at room temperature, and test the reaction amplitude at 0, 6, 12, 24, and 48 hours respectively. Measured using the CKMB kit, the test instrument is a Hitachi 7180 fully automatic biochemical analyzer, record the test results and calculate the deviation, the results are as follows:

[0047]

[0048] As can be seen from the above table, the reaction amplitudes of the three gradient antigens of CKMB low, medium and high prepared using the preservation solutions of Examples 1 to 4 at 6, 12, 24 and 48 hours after being placed at room temperature are less deviated from those at the 0th hour, indicating that the CKMB antigens prepared by the preservation solutions of Examples 1 to 4 are almost not degraded after being placed at room temperature for 48 hours or within 48 hours, and have good stability; while the reaction amplitudes of the CKMB antigens prepared by the preservation solutions of Comparative Examples 1 to 3 are increasingly deviated from those at the 0th hour after 6, 12, 24 and 48 hours at room temperature, indicating that the CKMB antigens prepared by the preservation solutions of Comparative Examples 1 to 3 are more degraded as the room temperature is placed for a longer time, and the protective effect of no antioxidant on the CKMB protein is the worst, followed by polyols and inert proteins, which comprehensively indicates that the preservation solutions of Examples 1 to 4 have a better effect of protecting the activity of the CKMB antigen.

[0049] Real-time stability at 2-8°C: Take 5 samples prepared with the preservation solutions of Examples 1-4 and Comparative Examples 1-3, place at 2-8°C, take out one of the samples at 0, 2, 4, 8, and 12 months, equilibrate to room temperature, and measure with CKMB kit. The test instrument is Hitachi 7180 fully automatic biochemical analyzer. Record the test results and calculate the deviation. The results are as follows:

[0050]

[0051]

[0052] As can be seen from the above table, the reaction amplitudes of the three gradient antigens of CKMB low, medium and high prepared using the preservation solutions of Examples 1 to 4 at 2, 4, 8 and 12 months after being placed at 2-8°C are less deviated from those at month 0, indicating that the CKMB antigens prepared by the preservation solutions of Examples 1 to 4 are almost not degraded after being stored at 2-8°C for one year, and have good stability; while the reaction amplitudes of the CKMB antigens prepared by the preservation solutions of Comparative Examples 1 to 3 at 2, 4, 8 and 12 months after being placed at 2-8°C are increasingly deviated from those at month 0, indicating that the longer the CKMB antigens prepared by the preservation solutions of Comparative Examples 1 to 3 are placed at 2-8°C, the more they are degraded, and the protective effect of no antioxidant on CKMB protein is the worst, followed by polyols and inert proteins, and the results are basically consistent with the stability of opening the bottle at room temperature. The overall description shows that the preservation solutions of Examples 1 to 4 have a better effect of protecting the activity of CKMB antigen under refrigerated conditions of 2-8°C.

[0053] Compared with the existing preservation formulas on the market, the CKMB preservation solution of the present invention uses a combination of reducing agent + antioxidant + chelating agent + amino acid + sugar + polyol + inert protein + preservative at the same time, after repeated experiments, and controls the concentration of each component. With the help of the implementation scheme provided by the present application, the use of this preservation solution formula can greatly reduce the degradation rate of CKMB antigen in low-temperature refrigeration (2-8°C) preservation, maintain biological activity, improve stability, and extend the conventional low-temperature storage time. At present, commonly used buffers usually contain only 3-5 components of the formula of the present invention, and usually need to be freeze-dried or ultra-low temperature (-80°C) for preservation. After freeze-drying and re-dissolving, it can usually only maintain stability for 7-28d, and repeated freezing and thawing after ultra-low temperature storage also greatly reduces the activity of CKMB antigen. The CKMB calibrator or quality control product prepared by the preservation solution designed by the present invention only needs to be stored at 2-8°C. Compared with ultra-low temperature and freeze-drying, the storage conditions are simple, and the activity of CKMB antigen decreases within 10% in 1 year.

[0054] The above description is only a preferred example of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A formula for stably preserving myocardial enzyme CK-MB, characterized in that: The formula comprises 10-500 mM buffer, 100-500 mM inorganic salt, 10-50% glycerol, 1-5 mM chelating agent, 1-10 mM reducing agent, 5-50 mM antioxidant, 10-100 mM amino acid, 10-200 g / L sugar, 10-200 g / L polyol, 10-100 g / L inert protein and 0.5-2 g / L preservative.

2. The formula for stably preserving myocardial enzyme CK-MB according to claim 1, characterized in that: The buffer is any one of phosphate buffer, tris(hydroxymethylaminomethane) buffer, 4-hydroxyethylpiperazineethanesulfonic acid buffer, glycine buffer and borate buffer; the pH of the buffer is 7.0-8.5, and the concentration of the buffer is 10-200 mM.

3. The formula for stably preserving myocardial enzyme CK-MB according to claim 1, characterized in that: The inorganic salt is any one or more of sodium chloride, magnesium chloride, potassium chloride, sodium sulfite, magnesium sulfate, and magnesium acetate, and the concentration of the inorganic salt is 100-500 mM.

4. The formula for stably preserving myocardial enzyme CK-MB according to claim 1, characterized in that: The chelating agent is any one or more of EDTA, EGTA and ammonium citrate, and the concentration is 1-5 mM.

5. The formula for stably preserving myocardial enzyme CK-MB according to claim 1, characterized in that: The reducing agent is any one or more of DTT, mercaptoethanol, TCEP, and THPP, and the concentration is 1-10 mM.

6. The formula for stably preserving myocardial enzyme CK-MB according to claim 1, characterized in that: The antioxidant is any one or more of vitamin C, sodium succinate, and reduced glutathione, and the concentration is 5 to 50 mM.

7. The formula for stably preserving myocardial enzyme CK-MB according to claim 1, characterized in that: The stabilizer is any one or more of sugars, polyols, small molecule amino acids, and large molecule inert proteins.

8. The formula for stably preserving myocardial enzyme CK-MB according to claim 1, characterized in that: The sugar is any one or more of sucrose, trehalose, mannose, and glucose, with a concentration of 10 to 200 g / L; the polyol is sorbitol or mannitol, with a concentration of 10 to 200 g / L; the small molecule amino acid is any one or more of glycine, proline, cysteine, and arginine, with a concentration of 10 to 100 mM; and the macromolecular inert protein is any one or more of bovine serum albumin, casein, and fish skin gelatin, with a concentration of 10 to 100 g / L.

9. The formula for stably preserving myocardial enzyme CK-MB according to claim 1, characterized in that: The preservative is any one or more of sodium azide, Proclin 300 and gentamicin, with a concentration of 0.5 to 2 g / L.

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