Creatine Kinase Detection Reagent, Kit and Detection Method

Abstract

The present application discloses a creatine kinase detection reagent, a kit and a detection method. The detection reagent for creatine kinase or at least one isoenzyme of creatine kinase comprises a first reagent containing glucose, hexokinase, ADP, NADP, AMP, diadenosine pentaphosphate and an activator, wherein the first reagent further contains 0.5 - 5 g / L of a thiol protecting agent, and the thiol protecting agent is one or more compounds selected from the following formula (I), wherein, A ‑ is -SO 3 ‑ or -COO ‑ ; M + is a monovalent metal ion; R 1 and R 2 are each independently selected from H, -NQ 1 Q 2 and -R-NQ 1 Q 2 , provided that R 1 and R 2 are not simultaneously H, wherein Q 1 and Q 2 are each independently selected from H, methyl and ethyl, R is a substituted or unsubstituted group selected from C1-C6 alkyl, C1-C6 alkoxy, C1-C6 acyl and -(OCH 2 CH 2 ) n -, n is an integer of 2 or 3, and the substitution is substituted by 1-3 groups selected from -OH, -NH 2 and -COOH. The detection reagent of the present invention has a significantly improved long-term storage period and still has good reactivity after being stored for 12 months.

Description

Technical Field

[0001] This application relates to a detection reagent for creatine kinase, and particularly to a detection reagent for creatine kinase that can be stored for a long time. Background Art

[0002] Creatine kinase is an important kinase in living organisms that participates in cell energy conversion, muscle contraction, and ATP regeneration. Existing methods for detecting creatine kinase include bioluminescence method, colorimetric method, enzyme coupling method, fluorescence method, etc. Among them, the enzyme coupling method is more commonly used and is the main test method recommended by the International Federation of Clinical Chemistry. In this method, a thiol compound is often added to avoid enzyme inactivation, and the influence of adenylate kinase is excluded by the mixed inhibition method of AMP and AP5A. Due to the instability of creatine kinase in serum, a thiol-containing substance must be added to the reagent. Currently, the commonly used activator of creatine kinase is N-acetylcysteine. However, N-acetylcysteine is relatively active and easily loses its activity during long-term storage, and a small amount of the oxide of N-acetylcysteine also inhibits the activity of creatine kinase, resulting in an obvious downward trend in the reactivity per unit concentration of the reagent during long-term storage, and it is easy to cause inaccurate low-value test results during clinical testing.

[0003] Therefore, there is a need for a detection reagent for creatine kinase that can be stored for a long time. Summary of the Invention

[0004] Therefore, the object of the present invention is to provide a substance that can protect the thiol-containing creatine kinase activator in the detection reagent for detecting creatine kinase by the enzyme coupling method, so as to obtain a detection reagent and kit for creatine kinase with a long shelf life (storage period).

[0005] To this end, a first aspect of the present invention provides a detection reagent for creatine kinase isoenzyme or at least one isoenzyme of creatine kinase. The detection reagent includes a first reagent containing glucose, hexokinase, ADP, NADP, AMP, diadenosine pentaphosphate, and an activator. Among them, the first reagent further contains a thiol protecting agent, and the thiol protecting agent is one or more compounds selected from the following formula (I):

[0006]

[0007] Wherein, A - is -SO 3 - or -COO - ;

[0008] M + is a monovalent metal ion;

[0009] R 1 and R2 Each independently selected from H, -NQ 1 Q 2 and -R-NQ 1 Q 2 , provided that R 1 and R 2 are not both H at the same time, where Q 1 and Q 2 are each independently selected from H, methyl and ethyl, R is a substituted or unsubstituted group selected from C1-C6 alkyl, C1-C6 alkoxy, C1-C6 acyl and -(OCH 2 CH 2 ) n -, n is an integer of 2 or 3, and the substitution is substituted by 1-3 groups selected from -OH, -NH 2 and -COOH.

[0010] According to one embodiment, the first reagent comprises:

[0011]

[0012] According to a preferred embodiment, in the compound of formula (I), A - is selected from -SO 3 - and -COO - ;

[0013] M + is sodium or potassium ion;

[0014] R 1 and R 2 are each independently selected from H, -NH 2 , -NHCH 3 , -N(CH 3 ) 2 , -R-NH 2 , -R-NHCH 3 and -R-N(CH 3 ) 2 , provided that R 1 and R 2 are not both H at the same time, where R is C1-C3 alkyl, C1-C3 alkyloxy or C1-C3 acyl.

[0015] More preferably, R 1 is H, R 2 is para-substituted; or R 1 and R 2 are both not H, and are respectively 2, 4-substituted.

[0016] According to a further preferred embodiment, in formula (I):

[0017] A - is -SO 3 - ; M + is Na + ;

[0018] R 1 is H;

[0019] R 2 is a para - or meta - substituent selected from -NH 2 , -NHCH 3 , -R-NH 2 and -R-NHCH 3 , provided that R 1 and R 2 are not both H at the same time, where R is an alkyl group of C1 - C3 or an alkoxy group of C1 - C3.

[0020] Specifically, the thiol protecting agent is selected from the group consisting of p - aminobenzenesulfonate, m - aminobenzenesulfonate, 2,4 - diaminobenzenesulfonate, N - methyl - p - aminobenzenesulfonate, N,N - dimethyl - p - aminobenzenesulfonate, N,N - diethyl - p - aminobenzenesulfonate, p - aminomethylbenzenesulfonate, p - aminoethylbenzenesulfonate, p - aminopropylbenzenesulfonate, 4-(3 - amino - 2 - methyl) - propylbenzenesulfonate, 4-(4 - amino - 2 - methyl)butylbenzenesulfonate, p - aminohexylbenzenesulfonate, and 2 - amino - 4 - aminoethylbenzenesulfonate.

[0021] More preferably, the content of the thiol protecting agent is 0.5 - 2.5 g / L.

[0022] The inventors of the present invention found that after adding the thiol protecting agent of the present invention, the storage stability of the first reagent is significantly improved and does not interfere with the original detection reaction and detection. After storage for 12 months, it still has good reactivity and can obtain accurate detection results. Specifically, the relative deviation of the reactivity of the detection reagent after storage for 12 months compared with the reactivity before storage can reach 15% or less, even 10% or less.

[0023] According to a preferred embodiment, the first reagent further comprises one or more of alkyl polyethylene glycol ethers of formula II with a concentration of 0.5 - 4 g / L:

[0024] CH 3 -(CH 2 ) p -(OCH 2 CH 2 ) q -OH (II)

[0025] wherein p is an integer from 7 to 18 and q is an integer from 5 to 10.

[0026] Preferably, the alkyl polyethylene glycol ether is lauryl alcohol polyethylene glycol ether. Preferably, the content of the alkyl polyethylene glycol ether of Formula II can be 1-4 g / L, more preferably 1-2 g / L.

[0027] Alkyl polyethylene glycol ether is a polyethylene glycol type non-ionic surfactant. It is usually used to improve the dispersibility and stability of diagnostic test reagent systems. However, at lower dosages, such surfactants do not have a significant protective effect on activators with sulfhydryl groups. At higher dosages, they have a certain protective effect on the activator, but this will lead to an increase in the viscosity of the reagent, which is not conducive to the automatic detection equipment sucking the reagent. The inventors surprisingly found that the combination of this surfactant with the sulfhydryl protecting agent of the present invention achieved remarkable results: the relative deviation of the reactivity of the test reagent after storage for 12 months compared to the reactivity before storage can reach below 5%.

[0028] According to a more preferred embodiment, the first reagent comprises components having the following concentrations:

[0029]

[0030] The activator for activating creatine kinase can be selected from the group consisting of N-acetylcysteine, reduced glutathione, L-cysteine, dithiothreitol, N-acetyl-L-cysteine, dithioerythritol, mercaptoethanol, and mercaptoacetic acid. Among them, dithiothreitol, dithioerythritol, and thioglycerol are more preferred. When using these activators, the reactivity of the test reagent after storage for 12 months is relatively higher.

[0031] Conventionally, the first reagent may further contain at least one selected from surfactants, preservatives, and stabilizers.

[0032] The surfactant that can be used in the first reagent can be one or more of an anionic surfactant, an amphiphilic surfactant, and a non-ionic surfactant.

[0033] The stabilizer can be selected from the sodium salt and potassium salt of ethylenediaminetetraacetic acid.

[0034] The preservative can be those conventionally used in diagnostic test reagents.

[0035] The test reagent for creatine kinase or at least one isoenzyme of creatine kinase further comprises a second reagent, which contains the following components:

[0036] Glucose 6-phosphate dehydrogenase 8-24 IU / mL,

[0037] Phosphocreatine 150-200 mM and

[0038] Second buffer 30-100 mM.

[0039] Since the mercapto protecting agent of the present invention has no influence on the reaction in the detection, any suitable components and concentrations in the prior art can be adopted for other components and concentrations in the detection reagent.

[0040] Conventionally, the second reagent may further contain a preservative.

[0041] The isoenzyme of creatine kinase may be selected from muscle type, brain type, hybrid type and mitochondrial type creatine kinase isoenzymes. The detection reagent of the present invention can be used to detect any one or more of the isoenzymes of creatine kinase (for example, by adding an antibody against a specific subunit to the detection reagent using the immunoinhibition method), and can also detect the total content of all creatine kinases.

[0042] The second aspect of the present invention provides a detection kit for creatine kinase or at least one isoenzyme of creatine kinase, and the kit includes the detection reagent for creatine kinase or at least one isoenzyme of creatine kinase as described above.

[0043] The third aspect of the present invention provides a method for detecting creatine kinase or at least one isoenzyme of creatine kinase. The method includes mixing the first reagent with a sample and incubating, then adding the second reagent and mixing, and then detecting the absorbance A1 of the mixture at a first time point and detecting the absorbance A2 of the mixture at a second time point.

[0044] The absorbance is detected at a main wavelength of 340 nm and a secondary wavelength of 546 nm.

[0045] The sample may be a blood sample from a mammal, preferably from a human, and more preferably a serum sample.

[0046] The third aspect of the present invention provides the use of the compound of formula I as defined above and optionally the compound of formula II as defined above in protecting an activator in an enzyme-coupled detection reagent for creatine kinase or at least one isoenzyme of creatine kinase.

[0047] The inventors of the present invention unexpectedly found that an organic phenyl salt containing an amino group / amine group can significantly protect a mercapto-containing creatine kinase activator, so that satisfactory detection accuracy can still be achieved after the creatine kinase detection reagent is stored for 12 months. In addition, further combination with a specific alkyl polyethylene glycol ether has obtained a further significantly improved storage effect. The creatine kinase detection reagent or kit of the present invention has a long shelf life and reduces the cost of clinical use. Detailed implementation mode

[0048] Next, in combination with specific embodiments of the present invention and the accompanying drawings, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0049] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of contradiction, this specification shall prevail.

[0050] As used herein, the "isoenzyme of creatine kinase" refers to any one of the four isoenzyme forms: hybrid type (MB), muscle type (MM), brain type (BB), and mitochondrial type (MiMi). Among them, the hybrid isoenzyme mainly exists in cardiomyocytes and is a dimer enzyme composed of M-type and B-type monomer subunits; the muscle isoenzyme mainly exists in various muscle cells and is a dimer enzyme composed of two M-type monomer subunits; the brain type mainly exists in brain cells and is a dimer enzyme composed of two B-type monomer subunits; the mitochondrial type mainly exists in the mitochondria of the heart and skeletal muscles and is a dimer enzyme composed of two Mi-type monomer subunits.

[0051] As used herein, the "creatine kinase", unless otherwise specified, refers to the sum of the four isoenzymes of creatine kinase.

[0052] Based on a specific thiol protecting agent effective for creatine kinase activators containing thiol discovered by the inventors, the present invention provides a creatine kinase isoenzyme detection reagent with an extended shelf life based on the enzyme coupling method.

[0053] The detection of creatine kinase in a sample by the enzyme coupling method utilizes the reversible catalytic reaction of creatine kinase (CK) on phosphocreatine and creatine, enabling phosphocreatine to transfer a phosphate to ADP to generate creatine and ATP; then ATP and glucose are catalyzed by hexokinase (HK) to generate ADP and glucose 6-phosphate; glucose 6-phosphate converts NADP to NADPH under the action of glucose 6-phosphate dehydrogenase (G6P-DH):

[0054]

[0055]

[0056]

[0057] This detection method calculates the amount of creatine kinase by monitoring the change rate of the conversion of NADP to NADPH (the absorbance difference (A2 - A1) at a certain time interval) according to the following formula:

[0058] Content of creatine kinase = Absorbance of sample (A2 - A1) × Concentration of calibration solution / Absorbance of calibration (A2 - A1).

[0059] In this detection method, a compound with a sulfhydryl group needs to be added to the detection reagent to activate creatine kinase in serum. These sulfhydryl compounds, such as commonly used N - acetylcysteine, are prone to inactivation due to their reducibility, resulting in the inability of such detection reagents to be stored for a long time and also easily leading to inaccurate detection results.

[0060] The inventors found that phenyl organic acid salts such as benzenesulfonates and benzoates with amino groups (amine groups or amide groups) have a significant effect on stabilizing sulfhydryl compounds and do not affect the normal reaction of the reagent, thus completing the present invention and providing a detection reagent for creatine kinase that can be stably stored for at least 12 months.

[0061] The detection reagent of the present invention is not only suitable for detecting the most common M subunit and B subunit dimer creatine kinase (hybrid isoenzyme), but also suitable for detecting other creatine kinase isoenzymes and the total amount of all creatine kinase isoenzymes.

[0062] Conventional creatine kinase detection reagents include a first reagent and a second reagent because the pH values required for different reaction components are different. The pH value of the first reagent is about 6 (can be 5.4 - 6.7), and it includes the necessary reagents ADP, glucose, and hexokinase for the above reaction, AMP and diadenosine pentaphosphate (AP5A) for eliminating the influence of adenylate kinase, a sulfhydryl compound as a creatine kinase activator, and necessary buffers and additives. The pH value of the second reagent is higher, about 9 (can be 8.4 - 9.7), and it includes glucose - 6 - phosphate dehydrogenase, phosphocreatine, and necessary buffers and additives for carrying out the above reaction.

[0063] The sulfhydryl protecting agent of the present invention is used to protect the sulfhydryl - containing creatine kinase activator, so it is also present in the first reagent. According to the specific examples described in detail below, the addition of the sulfhydryl protecting agent of the present invention does not affect the various enzymatic reactions and the final detection. Therefore, except for the sulfhydryl protecting agent of the present invention, the concentrations of other reagents in the first reagent can adopt any concentration suitable for detection.

[0064] The addition amount of the sulfhydryl protecting agent can be 0.5 - 5 g / L. According to a preferred embodiment, the addition amount of the sulfhydryl protecting agent can be 0.5 - 2.5 g / L.

[0065] The sulfhydryl protecting agent can be selected from one or more compounds of the following formula (I):

[0066]

[0067] Among them, A - is -SO 3 - or -COO - ;

[0068] M + is a monovalent metal ion;

[0069] R 1 and R 2 are each independently selected from H, -NQ 1 Q 2 and -R-NQ 1 Q 2 provided that R 1 and R 2 are not both H at the same time, where Q 1 and Q 2 are each independently selected from H, methyl and ethyl, and R is a substituted or unsubstituted group selected from C1-C6 alkyl, C1-C6 alkoxy, C1-C6 acyl and -(OCH 2 CH 2 ) n -, n is an integer of 2 or 3, and the substitution is substituted by 1-3 groups selected from -OH, -NH 2 and -COOH.

[0070] According to a preferred embodiment, in the compound of formula (I), A - is selected from -SO 3 - and -COO - ;

[0071] M + is sodium or potassium ion;

[0072] R 1 and R 2 are each independently selected from H, -NH 2 -, -NHCH 3 -, -N(CH 3 ) 2 -, -R-NH 2 -, -R-NHCH 3 - and -R-N(CH 3 ) 2 provided that R 1 and R 2 are not both H at the same time, where R is C1-C3 alkyl, C1-C3 alkoxy or C1-C3 acyl.

[0073] More preferably, R 1is H, R 2 is para-substituted; or R 1 and R 2 are both not H, and are respectively 2,4-substituted.

[0074] According to a further preferred embodiment, A in formula (I) - is -SO 3 - ; M + is Na + ;

[0075] R 1 is H; R 2 is a para- or meta-substituent, selected from -NH 2 , -NHCH 3 , -R-NH 2 and -R-NHCH 3 , provided that R 1 and R 2 are not both H at the same time, where R is an alkyl group of C1-C3 or an alkoxy group of C1-C3, which can play a good protective role for the activator.

[0076] Specific mercapto protectants that can be listed, for example: p-aminobenzenesulfonate, m-aminobenzenesulfonate, 2,4-diaminobenzenesulfonate, N-methyl-p-aminobenzenesulfonate, N,N-dimethyl-p-aminobenzenesulfonate, N,N-diethyl-p-aminobenzenesulfonate, p-aminomethylbenzenesulfonate, p-aminoethylbenzenesulfonate, p-aminopropylbenzenesulfonate, 4-(3-amino-2-methyl)-propylbenzenesulfonate, 4-(4-amino-2-methyl)butylbenzenesulfonate, p-aminohexylbenzenesulfonate and 2-amino-4-aminoethylbenzenesulfonate. The above-mentioned benzenesulfonates containing amino or amine groups can be sodium salts or potassium salts.

[0077] Preferably, the mercapto protectant can be sodium p-aminobenzenesulfonate, sodium m-aminobenzenesulfonate, sodium 2,4-diaminobenzenesulfonate, sodium p-methylaminobenzenesulfonate and sodium p-aminomethylbenzenesulfonate.

[0078] The activator used in the present invention is not limited to N-acetylcysteine, and can also be reduced glutathione, L-cysteine, dithiothreitol, N-acetyl-L-cysteine, dithioerythritol, mercaptoethanol, mercaptoacetic acid, mercapto glycerol, etc. Among them, dithiothreitol, dithioerythritol and mercapto glycerol are more preferred.

[0079] In the enzyme coupling method utilized in the present invention, the reversible reaction reacts rapidly in a neutral pH environment. Therefore, the pH of the system can be in the range of 5.5 to 7.5, preferably in the range of 6.0 to 7.3.

[0080] The present invention does not particularly limit the type of buffer, and any conventionally applicable buffer can be used in the first reagent of the present invention. The buffer solution can be selected from Tris buffer solution, Mopso buffer solution, imidazole buffer solution, phosphate buffer solution, carbonate buffer solution, malic acid buffer solution, glycine buffer solution, but is not limited thereto. The preferred buffer solution is Tris buffer solution.

[0081] Additives that can also be added to the first reagent can include surfactants, preservatives, stabilizers, etc. One, two or all of them can be added as needed.

[0082] Among them, except that cationic surfactants are not applicable, conventional and suitable anionic surfactants, amphiphilic surfactants and nonionic surfactants can be used. Preferred are nonionic surfactants, such as Triton series (Triton X-405, Triton X-100, etc.), tween series (such as Tween 20, etc.).

[0083] The present invention does not particularly limit the preservative, and any preservative conventionally used in detection reagents can be used in the present invention. Examples include: sodium azide, Proclin 300, BND, erythromycin, gentamicin, etc., but are not limited thereto. Preferred are sodium azide and Proclin 300.

[0084] There is also no particular limitation on the stabilizer, and any stabilizer conventionally used in detection reagents can be used in the present invention. It is mainly used to chelate heavy metal ions in the complex system, thereby playing a role in stabilizing the reagent system. Examples include: disodium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, etc.

[0085] The second reagent includes glucose-6-phosphate dehydrogenase, creatine phosphate and a necessary buffer, as well as optional additives.

[0086] Each component in the second reagent can use any suitable components and concentrations conventionally used in the enzyme coupling method for detecting creatine kinase.

[0087] The buffer is as defined above, and the pH of the second reagent is 8.4 to 9.7.

[0088] The necessary additive can be a preservative. The definition of the preservative is as defined above.

[0089] The detection reagent of the present invention can be used to detect the total content of creatine kinase, and can also detect one or two specific creatine kinase isoenzymes by methods such as immuno-inhibition method. For example, an anti-CK-M subunit antibody can be added to the detection reagent (such as the first reagent) to detect CK-MB. The present invention does not particularly limit the antibody for detecting isoenzymes, and can be selected according to needs and will not be elaborated here.

[0090] The present invention also provides a detection kit comprising the above-mentioned creatine kinase or at least one isoenzyme of creatine kinase as a detection reagent.

[0091] In addition to the necessary detection reagents, the kit may further include calibration products, quality control products and / or diluents, etc. In addition, the kit also includes an instruction manual for explaining its usage method.

[0092] The method for detecting the content of creatine kinase and / or its isoenzyme in a sample using the detection reagent of the present invention may include the following steps: adding a first reagent to the sample and mixing evenly, adding a second reagent and mixing evenly after incubating at 37°C for 5-10 minutes, reading the absorbance A1 after reacting at 37°C for about 3-5 minutes, and then reading the absorbance A2 after 2-3 minutes. Calculate the content of creatine kinase and / or its isoenzyme according to the above calculation formula. Among them, the main wavelength for determination is 340 nm and the secondary wavelength is 546 nm. Depending on different detection devices or samples, the method may include additional steps, such as steps for diluting the sample or pre-treating the sample, etc.

[0093] The sample of the present invention can be from mammals, preferably a blood sample from humans, and more preferably a serum sample.

[0094] The following illustrates the present invention through specific examples, but the scope of the present invention is not limited by these.

[0095] Example 1:

[0096] Prepare Reagent 1 and Reagent 2 according to the following components and contents.

[0097] Reagent 1

[0098]

[0099] Reagent 2

[0100]

[0101] Example 2:

[0102] Prepare the reagent according to Reagent 1 and Reagent 2 in Example 1, except that the content of sodium sulfanilate in Reagent 1 is replaced with 2.5 g / L.

[0103] Example 3:

[0104] Prepare the reagent according to Reagent 1 and Reagent 2 in Example 1, except that the content of sodium sulfanilate in Reagent 1 is replaced with 5.0 g / L.

[0105] Comparative Example 1:

[0106] Prepare Reagent 1 according to the following components and contents. Reagent 2 is the same as Reagent 2 in Example 1.

[0107] Reagent 1

[0108]

[0109] Comparative Example 2:

[0110] Prepare Reagent 1 with the following components and contents. Reagent 2 is the same as Reagent 2 in Example 1.

[0111] Reagent 1

[0112]

[0113] Comparative Example 3:

[0114] Prepare the reagent with Reagent 1 and Reagent 2 in Comparative Example 2, except that the content of Brij 35 in Reagent 1 is replaced with 4 g / L.

[0115] Comparative Example 4:

[0116] Prepare the reagent with Reagent 1 and Reagent 2 in Comparative Example 2, except that the content of Brij 35 in Reagent 1 is replaced with 8 g / L.

[0117] Example 4:

[0118] Prepare Reagent 1 with the following components and contents. Reagent 2 is the same as Reagent 2 in Example 1.

[0119] Reagent 1

[0120]

[0121]

[0122] Test Example:

[0123] Detect the same samples with the reagents in the above examples and comparative examples according to the following method. The detection equipment is the automatic biochemical analyzer BS 800 of Shenzhen Mindray Bio-Medical Electronics Co., Ltd.

[0124] Selection of test samples: All the test samples required in this experiment are from commercial Roche routine biochemical composite calibrators and biochemical composite certified reference materials. Process and aliquot the serum according to the following steps: 1: Add the corresponding deionized water to dissolve the calibrator and the control according to the instructions in the manual, shake on a vortex oscillator for 5 min to fully mix the samples, and aliquot the samples at a volume of 200 μL per tube. The aliquoted samples are stored in a -20°C refrigerator; when taking them, they must be thawed at 2 - 8°C and fully mixed before use.

[0125] The long-term stable validity monitoring of this experiment can be achieved through two ways: 1. Prepare reagents with different formulations and store them in a cold storage at 2-8°C. Test the basic performance of the reagents before and after storage respectively, mainly monitoring the reactivity deviation of the measured values of different samples from the measured values on the 0th day. 2. Quickly predict the shelf life of in vitro diagnostic reagents through the Arrhenius equation (one day at 37°C is equivalent to 1.5 months of storage in a cold storage at 2-8°C). The first scheme is selected for this test example.

[0126] Test method: Add the sample (i.e., the quality control product, and the calibration tube uses the calibration product as the sample) and reagent 1 and mix well. Incubate at 37°C for 5-10 minutes, then add reagent 2 and mix well. Read the absorbance A1 of the 27th measurement point 3-5 minutes later, and then read the absorbance (the reading period of the measurement point is 18 seconds) A2 of the 33rd measurement point 2-3 minutes later. According to the formula: CK = measured absorbance (A2 - A1) × calibration solution concentration / calibration absorbance (A2 - A1), calculate the content of CK. Among them, the main measurement wavelength is 340 nm, the secondary wavelength is 546 nm, the sample (quality control product / calibration product) dosage is 50 μL, the reagent 1 dosage is 200 μL, and the reagent 2 dosage is 50 μL. Taking the average value of the test reactivity of each sample (quality control product / calibration product) measured by the refrigerated reagent on the 0th day (2-8°C) as the standard, calculate the relative deviation of the average value of the test reactivity of the same batch of samples after the detection reagent is stored for 12 months (2-8°C) from the reagent reactivity on the 0th day. The long-term stability test results of the above examples and comparative examples are shown in Tables 1-8.

[0127] Table 1

[0128]

[0129]

[0130] Table 2

[0131]

[0132] Table 3

[0133]

[0134]

[0135] Table 4

[0136]

[0137] Table 5

[0138]

[0139] Table 6

[0140]

[0141]

[0142] Table 7

[0143]

[0144] Table 8

[0145]

[0146]

[0147] It can be seen from the results of the long-term stability experiments of the above reagents that within a certain content range, separately adding sodium sulfanilate or Brij 35 alone can both play a role in extending the shelf life of the reagent. Among them, the effect of separately adding sodium sulfanilate alone is better than that of separately adding Brij 35 alone (see Examples 1 to 3 and Comparative Examples 1 to 4, and Tables 1 to 7).

[0148] When sodium sulfanilate is used alone at a relatively low concentration, its protective effect on the activator is relatively weak. After recalibrating the reagent with a calibrator and then performing sample testing, accurate results can also be obtained.

[0149] Brij 35 is a surfactant. Adding a large amount of it will cause an increase in the viscosity of the reagent and is not suitable for fully automatic biochemical analyzers. Therefore, its addition amount should not be too large. Therefore, the scheme of separately adding Brij 35 is not applicable. According to Example 4 and Table 8, the combined addition of Brij 35 and sodium sulfanilate has a synergistic enhancement effect and can significantly increase the shelf life of the reagent.

[0150] The above is only the preferred embodiment of the present invention, and it does not limit the patent scope of the present invention accordingly. Any equivalent structural transformation made under the inventive concept of the present invention by using the content of the specification and drawings of the present invention, or directly / indirectly applied in other related technical fields, is included in the patent protection scope of the present invention.

Claims

1. A detection reagent for creatine kinase or at least one isoenzyme of creatine kinase, the detection reagent comprising a first reagent containing glucose, hexokinase, ADP, NADP, AMP, diadenosine pentaphosphate and an activator, wherein the activator is an activator for creatine kinase containing a sulfhydryl group or at least one isoenzyme of creatine kinase. Wherein, the first reagent further contains a sulfhydryl protecting agent, and the sulfhydryl protecting agent is one or more compounds selected from the following formula (I): Among them, A - is -SO 3 - or -COO - ; M + is a monovalent metal ion; R 1 and R 2 are each independently selected from H, -NQ 1 Q 2 and -R-NQ 1 Q 2 provided that R 1 and R 2 are not simultaneously H, where Q 1 and Q 2 are each independently selected from H, methyl and ethyl, R is a substituted or unsubstituted group selected from C1-C6 alkyl, C1-C6 alkoxy, C1-C6 acyl and -(OCH 2 CH 2 ) n - in which n is an integer of 2 or 3, and the substitution is substituted by 1-3 groups selected from -OH, -NH 2 and -COOH.

2. The detection reagent for creatine kinase or at least one isoenzyme of creatine kinase according to claim 1, wherein the first reagent comprises:

3. The detection reagent for creatine kinase or at least one isoenzyme of creatine kinase according to claim 1 or 2, wherein the sulfhydryl protecting agent is selected from the group consisting of p-aminobenzenesulfonate, m-aminobenzenesulfonate, 2,4-diaminobenzenesulfonate, N-methyl-p-aminobenzenesulfonate, N,N-dimethyl-p-aminobenzenesulfonate, N,N-diethyl-p-aminobenzenesulfonate, p-aminomethylbenzenesulfonate, p-aminoethylbenzenesulfonate, p-aminopropylbenzenesulfonate, 4-(3-amino-2-methyl)-propylbenzenesulfonate, 4-(4-amino-2-methyl)butylbenzenesulfonate, p-aminohexylbenzenesulfonate and 2-amino-4-aminoethylbenzenesulfonate.

4. The detection reagent for creatine kinase or at least one isoenzyme of creatine kinase according to claim 1 or 2, wherein the first reagent further comprises one or more of alkyl polyethylene glycol ethers selected from the following formula II with a concentration of 0.5 - 4 g / L: CH 3 -(CH 2 ) p -(OCH 2 CH 2 ) q -OH (II) Wherein, p is an integer from 7 to 18, and q is an integer from 5 to 10.

5. The detection reagent for creatine kinase or at least one isoenzyme of creatine kinase according to claim 4, wherein the alkyl polyethylene glycol ether of formula II is lauryl alcohol polyethylene glycol ether.

6. The detection reagent for creatine kinase or at least one isoenzyme of creatine kinase according to claim 4, wherein the first reagent comprises components with the following concentrations:

7. The detection reagent for creatine kinase or at least one isoenzyme of creatine kinase according to claim 1 or 2, wherein the activator is selected from the group consisting of N-acetylcysteine, reduced glutathione, L-cysteine, dithioerythritol, N-acetyl-L-cysteine, dithiothreitol, mercaptoethanol and thioglycolic acid.

8. The detection reagent for creatine kinase or at least one isoenzyme of creatine kinase according to claim 1 or 2, wherein the first reagent further contains at least one selected from surfactants, preservatives and stabilizers.

9. The detection reagent for creatine kinase or at least one isoenzyme of creatine kinase according to claim 8, wherein the surfactant is one or more of anionic surfactants, amphiphilic surfactants and nonionic surfactants.

10. The detection reagent for creatine kinase or at least one isoenzyme of creatine kinase according to claim 8, wherein the stabilizer is selected from the sodium salt and potassium salt of ethylenediaminetetraacetic acid.

11. The detection reagent for creatine kinase or at least one isoenzyme of creatine kinase according to claim 1 or 2, wherein the detection reagent further comprises a second reagent containing the following components: Glucose 6-phosphate dehydrogenase 8 - 24 IU / mL, Phosphocreatine 150 - 200 mM and A second buffer 30 - 100 mM.

12. The detection reagent for creatine kinase or at least one isoenzyme of creatine kinase according to claim 11, wherein the second reagent further contains a preservative.

13. The detection reagent for creatine kinase or at least one isoenzyme of creatine kinase according to claim 1 or 2, wherein the isoenzyme of creatine kinase is selected from muscle type, brain type, hybrid type, and mitochondrial type creatine kinase isoenzymes.

14. A detection kit for creatine kinase or at least one isoenzyme of creatine kinase, the kit comprising the detection reagent for creatine kinase or at least one isoenzyme of creatine kinase according to any one of claims 1 to 13.

15. A detection method for creatine kinase or at least one isoenzyme of creatine kinase, the method comprising mixing and incubating a sample with the first reagent defined in any one of claims 1 to 10, adding the second reagent defined in claim 11 or 12 and mixing, and then detecting the absorbance A1 of the mixture at a first time point and detecting the absorbance A2 of the mixture at a second time point.

16. The detection method according to claim 15, wherein the absorbance is detected at a main wavelength of 340 nm and a secondary wavelength of 546 nm.

17. The detection method according to claim 15, wherein the sample is a mammalian blood sample.

18. The detection method according to claim 17, wherein the sample is a human blood sample.

19. The detection method according to claim 17, wherein the sample is a human serum sample.

20. Use of the compound shown in formula I as an activator protector in the enzyme-coupled detection reagent for creatine kinase or at least one isoenzyme of creatine kinase, wherein the activator is an activator of creatine kinase or at least one isoenzyme of creatine kinase containing a thiol group, wherein the compound shown in formula I is: wherein, A - is -SO 3 - or -COO - ; M + is a monovalent metal ion; R 1 and R 2 are each independently selected from H, -NQ 1 Q 2 and -R-NQ 1 Q 2 provided that R 1 and R 2 are not both H at the same time, where Q 1 and Q 2 are each independently selected from H, methyl and ethyl, R is a substituted or unsubstituted group selected from C1-C6 alkyl, C1-C6 alkoxy, C1-C6 acyl and -(OCH 2 CH 2 ) n - in which n is an integer of 2 or 3, and the substitution is substituted by 1-3 groups selected from -OH, -NH 2 and -COOH.

21. Use of the compound shown in formula I and the compound shown in formula II as activator protectors in the enzyme-coupled detection reagent for creatine kinase or at least one isoenzyme of creatine kinase, wherein the activator is an activator of creatine kinase or at least one isoenzyme of creatine kinase containing a thiol group, wherein the compound shown in formula I is: wherein, A - is - SO 3 - or - COO - ; M + is a monovalent metal ion; R 1 and R 2 each independently selected from H, -NQ 1 Q 2 and -R-NQ 1 Q 2 provided that R 1 and R 2 are not simultaneously H, where Q 1 and Q 2 each independently selected from H, methyl and ethyl, R is a substituted or unsubstituted group selected from C1-C6 alkyl, C1-C6 alkoxy, C1-C6 acyl and -(OCH 2 CH 2 ) n -, n is an integer of 2 or 3, and the substitution is substituted by 1-3 groups selected from -OH, -NH 2 and -COOH; The compound shown in formula II is: CH 3 -(CH 2 ) p -(OCH 2 CH 2 ) q -OH (II) wherein, p is an integer from 7 to 18, and q is an integer from 5 to 10.

22. Use according to claim 20 or 21, wherein the compound of formula I is selected from the group consisting of p-aminobenzenesulfonate, m-aminobenzenesulfonate, 2,4-diaminobenzenesulfonate, N-methyl-p-aminobenzenesulfonate, N,N-dimethyl-p-aminobenzenesulfonate, N,N-diethyl-p-aminobenzenesulfonate, p-aminomethylbenzenesulfonate, p-aminoethylbenzenesulfonate, p-aminopropylbenzenesulfonate, 4-(3-amino-2-methyl)-propylbenzenesulfonate, 4-(4-amino-2-methyl)butylbenzenesulfonate, p-aminohexylbenzenesulfonate, and 2-amino-4-aminoethylbenzenesulfonate.

23. Use according to claim 21, wherein the compound of formula II is lauryl alcohol ethylene glycol ether.

24. Use according to claim 20 or 21, wherein the activator is selected from the group consisting of N-acetylcysteine, reduced glutathione, L-cysteine, dithiothreitol, N-acetyl-L-cysteine, dithioerythritol, mercaptoethanol, and mercaptoacetic acid.

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