An ischemia-modified albumin calibrator and its application

Abstract

The invention discloses a raw material of the calibration product, which includes a buffer, a chelating agent, a surfactant, a stabilizer and a preservative, wherein the chelating agent is any one or more of diethylenetriamine pentaacetic acid pentasodium, hydroxyethylethylenediaminetriacetic acid trisodium salt, sodium tartrate and calcium citrate. The calibration product of the invention has good stability, can be stored for a long time, does not have the matrix effect of the calibration product prepared by EDTA and the IMA reagent that has been on the market during calibration, and the detection result is more accurate, and has broad application prospects and huge market value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an ischemia-modified albumin calibrator and an application thereof. Background Art

[0002] Ischemia Modified Albumin (IMA) is one of the myocardial ischemia markers that is converted from human serum albumin (HSA) in the case of myocardial ischemia. The amino terminal sequence of human serum albumin is unique to humans and is a binding site for transition metal ions such as cobalt, copper and nickel. Normal albumin has a strong ability to bind to transition metals. During myocardial ischemia, reduced blood flow leads to insufficient tissue oxygen supply and a decrease in pH, forming superoxide radicals and hydrogen peroxide. In the presence of metals such as copper or iron, hydrogen peroxide undergoes a Fenton reaction to form hydroxyl radicals. Hydroxyl radicals are highly active and can change the amino acid at the amino terminal of the albumin molecule. Albumin with a changed amino terminal cannot bind to metal ions. This albumin that cannot bind to metal ions produced during ischemia is called ischemia modified albumin.

[0003] Cardiovascular disease is a leading cause of death worldwide, claiming approximately 17.9 million lives each year. Acute coronary syndrome (ACS) is a common cardiovascular disease in clinical practice. It refers to an acute ischemic syndrome of the heart caused by thrombosis caused by rupture or erosion of unstable atherosclerotic plaques in the coronary arteries. The manifestations of myocardial ischemia are varied, such as chest pain, upper abdominal discomfort, dyspnea, nausea and vomiting. However, these symptoms may be subtle and difficult to identify. Due to the variety of symptoms, rapid onset and high mortality rate, early diagnosis of patients with acute myocardial infarction is crucial.

[0004] In recent years, ischemic markers have developed rapidly. Traditional myocardial ischemic markers, such as myoglobin (Myo), troponin (cTn), and creatine kinase isoenzyme MB (CK-MB), have good specificity and sensitivity, but they only show a significant increase about 3-6 hours after myocardial cell damage occurs, at which time the patient has suffered irreversible pathological damage. Ischemia-modified albumin is the first biochemical marker approved by the U.S. Food and Drug Administration (FDA) for detecting myocardial ischemia. Its sensitivity for detecting myocardial ischemia in ACS patients is twice that of electrocardiogram (ECG) and four times that of troponin (cTn). Unlike traditional myocardial ischemic markers, the blood concentration of IMA can increase 5-10 minutes after the onset of myocardial ischemia, reach a peak value in 2-6 hours, and basically return to normal in 12-24 hours. Therefore, IMA can be used to evaluate early reversible myocardial ischemia and has important clinical significance for the early diagnosis of ACS, guiding treatment, improving patient prognosis, and reducing mortality.

[0005] At present, the detection method of IMA is mainly the indirect method of albumin cobalt binding test (ACB method). 2+ The albumin in the serum sample exists in an active form. After adding cobalt chloride solution, Co 2+ It can bind to albumin, and the remaining free Co in the solution 2+ It reacts with dithiothreitol to form a reddish brown product. Since the IMA content is proportional to the color degree, the absorbance at 505nm can be detected to indirectly determine the IMA quantitatively. After calibration with specially prepared calibrants, the results can be directly reported in the form of IMA.

[0006] The results of IMA determination by the ACB indirect method are greatly affected by the stability of human serum albumin and ischemic modified albumin. The traditional method uses serum matrix and ischemic modified albumin as raw materials for preparing calibrators, but it has been shown that human serum albumin and ischemic modified albumin are very unstable in the serum matrix. Under storage conditions of 2-8°C, the measured value decreases by about 20% after being placed for one month. Therefore, it also brings great pressure on storage and cold chain transportation. The Chinese patent with publication number CN109557322A discloses a method for preparing calibrators using EDTA chelating agent. Compared with the serum calibrators of the traditional method, it is more efficient, stable and simple, and at the same time reduces the cost. However, this calibrator prepared with EDTA will have a matrix effect when calibrated with the IMA reagent that has been on the market, which seriously affects the test results. Summary of the invention

[0007] In view of the deficiencies in the prior art and actual needs, the present invention provides an ischemic modified albumin calibrator and an application thereof, and improves the formula of the calibrator in the prior art. The obtained calibrator has good stability and can be stored for a long time. There is no matrix effect when calibrating the calibrator prepared with EDTA and the IMA reagent already on the market. The detection result is more accurate, and the calibrator has broad application prospects and huge market value.

[0008] The invention provides an ischemia modified albumin calibrator. The raw materials of the calibrator include a buffer, a chelating agent, a surfactant, a stabilizer and a preservative. The chelating agent is any one or more of diethylenetriamine pentaacetic acid pentasodium, hydroxyethylethylenediaminetriacetic acid trisodium salt, sodium tartrate and calcium citrate.

[0009] Furthermore, the surfactant is any one or more of nonylphenol polyoxyethylene ether, Emulgen 1118s-70, polystyrene cross-linked divinylbenzene, polyoxyethylene lauryl ether, Triton X-405, Tween 80, polyethylene glycol 4000, polyethylene glycol 6000, and polyethylene glycol 8000.

[0010] Furthermore, the stabilizer is any one or more of ethyl linolenate, methyl oleate, sodium sulfate, calcium chloride, methyl-β-cyclodextrin, 2-hydroxypropyl-α-cyclodextrin, bovine serum albumin, dimethyl sulfoxide, and glycerol.

[0011] Furthermore, the buffer is any one of piperazine-N,N'-bis(2-ethanesulfonic acid) buffer, piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) buffer, 2-morpholineethanesulfonic acid buffer, 3-morpholinepropanesulfonic acid buffer, tris(hydroxymethyl)aminomethane buffer, and N,N-dihydroxyethylglycine buffer, and the pH value of the buffer is 7.0-9.0.

[0012] Furthermore, the preservative is any one or more of sodium azide, sodium p-hydroxybenzoate, Proclin 200, Proclin 300, Proclin 950, and potassium sorbate.

[0013] Furthermore, the raw materials of the calibration substance include 1-500 mM buffer, 0.05-50 mM chelating agent, 0.01-2% surfactant, 0.1%-10% stabilizer and 0.05%-1% preservative.

[0014] Furthermore, the chelating agent is pentasodium diethylenetriamine pentaacetate.

[0015] Furthermore, the calibrators are level reagent 1, level reagent 2 and level reagent 3, and the level reagent 1, level reagent 2 and level reagent 3 have different concentrations of chelating agents.

[0016] The present invention also provides a method for detecting ischemia-modified albumin, wherein the method uses a chelating agent as a main raw material of a calibration substance, wherein the chelating agent is any one or more of diethylenetriamine pentaacetic acid pentasodium, hydroxyethylethylenediaminetriacetic acid trisodium salt, sodium tartrate and calcium citrate.

[0017] The present invention also provides a kit, which comprises the above-mentioned standard product;

[0018] Preferably, the kit further comprises reagent R1 and reagent R2;

[0019] Wherein, the components of the reagent R1 include cobalt chloride;

[0020] The components of the reagent R2 include dithiothreitol.

[0021] The present invention also provides a use of the above-mentioned calibrator and / or the above-mentioned kit for detecting ischemia-modified albumin.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The invention provides an ischemic modified albumin calibrator, wherein a chelating agent is any one or more of diethylenetriamine pentaacetic acid pentasodium, hydroxyethylethylenediaminetriacetic acid trisodium salt, sodium tartrate and calcium citrate, and a surfactant and a stabilizer are added. The obtained calibrator has better stability than a traditional serum matrix calibrator and can be stably stored in long-distance cold chain transportation. On the other hand, compared with an EDTA chelating agent calibrator, the calibrator has no matrix effect, thereby avoiding the influence of the matrix effect on the test result. The calibrator is used to detect ischemic modified albumin, and the detection sensitivity is high, the specificity is strong, and the cost is low, and accurate detection of ischemic modified albumin (IMA) can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0025] Figure 1 It is a linear regression graph of the test results of comparative example 1 and 20 fresh clinical samples;

[0026] Figure 2It is a linear regression graph of the test results of Example 1 and 20 fresh clinical samples. DETAILED DESCRIPTION

[0027] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The present invention is specifically introduced below in conjunction with the specific embodiments.

[0028] The embodiment of the present invention provides an ischemia modified albumin calibrator, wherein the raw materials of the calibrator include a buffer, a chelating agent, a surfactant, a stabilizer and a preservative, wherein the chelating agent is any one or more of diethylenetriamine pentaacetic acid pentasodium, hydroxyethylethylenediaminetriacetic acid trisodium salt, sodium tartrate and calcium citrate.

[0029] Among them, the chelating agent in the calibrator is the above-mentioned substance, and a surfactant and a stabilizer are added. On the one hand, the obtained calibrator has better stability than the traditional serum matrix calibrator, and it can be stably stored during long-distance cold chain transportation. On the other hand, compared with the EDTA chelating agent calibrator, it has no matrix effect, avoiding the influence of the matrix effect on the test results. The use of this calibrator to detect ischemia-modified albumin has high detection sensitivity, strong specificity, and low cost, and can achieve accurate detection of ischemia-modified albumin (IMA).

[0030] Specifically, the surfactant is any one or more of nonylphenol polyoxyethylene ether, Emulgen 1118s-70, polystyrene cross-linked divinylbenzene, polyoxyethylene lauryl ether, Triton X-405, Tween 80, polyethylene glycol 4000, polyethylene glycol 6000, and polyethylene glycol 8000.

[0031] Specifically, the stabilizer is any one or more of ethyl linolenate, methyl oleate, sodium sulfate, calcium chloride, methyl-β-cyclodextrin, 2-hydroxypropyl-α-cyclodextrin, bovine serum albumin, dimethyl sulfoxide, and glycerol.

[0032] Specifically, the buffer is any one of piperazine-N,N'-bis(2-ethanesulfonic acid) buffer, piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) buffer, 2-morpholineethanesulfonic acid buffer, 3-morpholinepropanesulfonic acid buffer, tris(hydroxymethyl)aminomethane buffer, and N,N-dihydroxyethylglycine buffer, and the pH value of the buffer is 7.0-9.0.

[0033] Specifically, the preservative is any one or more of sodium azide, sodium p-hydroxybenzoate, Proclin 200, Proclin 300, Proclin 950, and potassium sorbate.

[0034] Specifically, the raw materials of the calibration substance include 1-500 mM buffer, 0.05-50 mM chelating agent, 0.01-2% surfactant, 0.1%-10% stabilizer and 0.05%-1% preservative.

[0035] Preferably, the chelating agent is pentasodium diethylenetriamine pentaacetate.

[0036] Specifically, the calibrators are level reagent 1, level reagent 2 and level reagent 3, and the level reagent 1, level reagent 2 and level reagent 3 have different chelating agent concentrations.

[0037] An embodiment of the present invention also provides a method for detecting ischemia-modified albumin, wherein the method uses a chelating agent as the main raw material of a calibrant, and the chelating agent is any one or more of diethylenetriamine pentaacetic acid pentasodium, hydroxyethylethylenediaminetriacetic acid trisodium salt, sodium tartrate and calcium citrate.

[0038] The embodiment of the present invention further provides a kit, the kit comprising the above-mentioned standard;

[0039] Preferably, the kit further comprises reagent R1 and reagent R2;

[0040] Wherein, the components of the reagent R1 include cobalt chloride;

[0041] The components of the reagent R2 include dithiothreitol.

[0042] The embodiment of the present invention also provides a use of the above-mentioned calibrator and / or the above-mentioned kit for detecting ischemia-modified albumin.

[0043] The following is explained in conjunction with specific embodiments:

[0044] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified, and the techniques not described in detail are all performed according to standard methods well known to those skilled in the art.

[0045] Example 1

[0046] The formula of calibrator level reagent 1 is shown in Table 1.

[0047] Table 1

[0048] Serial number Calibrator Level Reagent 1 concentration 1 3-Morpholinepropanesulfonic acid 5mM 2 Pentasodium diethylenetriaminepentaacetate 2mM 3 Sodium sulfate 2mM 4 Ethyl linolenate 0.50％ 5 Sodium Azide 0.5％ 6 Nonylphenol polyoxyethylene ether 0.35％

[0049] The formula of calibrator level reagent 2 is shown in Table 2:

[0050] Table 2

[0051]

[0052]

[0053] The formula of calibrator level reagent 3 is shown in Table 3:

[0054] Table 3

[0055] Serial number Calibrator Level Reagent 3 concentration 1 3-Morpholinepropanesulfonic acid 5mM 2 Pentasodium diethylenetriaminepentaacetate 2.7mM 3 Sodium sulfate 2mM 4 Ethyl linolenate 0.50％ 5 Sodium Azide 0.5％ 6 Nonylphenol polyoxyethylene ether 0.35％

[0056] The preparation method of the calibrator level reagent 1, level reagent 2 and level reagent 3 of Example 1 is to measure the buffer solution (3-morpholinepropanesulfonic acid) according to the concentration of each raw material in the formula in Table 1, Table 2 and Table 3, weigh each raw material, add the raw materials to the buffer solution in sequence, add purified water after mixing, and adjust the pH value to 8.5, thereby obtaining the calibrator level reagent 1, level reagent 2 and level reagent 3 of Example 1.

[0057] Comparative Example 1

[0058] The formula of calibrator level reagent 1 is shown in Table 4:

[0059] Table 4

[0060] Serial number Calibrator Level Reagent 1 concentration 1 3-Morpholinepropanesulfonic acid 5mM 2 <![CDATA[Sodium edetate dihydrate]]> 3.6mM 3 NaCl 2mM 4 glycerin 0.50％ 5 Sodium Azide (preservative) 0.5％

[0061] The formula of calibrator level reagent 2 is shown in Table 5:

[0062] Table 5

[0063] Serial number Calibrator Level Reagent 2 concentration 1 3-Morpholinepropanesulfonic acid 5mM 2 <![CDATA[Sodium EDTA dihydrate]]> 11.5mM 3 NaCl 2mM 4 glycerin 0.50％ 5 Sodium Azide (preservative) 0.5％

[0064] The formula of calibrator level reagent 3 is shown in Table 6:

[0065] Table 6

[0066] Serial number Calibrator Level Reagent 3 concentration 1 3-Morpholinepropanesulfonic acid 5mM 2 Disodium EDTA·2H2O 4.8mM 3 NaCl 2mM 4 glycerin 0.50％ 5 Sodium Azide (preservative) 0.5％

[0067] The preparation method of the calibration level reagent 1, level reagent 2 and level reagent 3 of comparative example 1 is to measure the buffer solution (3-morpholinepropanesulfonic acid) according to the concentration of each raw material in the formula in Table 4, Table 5 and Table 6, weigh each raw material, add the raw materials to the buffer solution in sequence, add purified water after mixing, and adjust the pH value to 8.5, so as to obtain the calibration level reagent 1, level reagent 2 and level reagent 3 of comparative example 1.

[0068] Matrix Effect Assessment:

[0069] The detection system consisting of IMA test kits and matching calibration quality control from two different listed companies was named the evaluation method and the comparison method respectively. The calibration level reagents 1, 2 and 3 of Example 1, the calibration level reagents 1, 2 and 3 of the comparative example and 20 fresh clinical samples were randomly arranged, and all samples were measured using the evaluation method and the comparison method respectively, and the measurement was repeated 3 times, and each sample in each batch was measured once, and each batch was calibrated. The evaluation method and the comparison method were carried out simultaneously.

[0070] Calculation method:

[0071]

[0072] Using the above equation, the mean value determined by the comparison method is taken as the x value, and the 95% confidence interval of the y value of each prepared sample is calculated. If the mean value determined by the evaluation method falls within this interval, it means that the prepared sample has no matrix effect on the evaluation method; if the mean value determined by the evaluation method is outside the 95% confidence interval, it means that the prepared sample has a matrix effect on the evaluation method.

[0073] The test results of comparative example 1 and 20 fresh clinical samples are shown in Table 7:

[0074] Table 7

[0075]

[0076]

[0077] The results in Table 7 were subjected to linear regression. Figure 1 .

[0078] The matrix effect was evaluated according to the above method, and the results are shown in Table 8:

[0079] Table 8

[0080]

[0081] The test results of Example 1 and 20 fresh clinical samples are shown in Table 9:

[0082] Table 9

[0083]

[0084]

[0085] The results in Table 9 were subjected to linear regression. Figure 2 .

[0086] The matrix effect was evaluated according to the above method. The results are shown in Table 10:

[0087] Table 10

[0088]

[0089] From the above table and Figure 1-2 The experimental results were analyzed and it was found that the three level reagents of comparative example 1 all had matrix effects, while the three level reagents prepared in embodiment 1 had no matrix effects, indicating that the calibrator prepared by the method of the present invention solved the problem of matrix effects of IMA detection reagents and EDTA chelating agent calibrators in the test process.

[0090] Stability Assessment:

[0091] Three batches of calibrators prepared in Example 1 were measured using a detection system consisting of an IMA kit of a listed company and a matching calibration quality control. The calibrators were stored at 2-8°C and -20°C, respectively. The concentrations were then measured once on the 1st day, 3rd day, 7th day, 14th day, 1 month, 2 months, 3 months, 4 months, 6 months, 9 months, and 12 months using the IMA kit detection system of the listed company. The results are shown in Tables 11 and 12.

[0092] Table 11

[0093]

[0094]

[0095] Table 12

[0096]

[0097] It can be seen from Tables 11 and 12 that the calibrants prepared in the examples can be stored for at least one year under storage conditions of 2-8°C and -20°C, and have strong stability, which solves the problem of stable maintenance during cold chain transportation and the accuracy of the calibrants in practical applications.

[0098] 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 and improvements made within the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An ischemic modified albumin calibrator, characterized in that The raw materials of the calibration product include 5 mM buffer, 0.05-50 mM chelating agent, 0.35% surfactant, 0.5% stabilizer and 0.5% preservative. The buffer is a 3-morpholinepropanesulfonic acid buffer with a pH value of 7.0-9.0, the chelating agent is pentasodium diethylenetriaminepentaacetate, the surfactant is nonylphenol polyoxyethylene ether, the stabilizer is ethyl linolenate, and the preservative is sodium azide.

2. The ischemic modified albumin calibrator according to claim 1, characterized in that The calibrators are level reagent 1, level reagent 2 and level reagent 3, and the level reagent 1, level reagent 2 and level reagent 3 have different concentrations of chelating agents.

3. A method for detecting ischemic modified albumin using the calibrator according to claim 1, characterized in that: The method adopts a chelating agent as the main raw material of a calibration substance, and the chelating agent is pentasodium diethylenetriamine pentaacetate.

4. A kit, characterized in that: The kit comprises a calibrator according to any one of claims 1 to 2; The kit also includes reagent R1 and reagent R2; Wherein, the components of the reagent R1 include cobalt chloride; The components of the reagent R2 include dithiothreitol.

5. Use of the calibrator according to any one of claims 1 to 2 and / or the kit according to claim 4 for detecting ischemia-modified albumin.

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