Cardiac troponin T calibrator diluent composition, diluent, calibrator and preparation method and application thereof

Abstract

The invention discloses a cardiac troponin T calibrator diluent composition, a diluent, a calibrator as well as a preparation method and application of the calibrator. The composition comprises a basic diluent component, a potassium salt, a casein hydrolysate and dodecyl trimethyl ammonium bromide, and the mass ratio of the potassium salt to the casein hydrolysate to the dodecyl trimethyl ammonium bromide is (5-39): (1-20): (0.5-3). According to the provided cardiac troponin T calibrator diluent composition and diluent, by optimizing the variety and concentration in the formula, the stability of cardiac troponin T in a liquid state and a freeze-drying state is remarkably improved, adsorption and degradation of cardiac troponin T in a glass container are effectively inhibited, the cardiac troponin T can be stabilized in a room-temperature glass bottle, and the application range of the cardiac troponin T calibrator diluent composition is widened. And after redissolution, good concentration consistency is still maintained, and reliable stability guarantee is provided for preparation, freeze-drying and clinical detection of the cardiac troponin T calibrator.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of in vitro diagnostic detection, in particular to a calibrator diluent composition of cardiac troponin T, a diluent, a calibrator and a preparation method and application thereof. BACKGROUND

[0002] Cardiac troponin T (cTnT) has a molecular weight of about 37KD and is one of the constituent subunits of cardiac troponin. Under normal physiological conditions, cTnT in the blood is at a low concentration level. When myocardial injury occurs, cTnT is degraded from myocardial fibers into the blood, and its concentration increases slowly and stably, which is strongly correlated with the degree of myocardial injury, death risk and malignant cardiovascular events. In the fourth edition of the general definition of myocardial infarction published in 2018, cTnT is used as the preferred biomarker for diagnosing acute myocardial infarction and risk stratification. In addition, cTnT is also the main basis for diagnosing, risk stratification and prognosis of acute coronary syndrome, and high-sensitivity cTnT detection is recommended for routine clinical application.

[0003] For accurate diagnosis, calibrators and quality control products are essential components of in vitro immunodiagnostic reagents. Therefore, the stability of cTnT calibrators is an important guarantee for the accurate detection of cTnT detection reagents. Due to factors such as temperature, enzymatic degradation, microbial contamination, the stability of liquid calibrators is often not as good as that of lyophilized forms. Considering thermal stability, sealing, mechanical strength and cost control, glass is often the best material for lyophilization containers. Due to its own characteristics, cTnT is prone to degradation in liquid solution, and glass material has adsorption effect on cTnT, which causes the cTnT calibrator to gradually lose value after preparation in a glass bottle, which is not conducive to the preparation of lyophilized products. On the other hand, cTnT calibrators still need to contact glass bottles after reconstitution, which causes the concentration of cTnT in the reconstituted lyophilized product to decrease significantly during the detection period, resulting in a large deviation in the detection results. SUMMARY

[0004] Therefore, it is necessary to provide a calibrator diluent composition of cardiac troponin T, a diluent, a calibrator and a preparation method and application thereof.

[0005] The first aspect of the present application provides a calibrator diluent composition of cardiac troponin T, which comprises a basic diluent component, a potassium salt, casein hydrolysate and dodecyl trimethyl ammonium bromide, wherein the mass ratio of the potassium salt, the casein hydrolysate and the dodecyl trimethyl ammonium bromide is (5-39):(1-20):(0.5-3).

[0006] In some embodiments, the mass ratio of the potassium salt, the casein hydrolysate, and the dodecyltrimethylammonium bromide is (19~26):(2~10):(0.5~1).

[0007] In some embodiments, the base diluent component comprises a buffer, inorganic salt ions, a protein protective agent, and a surfactant.

[0008] In some embodiments, the cardiac troponin T calibrator diluent composition satisfies one or more of the following characteristics:

[0009] The buffer comprises one or more of a Tris buffer, a PBS buffer, and a HEPES buffer;

[0010] The inorganic salt ions comprise a sodium salt;

[0011] The protein protective agent in the base diluent component comprises bovine serum albumin;

[0012] The surfactant in the base diluent component comprises Tween 20;

[0013] The base diluent component comprises a buffer, a sodium salt, a protein protective agent, and a surfactant;

[0014] The base diluent component further comprises a preservative;

[0015] The cardiac troponin T calibrator composition comprises a buffer, a sodium salt, a potassium salt, a protein protective agent, a surfactant, and a preservative, wherein the protein protective agent in the cardiac troponin T calibrator composition comprises one or more of bovine serum albumin and casein hydrolysate, and the surfactant in the cardiac troponin T calibrator composition comprises one or more of Tween 20 and dodecyltrimethylammonium bromide.

[0016] A second aspect of the present application provides a cardiac troponin T calibrator diluent, comprising an aqueous solvent and the cardiac troponin T calibrator diluent composition of the first aspect of the present application;

[0017] Optionally, the aqueous solvent comprises water.

[0018] In some embodiments, the concentration of the potassium salt in the cardiac troponin T calibrator diluent is 125 mM~1000 mM;

[0019] The concentration of the casein hydrolysate in the cardiac troponin T calibrator diluent is 0.1% (w / v)~2.0% (w / v);

[0020] The concentration of the dodecyl trimethyl ammonium bromide in the troponin T calibrator diluent is 0.05% (w / v) to 0.3% (w / v).

[0021] In some embodiments, the troponin T calibrator diluent comprises the following components: 40 mM to 100 mM of a buffer, 0.5% (w / v) to 1.0% (w / v) of a sodium salt, 1% (w / v) to 5% (w / v) of bovine serum albumin, 0.04% (w / v) to 0.06% (w / v) of Tween 20, 125 mM to 1000 mM of a potassium salt, 0.1% (w / v) to 2.0% (w / v) of casein hydrolysate, and 0.05% (w / v) to 0.3% (w / v) of dodecyl trimethyl ammonium bromide, and the pH of the troponin T calibrator diluent is 7.4 ± 0.2.

[0022] In some embodiments, the troponin T calibrator diluent satisfies one or more of the following characteristics:

[0023] The buffer comprises one or more of a Tris buffer, a PBS buffer, and a HEPES buffer;

[0024] The sodium salt comprises sodium chloride, and optionally is sodium chloride;

[0025] The potassium salt comprises potassium chloride, and optionally is potassium chloride;

[0026] The troponin T calibrator diluent further comprises a preservative, and the concentration of the preservative in the troponin T calibrator diluent is 0 to 0.1% (w / v);

[0027] The troponin T calibrator diluent comprises water and the following components at the following amounts: 50 mM of a Tris buffer, 0.9% (w / v) of sodium chloride, 2% (w / v) of bovine serum albumin, 0.05% (w / v) of Tween-20, 0.05% (w / v) of ProClin 300, 500 mM of potassium chloride, 0.5% (w / v) of casein hydrolysate, and 0.05% (w / v) of dodecyl trimethyl ammonium bromide, and the pH of the troponin T calibrator diluent is 7.4 ± 0.1; and optionally, the water is the balance of the water.

[0028] A third aspect of the present application provides use of the troponin T calibrator diluent composition of the first aspect of the present application or the troponin T calibrator diluent of the second aspect of the present application in the preparation of a troponin T calibrator.

[0029] The fourth aspect of the present application provides a method for preparing a calibrator of cardiac troponin T, comprising mixing the cardiac troponin T calibrator diluent of the first aspect of the present application with cardiac troponin T to obtain the calibrator of cardiac troponin T; and optionally, further comprising lyophilizing after mixing to obtain a lyophilized calibrator of cardiac troponin T.

[0030] In some embodiments, the lyophilizing step comprises pre-freezing and drying;

[0031] Optionally, the pre-freezing conditions comprise pre-freezing at a temperature of -8℃ to -12℃ for 90 min to 150 min; pre-freezing at a temperature of -48℃ to -52℃ for 60 min to 120 min; pre-freezing at a temperature of -18℃ to -22℃ for 60 min to 120 min; and pre-freezing at a temperature of -54℃ to -56℃ for 3 h to 4 h.

[0032] Optionally, the drying comprises primary drying and secondary drying, and the primary drying conditions comprise a vacuum degree of 0.25 mbar to 0.35 mbar at a temperature of -54℃ to -56℃ for 8 min to 12 min; a vacuum degree of 0.08 mbar to 0.12 mbar at a temperature of -28℃ to -32℃ for 11 h to 13 h; and warming to a temperature of -8℃ to -12℃ for 2 h to 4 h.

[0033] The secondary drying conditions comprise a vacuum degree of 0.004 mbar to 0.006 mbar at a temperature of 8℃ to 12℃ for 6 h to 8 h.

[0034] The fifth aspect of the present application provides an immunological detection kit for cardiac troponin T, comprising the calibrator of cardiac troponin T of the fourth aspect of the present application.

[0035] The sixth aspect of the present application provides the use of the calibrator of cardiac troponin T of the fourth aspect of the present application or the immunological detection kit for cardiac troponin T of the fifth aspect of the present application in detecting cardiac troponin T.

[0036] The use comprises the step of contacting a detection sample with a glass container; wherein the detection sample comprises a sample to be detected and the calibrator of cardiac troponin T.

[0037] Optionally, the detection of cardiac troponin T comprises the steps of: detecting optical signals in the sample to be detected and the calibrator of cardiac troponin T respectively by using chemiluminescence; and comparing and analyzing the detection results of the sample to be detected and the calibrator of cardiac troponin T to obtain information about the content of cardiac troponin T.

[0038] The aforementioned cardiac troponin T calibrator diluent composition and diluent significantly improve the stability of cardiac troponin T in liquid and lyophilized states by optimizing the types and concentrations in the formula, effectively inhibit the adsorption and degradation of cardiac troponin T in glass containers, and enable the cardiac troponin T to be stably stored in a glass bottle at room temperature for at least 8 hours, and still maintain good concentration consistency after reconstitution, thereby providing reliable stability guarantee for the preparation, lyophilization and clinical detection of the cardiac troponin T calibrator. DETAILED DESCRIPTION

[0039] The present application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0040] Unless otherwise defined, 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 belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0041] In the present application, "optionally", "optional", "option" means optional, that is, selected from "yes" or "no" two parallel schemes. If there are multiple "options" in a technical solution, unless otherwise specified, and there is no contradiction or mutual restriction, each "option" is independent.

[0042] In the present application, "preferably", "better", "better", "as appropriate" only describe the effect of the embodiment or embodiment, and it should be understood that it does not constitute a limitation on the scope of protection of the present application.

[0043] The terms "have", "contain", "include" and "comprise" used in the present application are synonymous terms, which are inclusive or open, and do not exclude additional, unmentioned members or features. Members or features, such as materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features, such as actions, conditions, timing, state, etc.

[0044] In the present application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of listed contents, and also include open technical features or technical solutions containing listed contents.

[0045] In the present application, the unit related to the data range, if only the unit is added after the right end point, it means that the units of the left end point and the right end point are the same.

[0046] In the present application, unless otherwise explicitly stated, the execution of the steps involved in the method flow does not have strict order limitation, and can be executed in other order than described. Moreover, any step can include multiple sub-steps or multiple stages, which do not necessarily be executed at the same time, but can be executed at different times, and the execution order is not necessarily sequential, but can be executed alternately or simultaneously with other steps or sub-steps or stages of other steps.

[0047] In the present application, the exemplary description involving "in some embodiments" or "in one embodiment" or the like can cover but not limited to the following meanings: these schemes can be combined with other schemes in a suitable manner to form new technical schemes.

[0048] In the present application, the terms "first", "second", "third", "fourth" and the like in "first aspect", "second aspect", "third aspect", "fourth aspect" and the like are only for descriptive purposes and should not be construed as indicating or implying relative importance or quantity, nor should it be construed as implicitly indicating the importance or quantity of the technical features indicated. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration description, and should be understood as not constituting a closed limitation on the quantity.

[0049] In the present application, with respect to the numerical interval (i.e. numerical range), if no special instructions are given, the distribution of the optional values in the numerical interval is considered to be continuous, and includes the two numerical end points (i.e. the minimum value and the maximum value) of the numerical interval, and every value between the two numerical end points. If no special instructions are given, when the numerical interval only points to the integers in the numerical interval, including the two end point integers of the numerical range and every integer between the two end points, it is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe a feature or property, these numerical ranges can be combined. In other words, unless otherwise indicated, the numerical range disclosed herein should be understood to include any and all sub-ranges therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. The "numerical interval" allows a broad interpretation including percentage interval, ratio interval, value interval, etc.

[0050] In the present application, the term "room temperature" or "ambient temperature" generally refers to 4°C to 35°C, for example 20°C ± 5°C. In some embodiments of the present application, "room temperature" or "ambient temperature" refers to 10°C to 30°C.

[0051] In the present application, w / v means mass percentage. In some embodiments of the present application, w is measured in g, and v is measured in mL.

[0052] cTnT is prone to degradation in liquid solution due to its own characteristics, and glass materials have adsorption effect on it, leading to gradual drop in cTnT calibration product after preparation in glass bottles, which is not conducive to the preparation of freeze-dried products. On the other hand, cTnT calibration product still needs to contact glass bottles after freeze-drying and reconstitution, so that the concentration of cTnT in the freeze-dried product after reconstitution and before detection decreases significantly, resulting in a large deviation in the detection results.

[0053] Based on this, the embodiments of the present application at least provide a calibrator diluent composition of cardiac troponin T, a diluent, a calibrator, and a preparation method and application thereof.

[0054] The calibrator diluent of one embodiment comprises suitable buffer pairs (to maintain the pH of the solution), salt concentration (to maintain the ionic strength), protein protectant (to resist external damage), surfactant (to help dissolution), preservative (to inhibit bacteria), and the like.

[0055] In some embodiments, the present application provides a basic diluent formula: pH 7.4, 50 mM Tris, 0.9% NaCl, 2% BSA, 0.05% Tween 20, and 0.05% ProClin 300. NaCl is commonly used as a salt in the diluent, which can provide a suitable ionic strength and osmotic pressure for the protein solution, but it has a strong chaotropic effect, which can destroy the protein hydration layer. In contrast, KCl is a weak chaotropic ion, and it has a higher affinity for the carboxyl groups in the acidic amino acid residues of the protein, which can maintain the natural conformation of the protein. Casein hydrolysate is a mixture of short peptides and amino acids produced by enzymatic or acid hydrolysis of bovine casein. It is rich in hydrophilic amino acids and hydrophobic fragments, which can competitively bind to the surface hydrophobic region, reduce the aggregation or denaturation of the target protein (cTnT) caused by hydrophobic interaction, and at the same time, the hydrolyzed peptide fragments can be preferentially adsorbed on the surface of the container (such as glass, plastic), reducing the loss of cTnT caused by non-specific adsorption; dodecyltrimethylammonium bromide (DTAB) is a cationic surfactant that adsorbs to the negatively charged glass surface through electrostatic interaction, producing a charge shielding effect, reducing the electrostatic adsorption of cTnT to the glass bottle wall, thereby reducing protein loss. KCl, casein hydrolysate, and DTAB work synergistically through conformation maintenance + competitive adsorption + charge shielding, which not only ensures the stability of cTnT in solution, but also reduces the adsorption of glass materials to it. If one component is missing, it will have an adverse effect on the stability of the cTnT calibrator.

[0056] In the present application, the term "chaotropicity" refers to the ability of a substance to disrupt the ordered structure of biological macromolecules, manifested as disruption of the hydrogen-bond network of water molecules, weakening of the hydrophobic interactions within proteins, and promotion of the unfolded conformation.

[0057] In a first aspect of the present application, a cardiac troponin T calibrator diluent composition is provided, comprising a base diluent component, a potassium salt, a casein hydrolysate, and a dodecyltrimethylammonium bromide, wherein the mass ratio of the potassium salt, the casein hydrolysate, and the dodecyltrimethylammonium bromide is (5-39):(1-20):(0.5-3).

[0058] In some embodiments, the mass ratio of the potassium salt, the casein hydrolysate, and the dodecyltrimethylammonium bromide is (19-26):(2-10):(0.5-1). Without limitation, the mass ratio of the potassium salt, the casein hydrolysate, and the dodecyltrimethylammonium bromide can be, but is not limited to, 19:2:0.5, 22:2:0.5, 24:2:0.5, 26:2:0.5, 19:6:0.5, 22:6:0.5, 24:6:0.5, 26:6:0.5, 19:10:0.5, 22:10:0.5, 24:10:0.5, 26:10:0.5, 19:2:1, 22:2:1, 24:2:1, 26:2:1, 19:6:1, 22:6:1, 24:6:1, 26:6:1, 19:10:1, 22:10:1, 24:10:1, 26:10:1, or a ratio or range between any two of the above ratios.

[0059] In some embodiments, the base diluent component can be a diluent component conventional in the art, for example including but not limited to a buffer, an inorganic salt ion, a protein protective agent, and a surfactant.

[0060] In some embodiments, the buffer includes but is not limited to one or more of a Tris buffer, a PBS buffer, and a HEPES buffer.

[0061] In some embodiments, the inorganic salt ion in the base diluent component includes a sodium ion.

[0062] In some embodiments, the source of the inorganic salt ion in the base diluent component includes but is not limited to a sodium salt.

[0063] In some embodiments, the base diluent component can be a diluent component conventional in the art, for example including but not limited to a buffer, an inorganic salt, a protein protective agent, and a surfactant. Optionally, the inorganic salt in the base diluent component includes a sodium salt.

[0064] In some embodiments, the protein protective agent in the base diluent component includes but is not limited to bovine serum albumin.

[0065] In some embodiments, the surfactant in the base diluent component includes, but is not limited to, Tween 20.

[0066] In some embodiments, the base diluent component further includes a preservative, such as, but not limited to, ProClin 300.

[0067] In some embodiments, the troponin T calibrator composition includes a buffer, a sodium salt, a potassium salt, a protein protectant, a surfactant, and a preservative, wherein the protein protectant in the troponin T calibrator composition includes one or more of bovine serum albumin and casein hydrolysate, and the surfactant in the troponin T calibrator composition includes one or more of Tween 20 and dodecyltrimethylammonium bromide.

[0068] In a second aspect of the present application, a troponin T calibrator diluent is provided, including an aqueous solvent and the above-mentioned troponin T calibrator diluent composition, and further, the aqueous solvent can be water.

[0069] In some embodiments, the potassium salt has a concentration in the troponin T calibrator diluent of 125 mM to 1000 mM. Without limitation, the potassium salt can have a concentration in the troponin T calibrator diluent of, but not limited to, 125 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, or a value or a range between any two of the above-mentioned values.

[0070] In some embodiments, the potassium salt can be potassium chloride.

[0071] In some embodiments, the casein hydrolysate has a concentration in the troponin T calibrator diluent of 0.1% (w / v) to 2.0% (w / v). Without limitation, the casein hydrolysate can have a concentration in the troponin T calibrator diluent of, but not limited to, 0.1% (w / v), 0.5% (w / v), 1% (w / v), 1.5% (w / v), 2% (w / v), or a value or a range between any two of the above-mentioned values.

[0072] In some embodiments, the dodecyltrimethylammonium bromide has a concentration in the troponin T calibrator diluent of 0.05% (w / v) to 0.3% (w / v). Without limitation, the dodecyltrimethylammonium bromide can have a concentration in the troponin T calibrator diluent of, but not limited to, 0.05% (w / v), 0.1% (w / v), 0.15% (w / v), 0.2% (w / v), 0.25% (w / v), 0.3% (w / v), or a value or a range between any two of the above-mentioned values.

[0073] In some embodiments, the Troponin T calibrator diluent comprises the following components: 40 mM to 100 mM of a buffer, 0.5% (w / v) to 1.0% (w / v) of a sodium salt, 1% (w / v) to 5% (w / v) of bovine serum albumin, 0.04% (w / v) to 0.06% (w / v) of Tween 20, 125 mM to 1000 mM of a potassium salt, 0.1% (w / v) to 2.0% (w / v) of casein hydrolysate, and 0.05% (w / v) to 0.3% (w / v) of dodecyltrimethylammonium bromide, and the pH of the Troponin T calibrator diluent is 7.4 ± 0.2.

[0074] In some embodiments, the concentration of the buffer in the Troponin T calibrator diluent can be, but is not limited to, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or a value or a range between any two of the aforementioned values, for example, 40 mM to 100 mM.

[0075] In some embodiments, the concentration of the sodium salt in the Troponin T calibrator diluent can be, but is not limited to, 0.5% (w / v), 0.6% (w / v), 0.7% (w / v), 0.8% (w / v), 0.9% (w / v), 1.0% (w / v), or a value or a range between any two of the aforementioned values, for example, 1% (w / v) to 5% (w / v).

[0076] In some embodiments, the sodium salt comprises sodium chloride, and further can be sodium chloride.

[0077] In some embodiments, the concentration of the bovine serum albumin in the Troponin T calibrator diluent can be, but is not limited to, 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), or a value or a range between any two of the aforementioned values, for example, 1% (w / v) to 5% (w / v).

[0078] In some embodiments, the concentration of the Tween 20 in the Troponin T calibrator diluent can be, but is not limited to, 0.04% (w / v), 0.05% (w / v), 0.06% (w / v), or a value or a range between any two of the aforementioned values, for example, 0.04% (w / v) to 0.06% (w / v).

[0079] In some embodiments, the cardiac troponin T calibrator diluent further comprises a preservative, and the concentration of the preservative in the cardiac troponin T calibrator diluent is 0-0.1% (w / v). The preservative can be a conventional preservative in the art, for example but not limited to ProClin 300. Without limitation, the concentration of the preservative in the cardiac troponin T calibrator diluent can be, but is not limited to, 0, 0.01% (w / v), 0.05% (w / v), 0.1% (w / v), or a value or range between any two of the above values.

[0080] In some embodiments, the cardiac troponin T calibrator diluent comprises the following components: 50-55 mM of a buffer, 0.5-1.0% (w / v) of a sodium salt, 1-5% (w / v) of bovine serum albumin, 0.05-0.1% (w / v) of Tween 20, 0-0.1% (w / v) of a preservative, 125-1000 mM of a potassium salt, 0.1-2.0% (w / v) of casein hydrolysate, and 0.05-0.3% (w / v) of dodecyltrimethylammonium bromide, and the pH of the cardiac troponin T calibrator diluent is 7.4±0.2.

[0081] In some embodiments, the cardiac troponin T calibrator diluent comprises water and the following components: 50 mM of Tris buffer, 0.9% (w / v) of sodium chloride, 2% (w / v) of bovine serum albumin, 0.05% (w / v) of Tween-20, 0.05% (w / v) of ProClin 300, 500 mM of potassium chloride, 0.5% (w / v) of casein hydrolysate, and 0.05% (w / v) of dodecyltrimethylammonium bromide, and the pH of the cardiac troponin T calibrator diluent is 7.4±0.1; optionally, the water is the balance of water.

[0082] In the present application, by reasonably adjusting the types and concentrations of the components of the diluent formula, the components work synergistically to maintain the stability of the cTnT calibrator in a glass bottle. Compared with conventional diluents, the formula can enable the cTnT calibrator to be stably stored in a glass bottle for 8 h at room temperature, providing sufficient operation time for lyophilization, and making it possible for the lyophilized product to be stably used in the clinic.

[0083] In a third aspect of the present application, the use of the above-mentioned cardiac troponin T calibrator diluent composition or the above-mentioned cardiac troponin T calibrator diluent in the preparation of a cardiac troponin T calibrator is provided.

[0084] In a fourth aspect of the present application, a calibrator for cardiac troponin T is provided, which comprises the calibrator diluent for cardiac troponin T and cardiac troponin T; further, the concentration of cardiac troponin T in the calibrator can be within the measuring range of the reagent, for example, but not limited to, 1.5-10000 pg / mL. The concentration of cardiac troponin T can be, but is not limited to, 1.5 pg / mL, 5 pg / mL, 10 pg / mL, 100 pg / mL, 1000 pg / mL, 10000 pg / mL, or a value or range between any two of the above values.

[0085] In a fifth aspect of the present application, a method for preparing a calibrator for cardiac troponin T is provided, which comprises mixing the calibrator diluent for cardiac troponin T and cardiac troponin T to obtain the calibrator for cardiac troponin T.

[0086] In some embodiments, the calibrator diluent for cardiac troponin T is mixed with cardiac troponin T and then freeze-dried to obtain a freeze-dried calibrator for cardiac troponin T.

[0087] In some embodiments, the step of freeze-drying comprises pre-freezing and drying.

[0088] In some embodiments, the pre-freezing conditions comprise pre-freezing at a temperature of -8℃ to -12℃ for 90 min to 150 min; pre-freezing at a temperature of -48℃ to -52℃ for 60 min to 120 min; pre-freezing at a temperature of -18℃ to -22℃ for 60 min to 120 min; and pre-freezing at a temperature of -54℃ to -56℃ for 3 h to 4 h.

[0089] In some embodiments, the drying comprises primary drying and secondary drying, and the conditions of the primary drying comprise a vacuum degree of 0.25 mbar to 0.35 mbar at a temperature of -54℃ to -56℃ for 8 min to 12 min; a vacuum degree of 0.08 mbar to 0.12 mbar at a temperature of -28℃ to -32℃ for 11 h to 13 h; and warming to a temperature of -8℃ to -12℃ for 2 h to 4 h.

[0090] The conditions of the secondary drying comprise a vacuum degree of 0.004 mbar to 0.006 mbar at a temperature of 8℃ to 12℃ for 6 h to 8 h.

[0091] In a sixth aspect of the present application, an immunoassay kit for cardiac troponin T is provided, which comprises the calibrator for cardiac troponin T.

[0092] In some embodiments, the immunoassay kit for cardiac troponin T can be used for chemiluminescence detection.

[0093] In a seventh aspect of the present application, the use of the above-mentioned calibrator for cardiac troponin T or the above-mentioned immunological test kit for cardiac troponin T in detecting cardiac troponin T is provided.

[0094] In some embodiments, the above-mentioned use comprises the step of contacting the detection sample with the glass container; wherein the detection sample comprises the sample to be tested and the calibrator for cardiac troponin T.

[0095] In some embodiments, the detecting cardiac troponin T comprises the steps of: detecting the optical signal in the sample to be tested and the calibrator for cardiac troponin T respectively by chemiluminescence; and comparing the detection results of the sample to be tested and the calibrator for cardiac troponin T to obtain the content information of cardiac troponin T.

[0096] In some embodiments, the above-mentioned use is for non-disease diagnosis purposes, and can be used in scenarios such as scientific research verification, reagent performance evaluation, process optimization, or teaching demonstration.

[0097] In some embodiments, the above-mentioned use is for diagnosis purposes.

[0098] Some embodiments are provided below.

[0099] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods not specified in the following examples are preferably referred to the instructions given in the present application, and can also be performed according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the known experimental methods in the art.

[0100] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range if not otherwise specified. The acceptable deviations caused by the instrument testing accuracy or operation accuracy are allowed for the temperature and time parameters.

[0101] In the following examples, the percentage “%” refers to the mass / volume ratio if not otherwise specified.

[0102] The sources of the relevant materials in the following examples are shown in Table 1 below:

[0103] Table 1

[0104]

[0105] Example 1: Stability of the calibrator base diluent

[0106] Basic formula: pH 7.4, 50 mM Tris, 0.9% NaCl, 2% BSA, 0.05% Tween 20, 0.05% ProClin 300.

[0107] The cTnT antigens 1-6 were diluted to a specific concentration with the base diluent, and after testing the initial luminescence value (RLU) with the high-sensitivity troponin assay kit (chemiluminescence method), they were placed under the following conditions: cryotube room temperature, glass bottle room temperature, and after 4 h, the test was taken out and the deviation from the initial value was compared. The experimental results are shown in Table 2 below:

[0108] Table 2: Stability of cTnT antigens from different manufacturers in the base diluent

[0109]

[0110] From the above results, it can be seen that after 4 h of room temperature placement of all antigens in the base diluent, the RLU dropped by more than 10%, and after 4 h of room temperature placement in glass bottles, the drop was more than 60%, indicating that the cTnT antigen has poor stability in the base diluent, and the glass material has a further negative impact on its stability.

[0111] In summary, under the existing conditions, the stability of the cTnT antigen cannot meet the needs of production and clinical use. Through comparison, it is found that the drop of cTnT antigen 1 is the smallest, which can be used as the preferred antigen for subsequent optimization.

[0112] Example 2: Stability comparison of different calibrator diluent formulations

[0113] The calibrator diluent formulations in this example are as follows:

[0114] Base formulation: pH 7.4, 50 mM Tris, 0.9% NaCl, 2% BSA, 0.05% Tween 20, 0.05% ProClin 300;

[0115] Formulation 1: Base formulation + 500 mM KCl;

[0116] Formulation 2: Formulation 1 + 0.5% casein hydrolysate;

[0117] Formulation 3: Formulation 1 + 0.05% DTAB;

[0118] Formulation 4: Formulation 1 + 0.5% casein hydrolysate + 0.05% DTAB.

[0119] The preferred cTnT antigen in Example 1 was diluted to the same concentration with the above diluents, and after testing the initial value with the high-sensitivity troponin assay kit (chemiluminescence method), it was placed under the following conditions: cryotube room temperature, glass bottle room temperature, and at the set time point, the test was taken out and the deviation from the initial value was compared. The experimental results are shown in Table 3 below:

[0120] Table 3: Stability of preferred cTnT antigen in different formulation diluents

[0121]

[0122] According to the above results, compared with the base formulation, the freeze tube room temperature stability of formulation 1 showed obvious improvement, with a drop value within 10%, but its glass bottle room temperature stability did not show obvious improvement; Formulations 2 and 3 were based on formulation 1 and added casein hydrolysate and DTAB respectively, both of which maintained the freeze tube room temperature stability of formulation 1, while the glass bottle room temperature stability was obviously improved, but the drop value was still more than 10%, which did not meet the requirements of clinical application; Formulation 4 combined the performance of formulations 2 and 3, and added both casein hydrolysate and DTAB based on formulation 1, and its freeze tube and glass bottle room temperature 4 h drop values were both within 10%, meeting the requirements of clinical and production use.

[0123] In summary, the synergistic effect of KCl, casein hydrolysate and DTAB can ensure the overall stability of cTnT calibrators, making it possible to prepare them into lyophilized products.

[0124] Example 3: Key component concentration range study

[0125] The dilution formulation of the calibrator in this example is as follows:

[0126] Preferred formulation: pH 7.4, 50 mM Tris, 0.9% NaCl, 2% BSA, 0.05% Tween 20, 0.05% ProClin 300, 500 mM KCl, 0.5% casein hydrolysate, 0.05% DTAB;

[0127] After adjusting the concentration of the key components in the above preferred formulation diluent, dilute the preferred cTnT antigen in Example 1 to the same concentration, test the initial value with a high-sensitivity troponin assay kit (chemiluminescence method), and then place it under the following conditions: freeze tube room temperature, glass bottle room temperature, take out and test at set time points, and compare the deviation from the initial value.

[0128] The key component concentrations are adjusted as follows:

[0129] KCl concentration (mM): 125, 250, 500, 750, 1000;

[0130] Casein hydrolysate (%): 0.1, 0.2, 0.5, 1.0, 2.0;

[0131] DTAB (%): 0.05, 0.10, 0.20, 0.30.

[0132] The experimental results are shown in Tables 4-6 below:

[0133] Table 4: Effect of KCl concentration in diluent on stability of cTnT antigen

[0134]

[0135] From the above results, it can be found that when the concentration of KCl in the diluent is less than 500 mM, the drop value of cTnT antigen is more than 10% at room temperature for 4 hours in a glass bottle; as the concentration of KCl increases, the test signal value gradually decreases, it is speculated that when the concentration of KCl is too high, the ionic strength of the solution is increased, which weakens the binding force of antigen-antibody, thereby causing the signal to decrease, and when the concentration is 1000 mM, the signal is decreased by about 50% compared with 500 mM.

[0136] In summary, the preferred concentration range of KCl in the diluent is 500 mM-700 mM.

[0137] Table 5: Effect of casein hydrolysate concentration in diluent on stability of cTnT antigen

[0138]

[0139] From the above results, it can be found that when the concentration of casein hydrolysate in the diluent is less than 0.2%, the drop value of cTnT antigen is more than 10% at room temperature for 4 hours in a glass bottle, indicating that at this concentration, its role in maintaining the stability of cTnT antigen has been weakened; as the concentration of casein hydrolysate increases, the test signal value gradually decreases, it is speculated that since casein hydrolysate is a complex mixture of polypeptides / amino acids, high concentration may change the physicochemical properties of the diluent (such as ionic strength, viscosity, surface tension), interfere with the signal transmission or antigen-antibody binding efficiency of the detection system, thereby causing the signal to decrease, and when the concentration is 2.0%, the signal is decreased by about 50% compared with 0.5%.

[0140] In summary, the preferred concentration range of casein hydrolysate in the diluent is 0.2%-1.0%.

[0141] Table 6: Effect of DTAB concentration in diluent on stability of cTnT antigen

[0142]

[0143] From the above results, it can be found that the fluctuation of DTAB concentration in this experiment does not have a negative impact on the stability of cTnT antigen, but as the concentration of DTAB increases, the test signal also decreases, it is speculated that as a cationic surfactant, high concentration of DTAB can destroy the hydrophobic interaction and hydrogen bond of protein, leading to the unfolding of the native conformation of cTnT, so that it cannot be recognized by the antibody.

[0144] In summary, the preferred concentration range of the diluent DTAB is 0.05%~0.10%.

[0145] Example 4: Lyophilized calibrator reconstitution stability of preferred formulation

[0146] The calibrator diluent formulation in this example is as follows:

[0147] Preferred formulation: pH 7.4, 50 mM Tris, 0.9% NaCl, 2% BSA, 0.05% Tween 20, 0.05% ProClin 300, 500 mM KCl, 0.5% casein hydrolysate, 0.05% DTAB;

[0148] The preferred cTnT antigen in Example 1 was diluted to a specific concentration with the above diluent, and after testing the initial value with the high-sensitivity troponin assay kit (chemiluminescence method), it was lyophilized. The lyophilization procedure is as follows in Table 7:

[0149] Table 7: Lyophilization procedure of cTnT calibrator

[0150]

[0151] Because lyophilized calibrators need to be reconstituted before clinical use, it is necessary to evaluate the reconstitution stability of lyophilized products. To simulate the clinical use scenario, the reconstitution stability test of lyophilized calibrators includes: room temperature glass bottle stability, 2-8°C cryovial stability, -20°C cryovial stability. The specific experimental steps are as follows: take 3 cTnT lyophilized calibrators, test the initial value after reconstitution respectively, compare the deviation with the initial value before lyophilization, then place the reconstituted calibrators at room temperature for 8 h, test at different time points during the period; after 8 h, transfer the reconstituted calibrators to cryovials, test after 24 h storage at 2-8°C; then transfer each calibrator to -20°C for storage, test after thawing and mixing at different monitoring time points; all test values are compared with the reconstitution initial value. The experimental results are shown in Table 8 as follows:

[0152] Table 8: Reconstitution stability of lyophilized calibrator of preferred formulation

[0153]

[0154] From the above results, the deviation of the initial value of the reconstitution of the three calibration samples after freeze-drying from the deviation before freeze-drying is not more than 8%, indicating that the formula has no significant drop in the freeze-drying process; the deviation of the measurement value of the three calibration samples after reconstitution and placed in a glass bottle at room temperature for 8h from the initial reconstitution value is within 6%, indicating that the formula has good room temperature stability in the glass bottle; after placing at room temperature in the glass bottle, the samples are transferred to a frozen tube and continue to be placed in a 2-8℃ refrigerator for 24h, and then placed in a-20℃ refrigerator for 1 month, and the deviation of the monitoring value during the period is within 8%, indicating that the formula of the freeze-dried calibration sample can maintain good stability after reconstitution, storage at room temperature for 8h, storage at 2-8℃ for 24h, storage at-20℃ for 1 month, and freeze-thawing for 3 times.

[0155] In summary, the freeze-dried calibration sample of the preferred formula has good stability after reconstitution, and can maintain calibration function in different clinical use scenarios.

[0156] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not contradict, they should be considered within the scope of the present disclosure.

[0157] The above-described embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the description can be used to explain the content of the claims.

Claims

1. A cardiac troponin T calibrator diluent composition characterized in that, The basic diluent component, a potassium salt, a casein hydrolysate and dodecyl trimethyl ammonium bromide, wherein the mass ratio of the potassium salt, the casein hydrolysate and the dodecyl trimethyl ammonium bromide is (5-39):(1-20):(0.5-3).

2. The cardiac troponin T calibrator diluent composition of claim 1, wherein, The basic diluent component comprises a buffer, inorganic salt ions, a protein protective agent and a surfactant; Optionally, the cardiac troponin T calibrator diluent composition satisfies one or more of the following characteristics: The mass ratio of the potassium salt, the casein hydrolysate and the dodecyl trimethyl ammonium bromide is (19-26):(2-10):(0.5-1). The buffer comprises one or more of Tris buffer, PBS buffer and HEPES buffer; The inorganic salt ions comprise a sodium salt; The protein protective agent in the basic diluent component comprises bovine serum albumin; The surfactant in the basic diluent component comprises Tween 20; The basic diluent component comprises a buffer, a sodium salt, a protein protective agent and a surfactant; The basic diluent component further comprises a preservative; The cardiac troponin T calibrator diluent composition comprises a buffer, a sodium salt, a potassium salt, a protein protective agent, a surfactant and a preservative, wherein the protein protective agent in the cardiac troponin T calibrator diluent composition comprises one or more of bovine serum albumin and casein hydrolysate, and the surfactant in the cardiac troponin T calibrator diluent composition comprises one or more of Tween 20 and dodecyl trimethyl ammonium bromide.

3. A dilution of a cardiac troponin T calibrator, characterized in that, The cardiac troponin T calibrator diluent composition comprises an aqueous solvent and the cardiac troponin T calibrator diluent composition of claim 1 or 2. Optionally, the aqueous solvent comprises water.

4. The cardiac troponin T calibrator diluent of claim 3, wherein, The concentration of the potassium salt in the cardiac troponin T calibrator diluent is 125 mM-1000 mM; The concentration of the casein hydrolysate in the cardiac troponin T calibrator diluent is 0.1% (w / v)-2.0% (w / v); The concentration of the dodecyl trimethyl ammonium bromide in the cardiac troponin T calibrator diluent is 0.05% (w / v)-0.3% (w / v).

5. The cardiac troponin T calibrator diluent according to claim 3 or 4, characterized in that, The cardiac troponin T calibrator diluent comprises the following components: 40 mM-100 mM of a buffer, 0.5% (w / v)-1.0% (w / v) of a sodium salt, 1% (w / v)-5% (w / v) of bovine serum albumin, 0.04% (w / v)-0.06% (w / v) of Tween 20, 125 mM-1000 mM of a potassium salt, 0.1% (w / v)-2.0% (w / v) of casein hydrolysate and 0.05% (w / v)-0.3% (w / v) of dodecyl trimethyl ammonium bromide, and the pH of the cardiac troponin T calibrator diluent is 7.4±0.2; Optionally, the cardiac troponin T calibrator diluent satisfies one or more of the following characteristics: The buffer comprises one or more of Tris buffer, PBS buffer and HEPES buffer; The sodium salt comprises sodium chloride, and optionally, the sodium salt is sodium chloride. The potassium salt comprises potassium chloride, and optionally, the potassium salt is potassium chloride; The Troponin T calibrator diluent further comprises a preservative, and a concentration of the preservative in the Troponin T calibrator diluent is 0-0.1% (w / v); The Troponin T calibrator diluent comprises water and the following components: 50 mM Tris buffer, 0.9% (w / v) sodium chloride, 2% (w / v) bovine serum albumin, 0.05% (w / v) Tween-20, 0.05% (w / v) ProClin 300, 500 mM potassium chloride, 0.5% (w / v) casein hydrolysate, and 0.05% (w / v) dodecyltrimethylammonium bromide, and a pH of the Troponin T calibrator diluent is 7.4±0.1; and optionally, the water is a remainder of water.

6. A cardiac troponin T calibrator characterized in that, The Troponin T calibrator diluent comprises the Troponin T calibrator diluent according to any one of claims 3-5 and Troponin T. Optionally, a concentration of the Troponin T in the Troponin T calibrator diluent is 1.5 pg / mL-10000 pg / mL.

7. A method of preparing a cardiac troponin T calibrator, characterized by, The method comprises mixing the Troponin T calibrator diluent according to any one of claims 3-5 and Troponin T to obtain the Troponin T calibrator. Optionally, the method further comprises lyophilizing after the mixing to obtain a lyophilized Troponin T calibrator.

8. The method of preparing a cardiac troponin T calibrator according to claim 7, wherein, The lyophilizing comprises pre-freezing and drying. Optionally, the pre-freezing comprises pre-freezing at a temperature of -8℃ to -12℃ for 90 min to 150 min, pre-freezing at a temperature of -48℃ to -52℃ for 60 min to 120 min, pre-freezing at a temperature of -18℃ to -22℃ for 60 min to 120 min, and pre-freezing at a temperature of -54℃ to -56℃ for 3 h to 4 h. Optionally, the drying comprises primary drying and secondary drying, and the primary drying comprises a vacuum degree of 0.25 mbar to 0.35 mbar at a temperature of -54℃ to -56℃ for 8 min to 12 min, and a vacuum degree of 0.08 mbar to 0.12 mbar at a temperature of -28℃ to -32℃ for 11 h to 13 h, and a temperature rising to -8℃ to -12℃ for 2 h to 4 h. The secondary drying comprises a vacuum degree of 0.004 mbar to 0.006 mbar at a temperature of 8℃ to 12℃ for 6 h to 8 h.

9. A cardiac troponin T immunoassay kit, characterized by, The kit comprises the Troponin T calibrator according to claim 6.

10. The Troponin T calibrator according to claim 6 or the Troponin T immunoassay kit according to claim 9 is used for detecting Troponin T. The use includes the step of contacting the test sample with the glass container; wherein, The detecting sample comprises a sample to be detected and the Troponin T calibrator. Optionally, the detecting Troponin T comprises the following steps: detecting optical signals in the sample to be detected and the Troponin T calibrator by chemiluminescence, respectively; and comparing and analyzing detection results of the sample to be detected and the Troponin T calibrator to obtain content information of Troponin T.

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