Sample treating fluid, troponin complex detection kit and application

Abstract

The invention belongs to the field of in-vitro diagnosis, and particularly relates to a sample treating fluid, a troponin compound detection kit and application. The invention provides a sample treating fluid, which comprises a buffer solution, and a protein denaturant, a surfactant, a preservative and sodium chloride which are added in the buffer solution and have a specific concentration, and the sample treating fluid provided by the invention is used for treating a troponin compound in a sample, so that a troponin detection substance is more uniform in form and simpler in structure in the sample; identification is easier, interference is less, detection results are more accurate, cost is reduced, and manufacturability is improved. Meanwhile, the primary structure of the target protein cannot be damaged by adopting the sample liquid, the detection capability of a reagent cannot be influenced when the sample liquid is matched with the detection reagent for use, and the detection accuracy is improved.

Description

Technical Field

[0001] The present application belongs to the field of in vitro diagnosis, and specifically relates to a sample processing solution and a troponin complex detection kit and applications. Background Art

[0002] The troponin complex is composed of three subunits: troponin T (TnT), troponin I (TnI) and troponin C (TnC). It is a regulatory protein for muscle tissue contraction. Three different tissue-specific troponin I subtypes have been found in the human body, namely cardiac muscle type, slow skeletal muscle type and fast skeletal muscle type. Among them, cardiac troponin I (cTnI) and cardiac troponin T (cTnT) are specifically expressed in cardiomyocytes. Under normal physiological conditions, a small amount of cTn (2%~8%) exists in a free form in the cytoplasm and is soluble; most of it is fixed to the cardiac muscle fibers in the form of structural proteins and is not soluble.

[0003] When myocardial cells are damaged, cardiac troponin is degraded from myocardial fibers and released into the blood, mainly in the form of free cTnI, cTnT, cTnI-cTnC complexes, and full molecular weight and low molecular weight cTnT-cTnI-cTnC trimers in peripheral blood. Increased serum cTn levels reflect myocardial cell damage, which can be seen in acute myocardial infarction, unstable angina, pulmonary infarction, heart failure shock, and other diseases that cause myocardial damage such as connective tissue disease, severe diabetic ketoacidosis, pancreatitis, etc. After myocardial damage or myocardial infarction (MI), cTnI and cTnT can increase rapidly and last for several days, especially high-sensitivity troponin reagents, which can usually detect elevated cTnI levels within 3 hours after the onset of symptoms. Therefore, cTnI and cTnT are the preferred biomarkers for evaluating myocardial damage, and high-sensitivity hs-cTn is recommended for routine clinical use.

[0004] At present, studies have shown that after troponin enters the peripheral blood, the complete cTnI monomer is easily hydrolyzed into a variety of amino acid residues of different lengths and fragments, thus affecting the accuracy of its immunoassay. In the blood samples of AMI patients, the whole fragment cTnI and 11 major amino acid residues (aar.) can be detected, of which >80% of cTnI can be detected by antibodies that specifically recognize the aar.23-196 fragment, and 100% of cTnI can be detected by antibodies that specifically recognize the aar.34-126 fragment. However, the main binding sites of the cTnI-cTnC complex and the cTnT-cTnI-cTnC trimer are also in the aar.34-126 fragment, making the antibodies against this fragment susceptible to shielding by cTnC and cTnT.

[0005] At the same time, troponin released into the blood after myocardial injury may trigger an autoimmune reaction. Anti-troponin autoantibodies can be detected in the blood of some heart disease patients or healthy individuals, resulting in false negative results in the detection of cTnI and cTnT and delaying the treatment. Studies have shown that autoantibodies mainly bind to the IT spatial conformation in the cTnT-cTnI-cTnC trimer to form a troponin complex, thereby hindering the recognition of antigens by antibodies that recognize related sites in cardiac troponin detection reagents, resulting in false negative results.

[0006] At present, the determination of cardiac troponin I and T in the field of in vitro diagnosis is mainly based on the double antibody sandwich method. As capture antibodies and labeled antibodies, two or more complex combinations of antigenic epitopes, complex antigenic epitopes, etc. that recognize different monomers are usually used to avoid missed detection or false positives caused by site shielding, amino acid phosphorylation, skeletal muscle homologous sites, etc., so as to improve the sensitivity and specificity of its detection. However, the use of multiple antibodies also leads to high reagent costs, difficulty in stability optimization, poor manufacturability, and easy batch differences. At the same time, the different detection capabilities of different complexes also lead to large differences in test results between different reagents, which brings difficulties to clinical application. In addition, troponin has autoantibodies in the human body, which will form giant troponin complexes. The influence of giant troponin cannot be completely solved by conventional antibody recognition site optimization and combination, which will further lead to a decrease in the accuracy of troponin measurement.

[0007] In summary, there is currently a problem of low accuracy in the detection of calreticulin complexes. Summary of the invention

[0008] Based on this, an embodiment of the present application provides a sample processing solution and a troponin complex detection kit and application to improve the accuracy of troponin complex detection.

[0009] On the one hand, the present application provides a sample processing solution, including a buffer solution and a protein denaturant, a surfactant, a preservative and an osmotic pressure regulator added to the buffer solution.

[0010] In one embodiment, the pH of the buffer is 1-6 or 8-12.

[0011] In one embodiment, the final concentration of the protein denaturant in the sample processing solution is 0.2 mol / L~20 mol / L; the mass volume percentage of the surfactant in the sample processing solution is 0.01%~5%; the mass volume percentage of the preservative in the sample processing solution is 0.01%~1%; and the mass volume percentage of the osmotic pressure regulator in the sample processing solution is 0.5%~5%.

[0012] In one embodiment, the final concentration of the protein denaturant in the sample processing solution is 0.4 mol / L to 10 mol / L;

[0013] The surfactant has a mass volume percentage of 0.1% to 2% in the sample treatment solution;

[0014] The mass volume percentage of the preservative in the sample processing solution is 0.05% to 0.1%;

[0015] The mass volume percentage of the osmotic pressure regulator in the sample processing liquid is 0.5% to 2%.

[0016] In one embodiment, the protein denaturing agent includes urea, guanidine hydrochloride and guanidine isothiocyanate, SDS - , CTAB + and triton.

[0017] Optionally, the osmotic pressure regulator includes one or both of sodium chloride and potassium chloride.

[0018] In one embodiment, the surfactant includes one or both of an ionic surfactant and a non-ionic surfactant.

[0019] Optionally, the ionic surfactant includes one or more of a cationic surfactant, an anionic surfactant and an amphoteric surfactant.

[0020] Optionally, the nonionic surfactant includes one or more of Tween-20, Tween-80, TritonX-405 and Brij35.

[0021] In one embodiment, the preservative includes one or more of Proclin 300, sodium azide and 5-bromo-5-nitro-1,3-dioxane.

[0022] In one embodiment, the final concentration of the buffer medium in the buffer solution in the sample processing solution is 5 mmol / L to 500 mmol / L.

[0023] Optionally, the buffer medium includes an acidic buffer medium or an alkaline buffer medium.

[0024] Optionally, the acidic buffer medium includes one or more of citric acid-citrate, acetic acid-sodium acetate, disodium hydrogen phosphate-citric acid and glycine-hydrochloric acid.

[0025] Optionally, the alkaline buffer medium includes one or more of Tris-hydrochloric acid, borax-hydrochloric acid, glycine-sodium hydroxide, borax-sodium hydroxide, sodium carbonate-sodium bicarbonate and sodium carbonate-sodium hydroxide.

[0026] On the other hand, the present application provides a troponin complex detection kit, comprising the above-mentioned sample processing solution.

[0027] In one embodiment, a solid phase carrier is also included.

[0028] Optionally, the solid phase carrier comprises a magnetic bead coating, wherein the magnetic bead coating is coated with an anti-cardiac troponin antibody.

[0029] In one embodiment, a luminescent marker is also included.

[0030] In one embodiment, the luminescent marker is labeled with an anti-cardiac troponin antibody.

[0031] Optionally, the luminescent marker comprises one or more of adamantane, luminol, isoluminol, acridinium ester and acridinium ester derivatives.

[0032] On the other hand, the present application provides a method for detecting a troponin complex, comprising treating a sample to be tested with a sample treatment solution, adding a luminescent marker, and performing detection;

[0033] The sample processing liquid is a preformulated composition or a ready-made composition, and the sample processing liquid satisfies the above-mentioned definition of the sample processing liquid.

[0034] The present application provides a sample processing solution, including a buffer solution and a protein denaturant, a surfactant, a preservative and sodium chloride added to the buffer solution. The sample processing solution provided by the present application is used to treat the troponin complex in the sample, so that the troponin detection object in the sample has a more uniform morphology, a simpler structure, easier identification, less interference, and more accurate detection results, while reducing costs. At the same time, the sample solution of the present application will not destroy the primary structure of the target protein, and when used in conjunction with the detection reagent, it will not affect the detection ability of the reagent itself, thereby improving the accuracy of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0036] Figure 1The scatter plot is for the luminescence value detection of the control reagent;

[0037] Figure 2 A scatter plot of the luminescence value detected after the sample is treated with the sample treatment solution of the present application;

[0038] Figure 3 This is the scatter plot obtained from the luminescence value test of reagent 1;

[0039] Figure 4 This is the scatter plot obtained from the luminescence value test of reagent 2;

[0040] Figure 5 This is a scatter plot obtained from the luminescence value test of reagent 3. DETAILED DESCRIPTION

[0041] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0042] 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.

[0043] the term

[0044] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0045] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0046] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0047] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0048] The term "troponin complex" Troponin (Tn) is a regulatory protein for muscle tissue contraction. It is located on the thin filaments of contractile proteins and plays an important regulatory role in muscle contraction and relaxation. It contains three subtypes: fast-response type, slow-response type and cardiac troponin (cTn). The first two are related to skeletal muscle, while cardiac troponin only exists in myocardial cells. It is a complex composed of three subunits: troponin T (cTnT), troponin I (cTnI) and troponin C (cTnC). cTnT and cTnI are antigens specific to myocardial cells and are degraded from myocardial fibers when myocardial cells are damaged.

[0049] The term "protein denaturant" refers to an agent that denatures proteins. Denaturation is the process by which proteins are affected by physical or chemical factors, changing their internal molecular structure and properties. It is generally believed that changes or destruction of the secondary and tertiary structures of proteins are the result of denaturation.

[0050] The term "surfactant" Surfactant, also known as interfacial agent, is a compound that can significantly reduce the surface tension or interfacial tension between two liquids, between liquid and gas, and between liquid and solid. The molecular structure of surfactant is amphiphilic: one end is a hydrophilic group and the other end is a hydrophobic group; the hydrophilic group is often a polar group, such as carboxylic acid, sulfonic acid, sulfuric acid, amino or amine group and its salt, hydroxyl, amide, ether bond, etc. can also be used as polar hydrophilic groups; while the hydrophobic group is often a non-polar hydrocarbon chain, such as a hydrocarbon chain with more than 8 carbon atoms. Surfactants are divided into ionic surfactants (including cationic surfactants, anionic surfactants, amphoteric surfactants), non-ionic surfactants, compound surfactants, other surfactants, etc.

[0051] It is understood that the troponin complex mentioned in the present application also includes giant troponin complex and other similar complexes.

[0052] The present application provides a treatment solution for dissociating troponin complexes into monomers. The sample treatment solution reversibly dissociates cTnI-cTnC complexes and cTnT-cTnI-cTnC trimer complexes in blood by acidic treatment, alkaline treatment, or addition of substances that can destroy non-covalent bonds (such as hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.) between proteins, or a combination of these methods. The same purpose of dissociating troponin complexes is achieved by capturing antibody diluents and detecting antibody diluents.

[0053] At the same time, the primary structure of cTn is not destroyed, so that cTn exists mainly in the form of free monomers, which improves the recognition efficiency of coated and labeled antibodies for cTnI and cTnT, thereby improving the sensitivity and specificity of detection.

[0054] Since the troponin complex is mainly connected in the form of non-covalent bonding, such as hydrogen bonds, hydrophobic interactions, van der Waals forces and other forces, and non-covalent bonds in acidic, alkaline or in the presence of specific substances such as surfactants, denaturants, etc., the multi-level structure of the protein will be destroyed or changed, causing the troponin complex to dissociate into free monomers, thereby achieving the purpose of improving detection accuracy and specificity.

[0055] Acidic or alkaline conditions are often achieved by adjusting the pH with buffers or acidic solutions or alkaline solutions. Commonly used acidic buffers include citric acid-citrate buffer, acetic acid-sodium acetate buffer, disodium hydrogen phosphate-citric acid buffer, glycine-hydrochloric acid buffer, etc.; commonly used acidic solutions include hydrochloric acid solution, acetic acid solution, etc.; commonly used alkaline buffers include Tris-hydrochloric acid buffer, borax-hydrochloric acid buffer, glycine-sodium hydroxide buffer, borax-sodium hydroxide buffer, sodium carbonate-sodium bicarbonate buffer, sodium carbonate-sodium hydroxide buffer, etc.; commonly used alkaline solutions include sodium hydroxide solution, etc.

[0056] The present application only targets the processing steps of the complex in the sample test and does not limit the detection method or reaction steps. Therefore, the commonly used immunoassay methods for detecting troponin I, troponin T and high-sensitivity troponin I and high-sensitivity troponin T are all within the scope of the present application, such as immunoturbidimetry, immunofluorescence chromatography, microfluidic immunoassay, chemiluminescence, enzyme-linked immunosorbent assay, etc.

[0057] On the one hand, the present application provides a sample processing solution, including a buffer solution and a protein denaturant, a surfactant, a preservative and sodium chloride added to the buffer solution.

[0058] The pH of the buffer solution is 1-6 or 8-12.

[0059] The final concentration of the protein denaturant in the sample treatment solution is 0.2 mol / L~20 mol / L; for example, 1 mol / L, 2 mol / L, 3 mol / L, 4 mol / L, 5 mol / L, 6 mol / L, 7 mol / L, 8 mol / L, 9 mol / L, 10 mol / L, 11 mol / L, 12 mol / L, 13 mol / L, 14 mol / L, 15 mol / L, 16 mol / L, 17 mol / L, 18 mol / L, 19 mol / L, 20 mol / L.

[0060] The mass volume percentage of the surfactant in the sample processing liquid is 0.01%~5%, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, and 5%.

[0061] The mass volume percentage of the preservative in the sample processing solution is 0.01%~1%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%.

[0062] The mass volume percentage of sodium chloride in the sample treatment solution is 0.5%~5%. For example, 0.5%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%.

[0063] In a specific example, the protein denaturant includes, but is not limited to, one or more of urea, guanidine hydrochloride, and guanidine isothiocyanate. The protein denaturant also includes some surfactants, such as SDS-, CTAB+, and triton.

[0064] Optionally, the surfactant includes, but is not limited to, one or more of a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant.

[0065] Further optionally, the nonionic surfactant includes, but is not limited to, one or more of Tween-20, Tween-80, TritonX-405 and Brij35.

[0066] In a specific example, the preservative includes, but is not limited to, one or more of Proclin 300, sodium azide, and 5-bromo-5-nitro-1,3-dioxane.

[0067] In a specific example, the buffer medium includes an acidic buffer medium or an alkaline buffer medium, so that the entire buffer system is under non-neutral conditions.

[0068] Wherein, the final concentration of the buffer medium in the buffer solution in the sample processing solution is 5mmol / L~500mmol / L; for example, 5 mmol / L, 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L, 110 mmol / L, 120 mmol / L, 130 mmol / L, 140 mmol / L, 150 mmol / L, 160 mmol / L, 170 mmol / L, 180 mmol / L, 190 mmol / L, 200 mmol / L, 250 mmol / L, 300 mmol / L, 350 mmol / L, 400 mmol / L, 450 mmol / L, 500 mmol / L.

[0069] The acidic buffer medium includes one or more of citric acid-citrate, acetic acid-sodium acetate, disodium hydrogen phosphate-citric acid and glycine-hydrochloric acid.

[0070] The alkaline buffer medium includes one or more of Tris-hydrochloric acid, borax-hydrochloric acid, glycine-sodium hydroxide, borax-sodium hydroxide, sodium carbonate-sodium bicarbonate and sodium carbonate-sodium hydroxide.

[0071] On the other hand, the present application also provides a troponin complex detection kit, including the above-mentioned sample processing solution.

[0072] Optionally, the kit further comprises a magnetic bead coating, wherein the magnetic bead coating comprises magnetic microparticles coated with anti-cardiac troponin antibodies.

[0073] In one specific example, a luminescent marker is also included.

[0074] Optionally, the luminescent marker includes, but is not limited to, one or more of adamantane, luminol, isoluminol, acridinium ester and derivatives thereof.

[0075] Another party of the present application also provides a method for detecting a troponin complex, comprising treating a sample to be tested with a sample treatment solution, adding a luminescent marker, and performing detection.

[0076] It can be understood that the sample processing liquid can be a prefabricated composition or a ready-made composition, and the composition can satisfy the sample processing liquid provided in this application.

[0077] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0078] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0079] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0080] Example 1

[0081] 1. Reagent preparation:

[0082] 1. Magnetic bead working solution: After coating the anti-cTnI capture antibody and magnetic microparticles at a concentration of 20 μg / mg, dilute them with magnetic bead diluent to a working solution with a magnetic bead concentration of 0.25 mg / mL for later use. The magnetic bead diluent is a Tris buffer containing 0.5% BSA.

[0083] 2. Acridine working solution: After labeling the anti-cTnI labeled antibody with acridine ester at a concentration of 40 μg / mL, dilute it with acridine diluent to a working solution with a concentration of 0.4 μg / mL, and filter it with a 0.45 μm filter membrane for later use. The acridine diluent is PBS buffer containing 0.5% BSA.

[0084] 3. Sample treatment solution: The components of the sample treatment solution (100 mM citric acid buffer) used in this embodiment are shown in Table 1 below:

[0085] Table 1

[0086]

[0087] The prepared sample treatment solution was filtered through a 0.45µm filter membrane and stored at 2°C~8°C until use.

[0088] 4. The above three working solutions are combined into reagent A.

[0089] 5. Control reagent: A reagent that does not contain sample processing solution and only contains the above-mentioned magnetic beads and acridine working solution.

[0090] 2. Testing steps:

[0091] 1. The control reagent and reagent A were automatically detected using the iStar 500 series fully automatic chemiluminescence analyzer of Zhuorun Biotechnology Co., Ltd. The two reagents were used to simultaneously detect clinical serum samples with known concentration information, and the luminescence signal values ​​were collected and compared with the sample concentrations.

[0092] 2. Control reagent test steps: 100µL sample was mixed with 50µL magnetic bead working solution, incubated at 37℃ for 5 minutes, and then magnetically separated and washed 4 times. Then 50µL acridine working solution was added, incubated at 37℃ for 5 minutes, and then magnetically separated and washed 4 times. Pre-excitation solution and excitation solution were added to detect the luminescence value.

[0093] 3. Test steps for reagent A: 100 µL of sample was mixed with 50 µL of sample treatment solution and 50 µL of magnetic bead working solution, incubated at 37°C for 5 minutes, and then magnetically separated and washed 4 times. Then 50 µL of acridine working solution was added, incubated at 37°C for 5 minutes, and magnetically separated and washed 4 times. Pre-excitation solution and excitation solution were added to detect the luminescence value.

[0094] 4. The concentration information of clinical samples was obtained by testing in the hospital using the Abbott high-sensitivity troponin-I kit.

[0095] 5. The results of the luminescence signal value detection are shown in Table 2.

[0096] Table 2

[0097]

[0098] 6. Use the sample concentration as the X-axis and the signal values ​​obtained from the control reagent and reagent A tests as the Y-axis, and draw scatter plots such as Figure 1 and Figure 2 As shown, from Figure 1 and Figure 2 From the comparison, it can be seen that when the control reagent was used to test the sample, the sample was not processed, resulting in a poor comparison between the test result and the sample concentration, and the test results of some samples were low; when the reagent A with the sample processing solution added was used for detection, the correlation between the test result and the sample concentration was greatly improved, and the signal value did not decrease significantly. The deviation of the signal value in the experiment was mainly due to the increase in the volume of the final reaction system caused by the addition of the processing solution. This proves that treating the sample with the above-mentioned sample processing solution can effectively improve the accuracy of the reagent in detecting high-sensitivity troponin.

[0099] Comparative Example 1

[0100] 1. Reagent preparation:

[0101] 1. Magnetic bead working solution: After coating the anti-cTnT capture antibody and magnetic microparticles at a concentration of 20 μg / mg, dilute them with magnetic bead diluent to a working solution with a concentration of 0.18 mg / mL for later use. The magnetic bead diluent is a Tris buffer containing 0.5% BSA.

[0102] 2. Acridine working solution: After labeling the anti-cTnT labeled antibody with acridine ester at a concentration of 100 μg / mL, dilute it with acridine diluent to a working solution with a concentration of 1 μg / mL, and filter it with a 0.45μm filter membrane for later use. The acridine diluent is PBS buffer containing 0.5% BSA.

[0103] 3. Sample treatment solution: The components of the sample treatment solution 1 (50 mM Tris buffer) used in this embodiment are shown in Table 3 below:

[0104] Table 3

[0105]

[0106] 4. At the same time, the concentration of guanidine hydrochloride in the sample treatment solution was adjusted to 0.1 mol / L, and the other components remained unchanged to prepare sample treatment solution 2.

[0107] 5. At the same time, the concentration of guanidine hydrochloride in the sample treatment solution was adjusted to 21 mol / L, and the other components remained unchanged to prepare sample treatment solution 3.

[0108] 6. The above three sample treatment solutions were filtered through a 0.45µm filter membrane and stored at 2℃~8℃ for later use.

[0109] 7. The magnetic bead working solution and acridine working solution are respectively combined with sample processing solutions 1, 2, 3 to form reagents 1, 2, 3, wherein reagent 1 is a control reagent, and reagents 2 and 3 are protein denaturing agents, guanidine hydrochloride, which are test reagents beyond the scope of the claims.

[0110] 2. Testing steps:

[0111] 1. Reagents 1, 2, and 3 were fully automatically detected using the iStar 500 series fully automatic chemiluminescence analyzer of Zhuorun Biotechnology Co., Ltd. The three reagents were used to simultaneously detect clinical serum samples with known concentration information, and the luminescence signal values ​​were collected and compared with the sample concentrations.

[0112] 2. Test steps: 100µL sample was mixed with 50µL sample treatment solution, 50µL magnetic bead working solution and 50µL acridine working solution, incubated at 37°C for 10 minutes, magnetically separated and washed 4 times, and pre-excitation solution and excitation solution were added to detect the luminescence value.

[0113] 3. The concentration information of clinical samples was obtained by testing in the hospital using the Roche Troponin-T Kit.

[0114] 4. The results of the luminescence signal value detection are shown in Table 4.

[0115] Table 4

[0116]

[0117] 5. Use the sample concentration as the X-axis and the signal values ​​obtained from the tests of reagent 1, reagent 2 and reagent 3 as the Y-axis, and draw scatter plots such as Figure 3 , Figure 4 and Figure 5 As shown, by comparison, it can be seen that when the concentration of guanidine hydrochloride is reduced to below the effective range, the correlation between the test results and the sample concentration becomes worse.

[0118] 6. The test results of reagent 1 and reagent 3 were analyzed and it was found that when the concentration of guanidine hydrochloride increased to higher than the effective range, it would interfere with the detection performance of the reagent itself, resulting in the inability to perform normal detection.

[0119] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A sample processing solution, characterized in that: Including buffer and protein denaturant, surfactant, preservative and osmotic pressure regulator added to the buffer; The pH of the buffer solution is 1-6 or 8-12; The final concentration of the protein denaturant in the sample treatment solution is 0.2 mol / L to 20 mol / L; The surfactant has a mass volume percentage of 0.01% to 5% in the sample treatment solution; The mass volume percentage of the preservative in the sample processing solution is 0.01% to 1%; The mass volume percentage of the osmotic pressure regulator in the sample processing liquid is 0.5% to 5%.

2. The sample processing solution according to claim 1, characterized in that: The final concentration of the protein denaturant in the sample treatment solution is 0.4 mol / L to 10 mol / L; The surfactant has a mass volume percentage of 0.1% to 2% in the sample treatment solution; The mass volume percentage of the preservative in the sample processing solution is 0.05% to 0.1%; The mass volume percentage of the osmotic pressure regulator in the sample processing liquid is 0.5% to 2%.

3. The sample processing solution according to claim 1, characterized in that: The protein denaturants include urea, guanidine hydrochloride, guanidine isothiocyanate, SDS - , CTAB + and one or more of triton; Optionally, the osmotic pressure regulator includes one or both of sodium chloride and potassium chloride.

4. The sample processing solution according to claim 1, characterized in that: The surfactant includes one or both of an ionic surfactant and a non-ionic surfactant; Optionally, the ionic surfactant includes one or more of a cationic surfactant, an anionic surfactant and an amphoteric surfactant; Optionally, the nonionic surfactant includes one or more of Tween-20, Tween-80, TritonX-405 and Brij35.

5. The sample processing solution according to claim 1, characterized in that: The preservative includes one or more of Proclin 300, sodium azide and 5-bromo-5-nitro-1,3-dioxane.

6. The sample processing solution according to any one of claims 1 to 5, characterized in that: The final concentration of the buffer medium in the buffer solution in the sample treatment solution is 5 mmol / L to 500 mmol / L; Optionally, the buffer medium comprises an acidic buffer medium or an alkaline buffer medium; Optionally, the acidic buffer medium includes one or more of citric acid-citrate, acetic acid-sodium acetate, disodium hydrogen phosphate-citric acid and glycine-hydrochloric acid; Optionally, the alkaline buffer medium includes one or more of Tris-hydrochloric acid, borax-hydrochloric acid, glycine-sodium hydroxide, borax-sodium hydroxide, sodium carbonate-sodium bicarbonate and sodium carbonate-sodium hydroxide.

7. A troponin complex detection kit, characterized in that: The method comprises the sample processing solution according to any one of claims 1 to 6.

8. The troponin complex detection kit according to claim 7, characterized in that: Also included are solid phase supports; Optionally, the solid phase carrier comprises a magnetic bead coating, wherein the magnetic bead coating is coated with an anti-cardiac troponin antibody.

9. The troponin complex detection kit according to claim 7, characterized in that: Also included are luminescent markers; The luminescent marker is labeled with an anti-cardiac troponin antibody; Optionally, the luminescent marker comprises one or more of adamantane, luminol, isoluminol, acridinium ester and acridinium ester derivatives.

10. A method for detecting a troponin complex, characterized in that: The method comprises treating the sample to be tested with a sample treatment solution, adding a luminescent marker, and performing detection; The sample processing solution is a prefabricated composition or a ready-made composition, and the sample processing solution meets the definition of the sample processing solution according to any one of claims 1 to 6.

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