Storage liquid of acridinium ester marker, kit and preparation method and application of storage liquid

Abstract

The invention belongs to the field of chemiluminescence, and particularly relates to a preservation solution of an acridinium ester marker, a kit and a preparation method and application of the preservation solution. According to the preservation solution of the acridinium ester marker, the sodium caseinate and the BSA are adopted as protein stabilizers, and the specific surfactant, the preservative and the neutral salt range are matched to serve as the preservation solution, so that the stability of the acridinium ester and the marked object of the acridinium ester can be kept. In addition, when the preservation solution acridinium ester marker is adopted for dilution, the diluted reagent can be placed for at least 8 days at 37 DEG C, background signals can be effectively detected, and the detection sensitivity can be improved.

Description

Technical Field

[0001] The present application belongs to the field of chemiluminescence, and specifically relates to a storage solution, a kit, and a preparation method and application of an acridinium ester marker. Background Art

[0002] Immunoassay is a bioanalytical method based on antigen-antibody reaction, which can perform qualitative and quantitative analysis on specific molecules in biological samples. Immunoassay is widely used in clinical diagnosis, environmental monitoring, food testing, and biopharmaceutical analysis. To date, although a variety of markers and marker detection systems have been widely used in immunoassays, the most accurate and mature markers are currently recognized to be chemiluminescent markers and their detection systems.

[0003] Detecting the marker above the background noise and minimizing the nonspecific binding of the tracer are the primary requirements of the signal generation and detection system. Among them, alkaline phosphatase labeling, horseradish peroxidase labeling and acridinium ester labeling are three commonly used chemiluminescent labels. However, alkaline phosphatase / horseradish peroxidase is an active protein and is essentially indirect chemiluminescence, that is, the light signal is generated by catalyzing the corresponding substrate through two substances. Therefore, it is easily affected by interfering substances and changes in detection conditions during the signal generation stage.

[0004] Acridinium esters are a class of organic compounds that can directly generate photons under specific conditions. Their general structure can be expressed as RC(=O)O-C5H5. They have gradually become the mainstream labeling technology in the chemiluminescence market because their signal generation process does not require a catalyst, has a low background, a high signal-to-noise ratio, is less affected by external factors, and has simple labeling.

[0005] However, acridinium ester labeling also has some inherent defects, such as the instability of the active site of acridinium ester in alkaline and oxidative environments, and the product of acridinium ester labeling (mostly proteins) dissolved in water medium is a polymer solution with characteristics such as charge, viscosity, aggregation and aging. Aging is the precipitation phenomenon caused by spontaneous aggregation of polymer solutions during storage. It is affected by light, air, salts, pH, flocculants, radiation, etc., and is also a key factor affecting the long-term stability of protein substances.

[0006] Therefore, in the detection system using acridinium ester as a marker in the traditional technology, problems such as reduced signal-to-noise ratio and false positives due to high background, decreased stability and changed detection performance due to prolonged storage time may be encountered. Summary of the invention

[0007] Based on this, an embodiment of the present application provides a preservation solution for an acridinium ester marker. Using the preservation solution to preserve the acridinium ester marker can improve the stability of the acridinium ester marker, and effectively reduce the background luminescence value, thereby improving the signal-to-noise ratio.

[0008] On the one hand, the present application provides a preservation solution for an acridinium ester marker, the preservation solution comprising a basic buffer solution, and components having the following mass percentages in the basic buffer solution:

[0009] 0.86%~1.26% protein stabilizer, 0.04%~0.6% surfactant, 0.04%~0.06% preservative and 0.85%~0.95% neutral salt.

[0010] The protein stabilizer includes sodium caseinate and BSA.

[0011] In one embodiment, the mass percentage of the sodium caseinate in the protein stabilizer is 5% to 50%.

[0012] The mass percentage of the BSA in the protein stabilizer is 2.0% to 98%.

[0013] In one embodiment, the basic buffer comprises one or more of MES buffer, citrate buffer and Tris-HCl buffer.

[0014] In one embodiment, the concentration of the basic buffer is 10mM~500mM, and the pH is 5.0~7.0.

[0015] In one embodiment, the surfactant includes one or more of a nonionic surfactant and an amphoteric surfactant.

[0016] In one embodiment, the nonionic surfactant includes one or more of polysorbate and polyethylene glycol octylphenyl ether.

[0017] In one embodiment, the amphoteric surfactant comprises CHAPS.

[0018] In one embodiment, the preservative includes one or more of ProClin 300, ProClin 950 and BND-10.

[0019] In one embodiment, the neutral salt includes one or more of sodium chloride and potassium chloride.

[0020] On the other hand, the present application provides an acridinium ester chemiluminescence detection kit, which includes a storage solution for the acridinium ester marker.

[0021] On the other hand, the present application provides a method for preparing a preservative solution for an acridinium ester marker, comprising: adding a neutral salt to a buffer solution, adjusting the pH to 6.4-7.0, and then sequentially adding the protein stabilizer, the surfactant and the preservative, and mixing them evenly.

[0022] The preservation solution of acridinium ester markers was prepared by filtration using a filter membrane with a pore size of 0.20 μm to 0.24 μm.

[0023] On the other hand, the present application provides a method for detecting an acridinium ester marker, comprising:

[0024] The acridinium ester marker to be detected is diluted with the storage solution of the acridinium ester marker to prepare a working solution for detecting the working solution.

[0025] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] the term

[0029] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0030] 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").

[0031] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0032] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0033] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0034] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0035] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0036] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0037] 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.

[0038] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0039] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0040] 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 invention 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.

[0041] The term "acridinium ester" is a commonly used chemiluminescent marker, which is widely used in chemiluminescent immunoassay (CLIA), DNA analysis, enzyme activity determination, etc. They can emit light in alkaline H2O2 solution, and have the advantages of low background luminescence, high signal-to-noise ratio, high luminescence efficiency, and easy to bind to proteins.

[0042] The term "CHAPS" is (3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid inner salt).

[0043] On the one hand, the present application provides a preservation solution for an acridinium ester marker, the preservation solution comprising a basic buffer solution, and components having the following mass percentages in the basic buffer solution:

[0044] 0.86%~1.26% protein stabilizer, 0.04%~0.6% surfactant, 0.04%~0.06% preservative and 0.85%~0.95% neutral salt.

[0045] Protein stabilizers include sodium caseinate and BSA.

[0046] For example, in one specific embodiment, the mass percentage of the protein stabilizer is 0.86%, and in another specific embodiment, the mass percentage of the protein stabilizer is 1.26%; it can be understood that in other specific embodiments, the mass percentage of the protein stabilizer is 0.90%, 0.94%, 0.98%, 1.02%, 1.06%, 1.10%, 1.14%, 1.18%, 1.22% or other values.

[0047] For example, in one specific embodiment, the mass percentage of the surfactant is 0.04%, and in another specific embodiment, the mass percentage of the protein stabilizer is 0.6%; it can be understood that in other specific embodiments, the mass percentage of the protein stabilizer is 0.04%, 0.1%, 0.2%, 0.3%, 0.4%, 0.42%, 0.44%, 0.46%, 0.48%, 0.50%, 0.52%, 0.54%, 0.56%, 0.58% or other values.

[0048] For example, in one specific embodiment, the mass percentage of the preservative is 0.04%, and in another specific embodiment, the mass percentage of the protein stabilizer is 0.06%; it can be understood that in other specific embodiments, the mass percentage of the protein stabilizer is 0.042%, 0.044%, 0.046%, 0.048%, 0.050%, 0.052%, 0.054%, 0.056%, 0.058% or other values.

[0049] For example, in one specific embodiment, the mass percentage of neutral salt is 0.85%, and in another specific embodiment, the mass percentage of protein stabilizer is 0.95%; it can be understood that in other specific embodiments, the mass percentage of protein stabilizer is 0.86%, 0.87%, 0.88%, 0.89%, 0.90%, 0.91%, 0.92%, 0.93%, 0.94% or other values.

[0050] This application chooses to add sodium caseinate and BSA as protein stabilizers for acridinium ester markers. Among them, sodium caseinate is a soluble sodium salt formed by dissolving insoluble casein with alkaline NaOH, and is widely used as a thickener and emulsifier in the fields of food and coatings. Casein is the main protein in mammalian milk and is composed of single-chain polypeptides with different lengths and amino acid sequences. Depending on the amino acid sequence, casein can be divided into four subtypes: α, β, γ and κ. Because it contains a large number of hydrophobic proline residues unevenly distributed on the peptide chain, casein has many unique properties, such as hydrophobicity, rheology and excellent thermal stability (can withstand high temperature of 120°C for a long time without denaturation).

[0051] In addition, the first 40 to 50 amino acids at the N-terminus of casein are in a hydrophilic aggregated state, so casein and its sodium salt have unique amphiphilicity and exhibit good surfactant properties. In addition, there are a variety of biologically active peptide sequences in casein, which are embedded in proteins and exert functional activities such as antioxidant, antibacterial, and immunomodulatory. Therefore, the role of sodium caseinate in this application is multi-effective, and it may play the role of antioxidant, antibacterial, surfactant, and protein stabilizer.

[0052] BSA is a commonly used polymer solution protectant and an ideal protein protectant. It can withstand high temperatures of 65°C and can form reversible bonds with protein substances, thereby solubilizing and preventing protein aggregation and precipitation.

[0053] In a specific example, the base buffer has the following mass percentages of components:

[0054] 0.17%~0.21% sodium caseinate, 0.46%~0.50% BSA, 0.4%~0.6% surfactant, 0.04%~0.06% preservative and 0.85%~0.95% neutral salt.

[0055] In a specific example, the concentration of the basic buffer is 10 mM to 500 mM, and the pH is 5.0 to 7.0. For example, in a specific embodiment, the basic buffer is 10 mM, and in another specific embodiment, the mass percentage of the protein stabilizer is 500 mM; it is understood that in other specific embodiments, the mass percentage of the protein stabilizer is 10 mM, 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 120 mM, 140 mM, 160 mM, 180 mM, 200 mM, 300 mM, 400 mM, 500 mM, or other values.

[0056] Optionally, the basic buffer includes one or more of MES buffer, citrate buffer and Tris-HCL buffer. Basic buffer salt: Due to the instability of acridine itself to alkaline and oxidative environments, the buffer salt of the preservation solution selects a basic buffer of acidic to neutral pH (pH 5.0~7.0), specifically one of MES, citrate or Tris-HCL salt, and the concentration is 10mM~500mM. Among them, citrate is also a deflocculating agent, which can alleviate the aging characteristics of polymer solutions while providing buffering capacity. The buffer salt provides a stable pH environment for the acridine marker to avoid protein denaturation caused by pH changes during storage.

[0057] Further optionally, the surfactant includes a nonionic surfactant or an amphoteric surfactant.

[0058] In a specific example, the nonionic surfactant includes polysorbate or polyethylene glycol octylphenyl ether.

[0059] In one specific example, the amphoteric surfactant includes CHAPS.

[0060] Amphiphilicity is an inherent property of surfactants. Depending on whether the hydrophilic group is charged and the type of charge after dissolving in water, they can be divided into nonionic, anionic, cationic and diionic types. When anionic and cationic surfactants are introduced into polymer solutions, they will cause drastic changes in protein conformation through electrostatic interactions, while nonionic and diionic surfactants will not cause drastic changes in protein structure below the critical micelle concentration, so they are more suitable for the preservation of polymer solutions.

[0061] Nonionic surfactants have poor hydrophilicity and mainly interact with proteins through hydrophobic interactions. They occupy the hydrophobic sites of proteins to reduce the interactions between proteins, thereby preventing protein aggregation. At the same time, they can enhance the hydrophilicity of proteins and play a solubilizing role. Amphoteric surfactants can present different electrical properties under different conditions, and mainly interact with proteins through electrostatic and hydrophobic interactions, respectively binding to the polar and hydrophobic parts of proteins, thereby stabilizing proteins.

[0062] In the present application, one or more of nonionic surfactants (polysorbate and Triton X-100) and amphiphilic surfactants (CHAPS) are used to stabilize and solubilize acridine markers. In addition, CHAPS is often used as a lysing agent for membrane proteins to solubilize membrane proteins and interrupt protein-protein interactions. This property helps to reduce nonspecific adsorption in immune responses and reduce background.

[0063] In a specific example, the preservative includes but is not limited to one or more of ProClin 300, ProClin 950 and BND-10. In order to ensure the long-term stability of the preservation solution, the preservation solution of the present application further adds a preservative.

[0064] In a specific example, the neutral salt includes sodium chloride or potassium chloride, which mainly functions to adjust the isoelectric point and osmotic pressure of the acridinium ester marker.

[0065] On the other hand, the present application provides an acridinium ester chemiluminescence detection kit, comprising a storage solution for the acridinium ester marker mentioned above.

[0066] On the other hand, the present application also provides a method for preparing a preservation solution of an acridinium ester marker, comprising:

[0067] Provide 6.5%~7.5% protein stabilizer, 4%~6% surfactant, 0.4%~0.6 preservative, 8.5%~9.5% neutral salt and buffer.

[0068] Add neutral salt to the buffer, adjust the pH to 6.4-7.0, then add protein stabilizer, surfactant and preservative in sequence and mix well. For example, pH is 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0

[0069] The preservation solution of the acridinium ester marker is prepared by filtration using a filter membrane with a pore size of 0.20 μm to 0.24 μm. In one specific example, the pore size is 0.2 μm; in another specific example, the pore size is 0.24 μm; it is understood that the pore size can also be other values ​​such as 0.21 μm, 0.22 μm, 0.23 μm, etc.

[0070] The present application also provides a method for detecting an acridinium ester marker, comprising:

[0071] The sample to be tested is diluted with the preservation solution of the acridinium ester marker to prepare a working solution, the detection time is set, the detection is carried out, and the luminescence value is measured.

[0072] The present application provides a preservation solution for an acridinium ester marker, which uses sodium caseinate and BSA as protein stabilizers, and is combined with a specific surfactant, preservative, and a range of neutral salts as a preservation solution to maintain the stability of the acridinium ester and its labeled substance. In addition, the acridinium ester marker of the present application is diluted, and the diluted reagent can be placed at 37°C for at least 8 days, and can effectively reduce the background signal and improve the detection sensitivity during detection.

[0073] Furthermore, the preservation solution of the acridinium ester marker of the present application has a wide range of applications, and is applicable to markers that are antibodies, markers that are small molecule hapten derivatives, and markers that are natural antigens.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] Example 1

[0078] This embodiment provides a preservation solution for an acridinium ester marker, specifically a preservation solution for a calcitonin (CT) acridinium ester marker, wherein the marker is obtained by coupling an acridinium ester and an anti-CT antibody.

[0079] Among them, the basic buffer is MES buffer, the concentration is 100mM, the pH is 6.0, the protein stabilizer is sodium caseinate and BSA, the surfactant is CHAPS, the preservative is Proclin300, the neutral salt is NaCl, and based on 1kg of solvent, the components and their contents are shown in Table 1 below:

[0080] Table 1

[0081]

[0082] After conversion, the mass percentage of each component is shown in Table 2 below:

[0083] Table 2

[0084]

[0085] Example 2

[0086] This embodiment provides a preservation solution for an acridinium ester marker, specifically a preservation solution for an estradiol acridinium ester marker, wherein the marker is obtained by coupling an acridinium ester and an estradiol-BSA derived protein.

[0087] The basic buffer is citric acid monohydrate and trisodium citrate dihydrate buffer, the concentration is 120mM, the pH is 6.5, the protein stabilizer is sodium caseinate and BSA, the surfactant is Tween 20, the preservative is Proclin300, the neutral salt is NaCl, and based on 1kg of solvent, the components and their contents are shown in Table 3 below:

[0088] Table 3

[0089]

[0090] After conversion, the mass percentage of each component is shown in Table 4 below:

[0091] Table 4

[0092]

[0093] Example 3

[0094] This embodiment provides a preservation solution for an acridinium ester marker, specifically a preservation solution for a thyroglobulin acridinium ester marker, wherein the marker is obtained by coupling thyroglobulin and acridinium ester.

[0095] Among them, the basic buffer is Tris buffer and Tris-HCL buffer, the concentration is 100mM, the pH is 6.0, the protein stabilizer is sodium caseinate and BSA, the surfactant is CHAPS and Tween 20, the preservative is Proclin300, the neutral salt is NaCl, based on 1kg of solvent, the components and their contents are shown in Table 5 below:

[0096] Table 5

[0097]

[0098] After conversion, the mass percentage of each component is shown in Table 6 below:

[0099] Table 6

[0100]

[0101] Example 4

[0102] This embodiment provides a method for preparing a preservative solution of an acridinium ester marker, taking the components of Embodiment 1 as an example, comprising the following steps:

[0103] (1) Add 0.8 kg of purified water to a beaker, add the MES and NaCL described in Example 1 to the purified water in sequence, stir until completely dissolved, and adjust the pH to 6.5 with 4 M NaOH / HCL solution;

[0104] (2) Add sodium caseinate. After it is completely dissolved, continue to add BSA. After it is completely dissolved, add CHAPS and PC-300, and then add purified water to make up to 1.0 kg.

[0105] (3) Filter with a 0.22 μm filter membrane and place at 4°C until use.

[0106] Comparative Example 1

[0107] This comparative example refers to the embodiment of patent CN105628914A, and the dilution of the comparative example group is prepared as follows:

[0108] Add 2 g / L sucrose, 1 g / L trehalose, 10 g / L glycerol, 1 g / L glycine, 1 g / L BSA, 0.02 g / L DTPA, 2 g / L Tween-20, 0.5 g / L Proclin 300, 0.5 g / L PEG 6000, and 9 g / L sodium chloride to 50 mmol / L citrate buffer and stir until completely dissolved to obtain a diluted solution.

[0109] Comparative Example 2

[0110] Compared with Example 1, the difference between this comparative example and Example 1 is that the protein stabilizer is only sodium caseinate, and the rest is the same as Example 1.

[0111] This comparative example provides a preservation solution of an acridinium ester marker, specifically a preservation solution of a CT acridinium ester marker, wherein the marker is obtained by coupling an acridinium ester and an anti-CT antibody.

[0112] Among them, the basic buffer is MES buffer, the concentration is 100mM, the pH is 6.0, the protein stabilizer is sodium caseinate, the surfactant is CHAPS, the preservative is Proclin300, the neutral salt is NaCl, and based on 1kg of solvent, the components and their contents are shown in Table 7 below:

[0113] Table 7

[0114]

[0115] After conversion, the mass percentage of each component is shown in Table 8 below:

[0116] Table 8

[0117]

[0118] Comparative Example 3

[0119] The difference between this comparative example and Example 1 is that the protein stabilizer is only BSA, and the rest is the same as Example 1.

[0120] This comparative example provides a preservation solution of an acridinium ester marker, specifically a preservation solution of a CT acridinium ester marker, wherein the marker is obtained by coupling an acridinium ester and an anti-CT (calcitonin) antibody.

[0121] Among them, the basic buffer is MES buffer, the concentration is 100mM, the pH is 6.0, the protein stabilizer is BSA, the surfactant is CHAPS, the preservative is Proclin300, the neutral salt is NaCl, and based on 1kg of solvent, the components and their contents are shown in Table 9 below:

[0122] Table 9

[0123]

[0124] After conversion, the mass percentage of each component is shown in Table 10 below:

[0125] Table 10

[0126]

[0127] Comparative Example 4

[0128] The difference between the comparative example and the embodiment 1 is that the added amounts of sodium caseinate and BSA are different, and the rest is the same as the embodiment 1.

[0129] Specifically, it is a preservation solution of CT acridinium ester marker, which is obtained by coupling acridinium ester and anti-CT antibody.

[0130] Among them, the basic buffer is MES buffer, the concentration is 100mM, the pH is 6.0, the protein stabilizer is sodium caseinate and BSA, the surfactant is CHAPS, the preservative is Proclin300, the neutral salt is NaCl, and based on 1kg of solvent, the components and their contents are shown in Table 11 below:

[0131] Table 11

[0132]

[0133] After conversion, the mass percentage of each component is shown in Table 12 below:

[0134] Table 12

[0135]

[0136] The settings of Comparative Examples 5 to 10 are shown in Tables 13 and 14:

[0137] Table 13

[0138]

[0139] Table 14

[0140]

[0141] The results of Example 1 and Comparative Examples 1 to 10 are verified:

[0142] The labeled acridinium ester marker was added to the diluents of Examples 1 to 3 and Comparative Examples 1 to 10 to prepare working solutions according to the proportions, and the diluent and sample tests and accelerated stability tests were performed. The diluent and sample test reagents can be performed after preparation, and the accelerated stability reagent is taken out after being placed in a 37-degree constant temperature and humidity chamber for 7 days and tested after balancing at 2-8 degrees for 4 hours.

[0143] The Shine i2910 fully automatic chemiluminescence immunoassay analyzer was used to set the test items and test parameters, and then measure the luminescence value. The following are the test results obtained in the specific examples of this application.

[0144] 1. Effects of different storage solutions on test background and signal-to-noise ratio

[0145] In this experiment, the preservation solution of Example 1 and Comparative Examples 1 to 4, Example 2 and Comparative Example 1, Comparative Examples 5 to 7, Example 3 and Comparative Example 1, and Comparative Examples 8 to 10 were used to test the preservation solution background and the signal values ​​of 10 clinical samples with different concentrations. The signal-to-noise ratio was calculated based on the ratio of the signal value to the background. The results are shown in Table 15:

[0146] Table 15

[0147]

[0148] It is understandable that the difficulty in developing CT detection reagents lies in the relatively low detection limit and blank limit of the reagents. Therefore, if the background is slightly increased, clinical samples with relatively low concentrations will be judged as negative samples because of the value lower than the zero value matrix. The above results show that for CT items with low detection limits, Comparative Example 1 and the diluent containing only BSA without sodium caseinate cannot detect low-value samples (signal-to-noise ratio is less than 1), that is, the clinical sample signal is lower than the zero value matrix test value, and the signal-to-noise ratios of the remaining groups are all higher than 1.

[0149] Therefore, under low background conditions, the marker diluted with the preservation solution of the present application has a higher signal-to-noise ratio and better sensitivity when measuring low-concentration samples, reducing the risk of false negative results for some projects with low detection limits.

[0150] Example 2 The marker is an E2-protein derivative, which is generally tested by competition method, so low-concentration samples have higher signals. The difficulty in developing reagents for this project is that the antibody only recognizes a small epitope site of E2, and the affinity of commercially available antibodies is generally not very high. Therefore, the requirements for instruments and reagents are high when testing low-concentration samples, and the signal-to-noise ratio between the zero-value matrix and the sample is generally low.

[0151] The sample test results show that the zero-value matrix signals of Example 2 and the 4 groups of comparative examples are basically the same, but for the samples with values, Example 2 has a higher signal-to-noise ratio, and the comparative examples with sodium caseinate or BSA added alone are not as good as Example 2. The results are shown in Table 16:

[0152] Table 16

[0153]

[0154] Therefore, the marker diluted with the preservation solution of the present application also has a higher signal-to-noise ratio for the competitive method and has better sensitivity when measuring low-concentration samples.

[0155] The marker of Example 3 is a natural protein. Compared with the diluents of Comparative Examples 1 and 8-10, the diluent provided by the present application can also obtain significantly beneficial low background and high signal-to-noise ratio, and therefore also has better detection sensitivity, as shown in Table 17:

[0156] Table 17

[0157]

[0158] 2. Effect of storage solution on the stability of acridinium ester markers

[0159] In this experiment, Examples 1 to 3 of different projects were diluted with several of Comparative Examples 1 to 10 in the same proportion, and divided into two equal parts, one of which was stored at 2°C to 8°C, and the other was stored in a constant temperature and humidity chamber at 37°C. After 7 days, the diluted acridinium ester marker was taken out from the constant temperature and humidity chamber, and placed at 2°C to 8°C for more than 4 hours, and 4 different samples were measured at the same time: the zero-value matrix of Examples 1 to 3, horse serum, and low-value (sample 1) as a negative control and high-value clinical (sample 2) as a positive control. The results are shown in Tables 18 to 20 below:

[0160] Table 18

[0161]

[0162] Table 19

[0163]

[0164] Table 20

[0165]

[0166] From the data in the above table, it can be seen that compared with the preservation solution of comparative example 1, the preservation solutions of the three different acridinium ester markers can maintain the stability of the test signal under the high temperature challenge of 37°C, and the signal deviation is within 10%. In the preservation solution of the control group, the signal deviations of the three different markers have exceeded 10%.

[0167] It can be seen from the above experiments that the diluted reagent using the preservation solution of the acridinium ester marker provided by the present application has a lower background and sensitivity (signal-to-noise ratio) and also has better stability.

[0168] 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 can be used to interpret the content of the claims.

Claims

1. A storage solution for an acridinium ester labeled substance, characterized in that: The preservation solution includes a basic buffer, and components having the following mass percentages in the basic buffer: 0.86% to 1.26% of a protein stabilizer, 0.04% to 0.6% of a surfactant, 0.04% to 0.06% of a preservative, and 0.85% to 0.95% of a neutral salt; The protein stabilizer includes sodium caseinate and BSA.

2. The storage solution for acridinium ester labeled substances according to claim 1, characterized in that The mass percentage of the sodium caseinate in the protein stabilizer is 5% to 50%; The mass percentage of the BSA in the protein stabilizer is 2.0% to 98%.

3. The storage solution for acridinium ester labeled substances according to claim 1, characterized in that: The basic buffer comprises one or more of a MES buffer, a citrate buffer and a Tris-HCl buffer.

4. The storage solution for acridinium ester labeled substances according to claim 1, characterized in that: The concentration of the basic buffer is 10mM-500mM, and the pH is 5.0-7.

0.

5. The storage solution for an acridinium ester labeled substance according to any one of claims 1 to 4, characterized in that The surfactant includes one or more of a nonionic surfactant and an amphoteric surfactant; Optionally, the nonionic surfactant includes one or more of polysorbate and polyethylene glycol octylphenyl ether; Optionally, the amphoteric surfactant comprises CHAPS.

6. The storage solution for an acridinium ester labeled substance according to any one of claims 1 to 4, characterized in that The preservative includes one or more of ProClin 300, ProClin 950 and BND-10.

7. The storage solution for an acridinium ester labeled substance according to any one of claims 1 to 4, characterized in that The neutral salt includes one or more of sodium chloride and potassium chloride.

8. An acridinium ester chemiluminescent detection kit, characterized in that: The kit comprises a preservation solution for the acridinium ester marker according to any one of claims 1 to 7.

9. The method for preparing a storage solution for an acridinium ester labeled substance according to any one of claims 1 to 7, characterized in that: include: Adding neutral salt to the buffer solution to adjust the pH to 6.4-7.0, and then sequentially adding the protein stabilizer, the surfactant and the preservative, and mixing them evenly; as well as, The preservation solution of acridinium ester markers was prepared by filtration using a filter membrane with a pore size of 0.20 μm to 0.24 μm.

10. A method for detecting an acridinium ester marker, characterized in that: include: The acridinium ester marker to be detected is diluted with the storage solution of the acridinium ester marker according to any one of claims 1 to 7 to prepare a working solution, and the working solution is detected.

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