Chemiluminescence substrate solution and chemiluminescence detection method

Abstract

The invention relates to a chemiluminescence substrate solution and a chemiluminescence detection method. The chemiluminescent substrate liquid comprises a chemiluminescent substrate, fluorescein and a water-soluble polymer quaternary ammonium salt, wherein the chemiluminescent substrate is selected from a chlorinated dioxetane compound with a spiro-adamantane substituent group. The chemiluminescent substrate solution has a wider linear detection range.

Description

Technical Field

[0001] The present disclosure relates to the field of in vitro diagnostic immunoassays, and particularly to an alkaline phosphatase chemiluminescent substrate solution and a chemiluminescent detection method. Background Art

[0002] Chemiluminescence refers to a phenomenon in which, during a chemical reaction, a specific substance absorbs part of the chemical energy to reach an excited state, and the energy released during the return to the ground state is released in the form of photons, thereby generating luminescence. Chemiluminescence immunoassay (CLIA) refers to an analytical technique that combines highly sensitive chemiluminescence technology with highly specific immunoreactions for detecting antigens, antibodies, hormones, fatty acids, vitamins, drugs, etc. Chemiluminescence immunoassay has both the high sensitivity of chemiluminescence and the characteristics of simple operation and rapid reaction of enzyme-linked immunoassay, and is easy to standardize experimental operations. It has now been widely used in biological research, medical research, and clinical diagnosis.

[0003] In current immunoassay detections, the concentrations of some analytes in clinical samples vary greatly, and can even span several orders of magnitude from 10 5 to 10 6 Currently, the general method is to first dilute the high-concentration samples and then test them, which has disadvantages such as long test cycles, high test costs, large dilution interferences, and cumbersome operations.

[0004] Therefore, there is a need for further improvement in the detection methods for such clinical detection items. Summary of the Invention

[0005] The purpose of the present disclosure is to provide a chemiluminescent substrate solution with a wide linear range and a chemiluminescent detection method.

[0006] To this end, the first aspect of the present disclosure provides a chemiluminescent substrate solution, comprising a chemiluminescent substrate, a fluorophore, and a water-soluble polymeric quaternary ammonium salt, wherein the chemiluminescent substrate is selected from chlorinated dioxetane compounds having a spiro-adamantyl substituent.

[0007] According to some embodiments, the chlorinated dioxetane compounds having a spiro-adamantyl substituent are ADP-STAR and CDP-STAR.

[0008] According to some embodiments, the water-soluble polymeric quaternary ammonium salt is selected from quaternary ammonium salts containing polyvinyl groups. Preferably, the water-soluble polymeric quaternary ammonium salt is selected from at least one of polyvinylbenzyl-trimethylammonium chloride, polyvinylbenzyl-benzyldimethylammonium chloride, and polyvinylbenzyl-tributylammonium chloride.

[0009] According to some embodiments, the ratio of the number of photons generated per second between the upper limit and the lower limit of the linear detection of the chemiluminescent substrate solution is above 30,000, preferably above 50,000.

[0010] According to some embodiments, the lower limit of the linear detection of the chemiluminescent substrate solution is less than or equal to 3000 photons per second, and the upper limit of the linear detection is greater than or equal to 100M photons per second.

[0011] According to some preferred embodiments, the lower limit of the linear detection of the chemiluminescent substrate solution is 1000 - 3000 photons per second, preferably 1000 - 2000 photons per second; the upper limit of the linear detection of the chemiluminescent substrate solution is 100M - 200M photons per second, preferably 120M - 200M photons per second.

[0012] According to some embodiments, the chemiluminescent substrate solution includes 50 - 500 mg / L of a chemiluminescent substrate, 30 - 500 mg / L of fluorescein, and 1 - 10 g / L of a water-soluble polymer quaternary ammonium salt.

[0013] According to some embodiments, the chemiluminescent substrate solution includes 150 - 250 mg / L of a chemiluminescent substrate, 150 - 250 mg / L of fluorescein, and 3 - 7 g / L of a water-soluble polymer quaternary ammonium salt.

[0014] According to some embodiments, the chemiluminescent substrate solution further includes a buffer and a preservative.

[0015] According to some embodiments, the buffer is selected from at least one of the Tris-HCl, AMP-HCl, AMPD-HCl, DEA-HCl, CHES-HCl, boric acid - NaOH, glycine - NaOH buffer systems. Preferably, the buffer is selected from the AMP-HCl buffer system.

[0016] According to some embodiments, the preservative is selected from at least one of sodium azide, Proclin series, potassium sorbate, and sodium benzoate.

[0017] According to some embodiments, the chemiluminescent substrate solution chemiluminesces through the catalysis of alkaline phosphatase.

[0018] According to some embodiments, the concentration of the alkaline phosphatase is as low as 10 -19 mol / L.

[0019] According to some embodiments, the chemiluminescent substrate solution is used to detect human chorionic gonadotropin HCG in a sample to be tested.

[0020] According to some embodiments, the concentration range of human chorionic gonadotropin (HCG) in the sample to be tested is 1 to 200,000 mIU / mL. Preferably, the sample to be tested is a blood sample.

[0021] According to some embodiments, the chemiluminescent substrate solution is used to detect human chorionic gonadotropin (HCG) in the sample to be tested by the double antibody sandwich method.

[0022] According to some embodiments, in the double antibody sandwich method, superparamagnetic microparticles coated with HCG antibody are used as the capture reagent, and HCG antibody labeled with alkaline phosphatase is used as the label.

[0023] According to a second aspect of the present disclosure, there is provided a chemiluminescent detection method, comprising the following steps:

[0024] Mix the sample to be tested with a detection reagent, the detection reagent includes a capture reagent capable of binding to the analyte and an alkaline phosphatase label, so as to obtain an alkaline phosphatase-labeled immune complex;

[0025] Mix the alkaline phosphatase-labeled immune complex with the chemiluminescent substrate solution according to any of the embodiments in the first aspect above, so as to obtain a mixture;

[0026] Measure the optical signal of the mixture, and obtain the analysis result of the analyte in the sample to be tested based on the optical signal.

[0027] According to a third aspect of the present disclosure, there is provided a chemiluminescent detection method, comprising the following steps:

[0028] Mix the sample to be tested with a detection reagent, the detection reagent includes a capture reagent capable of binding to the analyte and an alkaline phosphatase label, so as to obtain an alkaline phosphatase-labeled immune complex;

[0029] Mix the alkaline phosphatase-labeled immune complex with a chemiluminescent substrate solution, so as to obtain a mixture;

[0030] Measure the optical signal of the mixture, and obtain the analysis result of the sample to be tested based on the optical signal,

[0031] wherein the chemiluminescent substrate solution includes a chemiluminescent substrate, a fluorescent agent and a surfactant,

[0032] wherein the ratio of the number of photons generated per second between the linear detection upper limit and the linear detection lower limit of the chemiluminescent substrate solution is above 30,000, or the linear detection lower limit of the chemiluminescent substrate solution is less than or equal to 2000 photons / second, and the linear detection upper limit is greater than or equal to 100 M photons / second.

[0033] According to some embodiments, the ratio of the number of photons generated per second between the upper limit and the lower limit of the linear detection of the chemiluminescent substrate solution is above 50,000, or the lower limit of the linear detection of the chemiluminescent substrate solution is 1000 - 3000 photons per second, preferably 1000 - 2000 photons per second; the upper limit of the linear detection of the chemiluminescent substrate solution is 100M - 200M photons per second, preferably 120M - 200M photons per second.

[0034] According to some embodiments, the optical signal of the assay mixture includes measuring the optical signal of the mixture with a photometer, the lower limit of the linear detection of the photometer is less than or equal to 2000 photons per second, and the upper limit of the linear detection of the photometer is greater than or equal to 100M photons per second.

[0035] According to some embodiments, the chemiluminescent substrate is a dioxetane compound. Preferably, the dioxetane compound is selected from chloro-substituted dioxetane compounds with spiro-adamantane substituents, the fluorescent agent is selected from fluorescein and / or carboxyl-substituted fluorescein, and the surfactant is selected from water-soluble polymeric quaternary ammonium salt surfactants.

[0036] According to some preferred embodiments, the chloro-substituted dioxetane compounds with spiro-adamantane substituents are ADP-STAR and CDP-STAR; the carboxyl-substituted fluoresceins are 5-carboxyfluorescein, 6-carboxyfluorescein, and 5(6)-carboxyfluorescein.

[0037] According to some preferred embodiments, the water-soluble polymeric quaternary ammonium salt is a quaternary ammonium salt containing vinyl groups, preferably at least one of polyvinylbenzyl-trimethylammonium chloride, polyvinylbenzyl-benzyldimethylammonium chloride, and polyvinylbenzyl-tributylammonium chloride.

[0038] According to some embodiments, the chemiluminescent substrate solution includes 50 - 500 mg / L of the chemiluminescent substrate, 30 - 500 mg / L of the fluorescent agent, and 1 - 10 g / L of the water-soluble polymeric quaternary ammonium salt. Preferably, the chemiluminescent substrate solution includes 150 - 250 mg / L of the chemiluminescent substrate, 150 - 250 mg / L of the fluorescent agent, and 3 - 7 g / L of the water-soluble polymeric quaternary ammonium salt.

[0039] According to some embodiments, in the above method, the analyte is human chorionic gonadotropin HCG. Preferably, the concentration range of HCG in the test sample is 1 - 200000 mIU / mL. More preferably, the method is a sandwich immunoassay. Particularly preferably, the capture reagent is superparamagnetic microparticles coated with an HCG antibody.

[0040] The chemiluminescent substrate solution of the present disclosure, through the combination of a chemiluminescent substrate, a fluorescent agent, and a surfactant, can achieve a ratio of the number of photons generated per second between the upper detection limit and the lower detection limit of more than 50,000, and in a preferred embodiment, even more than 60,000, with a wide linearity. Thus, an enzyme-catalyzed chemiluminescent immunoassay system such as alkaline phosphatase can be used to accurately detect low-concentration samples and high-concentration samples without first identifying whether the concentration of the analyte in the sample exceeds the detection limit and then diluting it, simplifying the detection steps and improving the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figures 1A to 1D It is the linear fitting curve of the luminescence values of chemiluminescent substrate solutions 1-1 to 1-4 in Example 1 in the alkaline phosphatase system.

[0042] Figures 2A to 2D It is the linear fitting curve of the luminescence values of chemiluminescent substrate solutions 2-1 to 2-4 in Example 2 in the alkaline phosphatase system.

[0043] Figures 3A to 3D It is the linear fitting curve of the luminescence values of chemiluminescent substrate solutions 3-1 to 3-4 in Example 3 in the alkaline phosphatase system.

[0044] Figures 4A to 4D It is the linear fitting curve of the luminescence values of chemiluminescent substrate solutions 4-1 to 4-4 in Example 4 in the alkaline phosphatase system.

[0045] Figures 5A to 5D It is the linear fitting curve of the luminescence values of chemiluminescent substrate solutions 5-1 to 5-4 in Example 5 in the alkaline phosphatase system. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0046] Next, the technical solutions of the present disclosure will be clearly and completely described in conjunction with specific embodiments of the present disclosure and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present disclosure without making creative efforts belong to the scope of protection of the present disclosure.

[0047] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present disclosure pertains. In case of contradiction, this specification shall prevail.

[0048] In this text, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, such that a method or product comprising a series of elements includes not only the explicitly recited elements, but also other elements not explicitly listed, or elements inherent to the implementation of the method or product.

[0049] Unless otherwise specified, the singular forms "a / an" and "the" as used herein include the plural of the nouns referred to.

[0050] Chemiluminescence Immunoassay (CLIA) is an immunoassay technique that combines a chemiluminescence system with an immune reaction for detecting trace antigens or antibodies, and has high sensitivity. However, it is always difficult to achieve both high sensitivity and a wide linear range in measurement.

[0051] As mentioned above, in current immunoassay detections, the concentrations of some analytes in clinical samples vary greatly, and can even span several orders of magnitude, such as 10 5 ~10 6 For example, human chorionic gonadotropin (HCG). The content of HCG in non-pregnant and pregnant populations can vary from less than 3 mIU / ml to approximately 2,000,000 mIU / ml. The lower limit of the detection range of the mainstream products of chemiluminescence quantitative detection HCG kits is between 0.1 and 2 mIU / ml, and the upper limit of the detection range is between 1000 and 15000 mIU / ml. By investigating the concentration distribution of HCG samples in multiple hospitals over one year, it was found that the proportion of clinical HCG detection samples with a concentration greater than 15000 mIU / ml was as high as 40%. In order to balance the detection accuracy of low-concentration samples, if the samples are first tested undiluted, then approximately 40% of the samples exceed the detection upper limit of the existing method. When such samples are found, they need to be diluted and retested. This seriously leads to a large consumption of testing time, manpower, and reagents, low testing efficiency, and high testing costs.

[0052] Therefore, in clinical detections of items such as HCG, it is highly necessary to provide a detection reagent and method with a wide linear range to make the detection more simple, efficient, and accurate.

[0053] In order to provide an immunoassay method with a wide linear range for samples with a large span of analyte concentration ranges, the present disclosure provides a chemiluminescent substrate solution with a wide linear luminescence value, as well as a chemiluminescence immunoassay method.

[0054] According to a first aspect of the present disclosure, there is provided a chemiluminescent substrate solution, characterized in that it includes a chemiluminescent substrate, a fluorophore, and a water-soluble polymeric quaternary ammonium salt, wherein the chemiluminescent substrate is selected from chlorinated dioxetane compounds having a spiro-adamantane substituent.

[0055] A chemiluminescent substrate refers to a compound that participates in energy transfer in a chemiluminescent reaction and finally releases energy in the form of emitting photons, and is also called a chemiluminescent agent or a luminescent substrate. Dioxetane compounds are enzyme-catalyzed glow-type chemiluminescent substrates and are a class of substrates for ultrasensitive alkaline phosphatase (AP). In a suitable buffer solution, with the catalytic hydrolysis of alkaline phosphatase, the signal emitted by the decomposition of dioxetane compounds can last for more than 20 hours, making them ideal chemiluminescent substances.

[0056] The inventor of the present invention has found that a chlorinated dioxetane compound with a spiro-adamantane substituent, in combination with a specific chemiluminescent enhancer such as fluorescein or its carboxylic acid derivative, in the presence of a water-soluble polymer quaternary ammonium salt cationic surfactant, can obtain a chemiluminescent substrate solution with a ratio of the number of photons generated per second between the upper detection limit and the lower detection limit of more than 30,000, and even more than 50,000, so that it can be conveniently applied to immunoassays where the concentration span of analytes in samples such as HCG is as high as 10 5 ~10 6 orders of magnitude.

[0057] The "dioxetane compound with a spiro-adamantane substituent" mentioned in this article refers to a class of compounds based on the following structure:

[0058]

[0059] For example, it includes the following compounds, but is not limited to:

[0060] AMPPD - (3-(2'-spiroadamatane)-4-methoxy-4-(3"-phosphoryloxy)-phenyl-1,2-dioxetane, CAS = 122341-56-4),

[0061] CSPD - (3-(2'-(spiro-5-chloroadamantane))-4-methoxy-4-(3"-phosphoryloxy)-phenyl-1,2-dioxetane, CAS = 142456-88-0),

[0062] ADP-STAR - (3-(2'-spiroadamatane)-4-methoxy-4-(3"-phosphoryloxy-4"-chloro)-phenyl-1,2-dioxetane, CAS = 189942-84-5),

[0063] CDP-STA-(3-(2'-(spiro-5-chloroadamantane))-4-methoxy-4-(3"-phosphoryloxy-4"-chloro)-phenyl-1,2-dioxetane, CAS = 160081-62-9),

[0064] TFE-AMPPD-(3-(2'-spiroadamatane)-4-trifluoroethoxy-4-(3"-phosphoryloxy)-phenyl-1,2-dioxetane).

[0065] The "chlorinated dioxetane compounds with spiro - adamantane substituents" mentioned in this article refer to those containing at least one chlorine atom in the structure of AMPPD.

[0066] In some embodiments, the chlorinated dioxetane compounds with spiro - adamantane substituents are one of ADP - STAR and CDP - STAR.

[0067]

[0068] In a specific embodiment, in the chemiluminescent substrate solution, the chemiluminescent substrate may exist in the form of a salt of the above - mentioned compound. According to some embodiments, the salt may be an alkali metal salt, such as sodium salt.

[0069] These dioxetane compounds with chlorinated groups, especially ADP - STAR and CDP - STAR, in combination with fluorescein or its carboxyl derivatives and water - soluble polymeric quaternary ammonium salts in an alkaline phosphatase catalytic system, unexpectedly can provide a sufficiently low sensitivity and at the same time can maintain linearity within a relatively high luminescence value range.

[0070] Fluorescent agents and surfactants can improve the efficiency of chemiluminescence. Among them, surfactants form micelles in the solution to protect the chemiluminescent substrate, thereby reducing the quenching reaction of the chemiluminescent substrate in aqueous solution. Fluorescent agents, as photon acceptors, receive the photon energy in the system through energy transfer effects and are excited to generate light signals, thereby enhancing the luminescence efficiency.

[0071] The fluorescent agent in the chemiluminescent substrate solution of the present disclosure is at least one of fluorescein and carboxyl - substituted fluorescein.

[0072] The carboxyl - substituted fluorescein includes compounds selected from those shown in General Formula I:

[0073]

[0074] Specifically, the carboxyfluorescein represented by General Formula I can be 5-carboxyfluorescein, 6-carboxyfluorescein, or 5(6)-carboxyfluorescein. Among them,

[0075] the 5-carboxyfluorescein is

[0076]

[0077] the 6-carboxyfluorescein is

[0078]

[0079] the 5(6)-carboxyfluorescein is a mixture of the 5-carboxyfluorescein and the 6-carboxyfluorescein in any proportion.

[0080] In the chemiluminescent substrate solution, the fluorescent agent is fluorescein. In other embodiments, the fluorescent agent can be any carboxy-substituted fluorescein, especially one of 5-carboxyfluorescein, 6-carboxyfluorescein, and 5(6)-carboxyfluorescein.

[0081] Fluorescein and its carboxy derivatives can improve the chemiluminescent efficiency of the chemiluminescent system. In addition to effectively improving the chemiluminescent efficiency of the chemiluminescent system, the above carboxy derivatives can also reduce the time required to reach the plateau phase and improve the sensitivity.

[0082] In the chemiluminescent substrate solution, the surfactant is a water-soluble polymeric quaternary ammonium salt cationic surfactant. According to one embodiment, the water-soluble polymeric quaternary ammonium salt is selected from quaternary ammonium salts containing vinyl groups. Such quaternary ammonium salts, for example, are vinylbenzyl-trialkylammonium halides. The three alkyl groups substituted on the N atom can be the same or different, and can be independently selected from, for example, methyl, ethyl, propyl, butyl, pentyl, benzyl, etc. In a specific embodiment, the water-soluble polymeric quaternary ammonium salt is selected from at least one of vinylbenzyl-trimethylammonium chloride, vinylbenzyl-benzyldimethylammonium chloride, and vinylbenzyl-tributylammonium chloride.

[0083] The water-soluble polymeric quaternary ammonium salt cationic surfactant can also form micelles in an aqueous solution. The chemiluminescent substrate is inside the hydrophobic micelles, and the fluorescent agent is in the hydrophilic part outside the micelles, which is more conducive to the chemiluminescent substrate transferring energy to the fluorescent agent to enhance the light signal. Compared with small molecule quaternary ammonium salts, due to the longer alkane chain of the polymeric quaternary ammonium salt, it can more effectively prevent the chemiluminescent substrate from quenching in an aqueous solution. In addition, the polymeric quaternary ammonium salt can also suppress the background signal and enhance the detection signal. Under the same conditions, the small molecule quaternary ammonium salt has a high background signal value and a low detection signal, and cannot achieve a wide linear detection range.

[0084] In a specific embodiment, the chemiluminescent substrate solution includes ADP-STAR or CDP-STAR as the chemiluminescent substrate, fluorescein, and a water-soluble polymeric quaternary ammonium salt.

[0085] In another specific embodiment, the chemiluminescent substrate solution includes ADP-STAR, fluorescein or carboxyl-substituted fluorescein as the fluorescent agent, and a water-soluble polymeric quaternary ammonium salt.

[0086] The specific combination of the above chemiluminescent substrate, fluorescent agent, and surfactant provides a chemiluminescent substrate solution with a wide linear luminescence value. The chemiluminescent substrate solution can obtain a linear range with a lower limit of less than 3,000 photons per second and an upper limit of more than 100,000,000 (100M) photons per second in an alkaline phosphatase system. Exemplarily, the chemiluminescent substrate solution can obtain a linear range with a lower limit of 1,000 - 3,000 photons per second and an upper limit of 100,000,000 (100M) - 200,000,000 (200M) photons per second in an alkaline phosphatase system. As shown in the following examples, the chemiluminescent substrate solution can obtain a background luminescence value of 1,000 - 2,000 photons per second and a linear range with an upper limit of 120,000,000 (120M) - 200,000,000 (200M) photons per second in an alkaline phosphatase system. It should be understood that the above lower limit range and upper limit range are only exemplary, and by adjusting the detection device or signal processing method, etc., a lower lower limit of photons per second or a higher upper limit of photons per second can be obtained.

[0087] In some embodiments, the ratio of the number of photons generated per second between the detection upper limit and the detection lower limit of the chemiluminescent substrate solution is above 30,000, for example, above 50,000, or even above 60,000. In some embodiments, the ratio of the number of photons generated per second between the detection upper limit and the detection lower limit of the chemiluminescent substrate solution is below 1,000,000, for example, below 800,000, 600,000, 500,000, or even below 400,000. Exemplarily, the ratio of the number of photons generated per second between the detection upper limit and the detection lower limit of the chemiluminescent substrate solution is 30,000 - 1,000,000, 30,000 - 800,000, 30,000 - 500,000, 50,000 - 1,000,000, 50,000 - 800,000, 50,000 - 500,000, etc.

[0088] In some embodiments, the chemiluminescent substrate solution comprises 50-500 mg / L of a chemiluminescent substrate. Preferably, the chemiluminescent substrate solution comprises 150-250 mg / L of a chemiluminescent substrate, and more preferably, 200 mg / L of a chemiluminescent substrate. Exemplarily, the chemiluminescent substrate solution comprises 150 mg / L, 160 mg / L, 170 mg / L, 180 mg / L, 190 mg / L, 200 mg / L, 210 mg / L, 220 mg / L, 230 mg / L, 240 mg / L, 250 mg / L of a chemiluminescent substrate.

[0089] In some embodiments, the chemiluminescent substrate solution comprises 30-500 mg / L of a fluorescent agent. Preferably, the chemiluminescent substrate solution comprises 150-250 mg / L of a fluorescent agent, and more preferably, 200 mg / L of a fluorescent agent. Exemplarily, the chemiluminescent substrate solution comprises 150 mg / L, 160 mg / L, 170 mg / L, 180 mg / L, 190 mg / L, 200 mg / L, 210 mg / L, 220 mg / L, 230 mg / L, 240 mg / L, 250 mg / L of a fluorescent agent.

[0090] In some embodiments, the chemiluminescent substrate solution comprises 1-10 g / L of a water-soluble polymeric quaternary ammonium salt. Preferably, the chemiluminescent substrate solution comprises 3-7 g / L of a water-soluble polymeric quaternary ammonium salt, and more preferably, 5 g / L of a water-soluble polymeric quaternary ammonium salt. Exemplarily, the chemiluminescent substrate solution comprises 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L of a water-soluble polymeric quaternary ammonium salt.

[0091] In a specific embodiment, the chemiluminescent substrate solution comprises 50-500 mg / L, preferably 150-250 mg / L of ADP-STAR or CDP-STAR, 30-500 mg / L, preferably 150-250 mg / L of fluorescein, and 1-10 g / L, preferably 3-7 g / L of a water-soluble polymeric quaternary ammonium salt.

[0092] In another specific embodiment, the chemiluminescent substrate solution comprises 50-500 mg / L, preferably 150-250 mg / L of ADP-STAR, 30-500 mg / L, preferably 150-250 mg / L of fluorescein or carboxyl-substituted fluorescein, and 1-10 g / L, preferably 3-7 g / L of a water-soluble polymeric quaternary ammonium salt.

[0093] The chemiluminescent substrate solution further contains additives such as a buffer and a preservative.

[0094] The present disclosure does not particularly limit the type of buffer, and any buffer conventionally applicable can be used in the chemiluminescent substrate solution of the present disclosure. In some embodiments, the buffer can be selected from Tris buffer, AMP buffer (AMP is 2-amino-2-methylpropanol), AMPD buffer (AMPD is 2-amino-2-methyl-1,3-propanediol), DEA buffer (DEA is diethanolamine), CHES buffer (CHES is 2-(N-cyclohexylamino)ethanesulfonic acid), Mopso buffer, imidazole buffer, phosphate buffer, carbonate buffer, malic acid buffer, glycine buffer, but not limited thereto. Exemplarily, the buffer can be selected from at least one of Tris-HCl, AMP-HCl, AMPD-HCl, DEA-HCl, CHES-HCl, boric acid-NaOH, glycine-NaOH buffer systems. Preferably, the buffer solution is selected from the AMP-HCl buffer system. The AMP-HCl buffer system can play a better buffering role in the range of pH 9.0 to 10.0, and it is found that it is beneficial to the stability of chemiluminescent substrate molecules.

[0095] According to some embodiments, the pH of the buffer is 7.1 to 10.6, preferably about 9.0 to about 10.0, and particularly preferably about 9.5.

[0096] The present disclosure does not particularly limit the amount of the buffer, which can be determined according to the selected buffer system.

[0097] The present disclosure does not particularly limit the type of preservative, and any preservative conventionally used in detection reagents can be used in the present disclosure. Examples include sodium azide, Proclin series, potassium sorbate, sodium benzoate, BND, erythromycin, gentamicin, etc., but not limited thereto. Preferably, they are sodium azide and Proclin series.

[0098] The present disclosure does not particularly limit the amount of the buffer, which can be determined according to the selected type of preservative.

[0099] In some embodiments, the chemiluminescent substrate solution may further include magnesium chloride. For example, it may include 0.1 to 2 mM of magnesium chloride.

[0100] According to a specific embodiment, the chemiluminescent substrate solution comprises ADP-STAR or CDP-STAR at 50 - 500 mg / L, preferably 150 - 250 mg / L; fluorescein at 30 - 500 mg / L, preferably 150 - 250 mg / L; a water-soluble polymeric quaternary ammonium salt at 1 - 10 g / L, preferably 3 - 7 g / L; an appropriate amount of (such as 30 - 500 mM) AMP-HCl buffer; an appropriate amount of (such as 0.5 - 2 g / L) sodium azide, with a pH of about 9.0 to about 10.0, such as about 9.5.

[0101] In another specific embodiment, the chemiluminescent substrate solution comprises ADP-STAR at 50 - 500 mg / L, preferably 150 - 250 mg / L; fluorescein or carboxyl-substituted fluorescein at 30 - 500 mg / L, preferably 150 - 250 mg / L; a water-soluble polymeric quaternary ammonium salt at 1 - 10 g / L, preferably 3 - 7 g / L; an appropriate amount of (such as 30 - 500 mM) AMP-HCl buffer; an appropriate amount of (such as 0.5 - 2 g / L) sodium azide, with a pH of about 9.0 to about 10.0, such as about 9.5.

[0102] According to some embodiments, the chemiluminescent substrate solution of the present disclosure has a low background luminescence value, high luminescence efficiency, a wide linear range, is suitable for the alkaline phosphatase-catalyzed chemiluminescence system, and is particularly suitable for chemiluminescent immunoassay.

[0103] According to some specific embodiments, the chemiluminescent substrate solution provided by the present disclosure reacts with alkaline phosphatase in the concentration range of 0.03 - 400 ng / mL, and the luminescence value shows good linearity (R 2 ≥ 0.99). As shown in the examples, the background value of the chemiluminescent substrate solution can be as low as below 3,000 photons per second, and it can detect an ultra-low concentration of alkaline phosphatase as low as 10 -19 mol / L, improving the detection sensitivity; at the same time, the detection value of the chemiluminescent substrate solution can be as high as above 100 M photons per second, such as 100 M - 200 M photons per second, 120 M - 200 M photons per second, showing a significantly wider linearity. Without additional dilution of the sample, the above chemiluminescent substrate solution can meet the detection of samples with high analyte concentrations.

[0104] The chemiluminescent substrate solution of the present disclosure is particularly suitable for detecting human chorionic gonadotropin HCG in a test sample.

[0105] HCG is a glycoprotein mainly secreted by placental trophoblast cells. Its content is very low in non-pregnant people, usually less than 3 mIU / ml, while during pregnancy, the HCG concentration can be as high as about 2,000,000 mIU / ml. Clinically, the HCG test results are used to assist in judging pregnancy. At the same time, the detection of HCG during pregnancy also has important significance. As mentioned above, the chemiluminescent substrate solution of the present disclosure has a wide linear luminescence characteristic and is suitable for samples with a large span of analyte concentration changes such as HCG.

[0106] According to some embodiments, the concentration range of HCG in the sample is 1 to 200,000 mIU / mL.

[0107] In some embodiments, the detection of HCG is by the sandwich immunoassay. According to specific embodiments, superparamagnetic microparticles coated with HCG antibody are used as the capture reagent in the detection, and an HCG antibody labeled with alkaline phosphatase is used as the label. In some embodiments, the HCG antibody is a monoclonal antibody.

[0108] The chemiluminescent substrate solution of the present disclosure combined with the sandwich immunoassay for HCG detection has high sensitivity, a wide linear range, does not require additional dilution of the sample, simplifies the operation, saves detection reagents, and avoids interference and decreased accuracy caused by additional dilution.

[0109] In the present disclosure for the use of the chemiluminescent substrate solution in the immunoassay of HCG, there are no particular limitations on the specific forms and dosages of corresponding immunoassay reagents such as HCG antibodies, specific capture reagents, alkaline phosphatase labels, etc., and any suitable reagents can be used.

[0110] The "sample" mentioned herein, unless otherwise specified, refers to a biological sample, which can be from a mammal, preferably a blood sample from a human, and more preferably a serum sample.

[0111] In addition to HCG, the chemiluminescent substrate solution of the present disclosure is also suitable for samples with a large span of other analytes, such as the detection of HBsAg, TSH, etc., and is not limited to the detection of HCG.

[0112] In a third aspect of the present disclosure, a chemiluminescent detection method using the above chemiluminescent substrate solution is further provided. According to one embodiment, the chemiluminescent detection method includes the following steps:

[0113] Mix the test sample with the detection reagent, and the detection reagent includes a capture reagent capable of binding to the analyte and an alkaline phosphatase label, so as to obtain an alkaline phosphatase-labeled immune complex;

[0114] Mix the alkaline phosphatase-labeled immune complex with the chemiluminescent substrate solution in any of the above embodiments to obtain a mixed solution;

[0115] Measure the optical signal of the mixed solution and obtain the analysis result of the sample to be tested based on the optical signal.

[0116] In some specific embodiments, the chemiluminescent substrate solution includes ADP-STAR or CDP-STAR as the chemiluminescent substrate, fluorescein, and water-soluble polymeric quaternary ammonium salt.

[0117] In another specific embodiment, the chemiluminescent substrate solution includes ADP-STAR, fluorescein or carboxyl-substituted fluorescein as the fluorescent agent, and water-soluble polymeric quaternary ammonium salt.

[0118] In some embodiments, the chemiluminescent substrate solution includes 50 - 500 mg / L of ADP-STAR or CDP-STAR. Preferably, the chemiluminescent substrate solution includes 150 - 250 mg / L, more preferably 200 mg / L of ADP-STAR or CDP-STAR.

[0119] In some embodiments, the chemiluminescent substrate solution includes 30 - 500 mg / L of fluorescein. Preferably, the chemiluminescent substrate solution includes 150 - 250 mg / L of fluorescein, more preferably, includes 200 mg / L of fluorescein.

[0120] In some embodiments, the chemiluminescent substrate solution includes 1 - 10 g / L of water-soluble polymeric quaternary ammonium salt. Preferably, the chemiluminescent substrate solution includes 3 - 7 g / L of water-soluble polymeric quaternary ammonium salt, more preferably, includes 5 g / L of water-soluble polymeric quaternary ammonium salt. The water-soluble polymeric quaternary ammonium salt is as described above.

[0121] Similarly, the chemiluminescent substrate solution also contains additives such as buffers and preservatives. The buffers, preservatives and other additives are as described above and will not be elaborated here.

[0122] According to a specific embodiment, the chemiluminescent substrate solution includes 50 - 500 mg / L, preferably 150 - 250 mg / L of ADP-STAR or CDP-STAR; 30 - 500 mg / L, preferably 150 - 250 mg / L of fluorescein; 1 - 10 g / L, preferably 3 - 7 g / L of water-soluble polymeric quaternary ammonium salt; an appropriate amount of (such as 30 - 500 mM) AMP-HCl buffer; an appropriate amount of (such as 0.5 - 2 g / L) sodium azide, with a pH of about 9.0 - about 10.0, such as about 9.5.

[0123] In another specific embodiment, the chemiluminescent substrate solution comprises ADP-STAR at 50 - 500 mg / L, preferably 150 - 250 mg / L; fluorescein or carboxyl-substituted fluorescein at 30 - 500 mg / L, preferably 150 - 250 mg / L; water-soluble polymeric quaternary ammonium salt at 1 - 10 g / L, preferably 3 - 7 g / L; an appropriate amount of (such as 30 - 500 mM) AMP-HCl buffer; an appropriate amount of (such as 0.5 - 2 g / L) sodium azide, with a pH of about 9.0 - about 10.0, such as about 9.5.

[0124] In a fourth aspect of the present disclosure, there is also provided a chemiluminescent detection method of another embodiment. The chemiluminescent detection method comprises the following steps:

[0125] Mix the sample to be tested with a detection reagent, which includes a capture reagent capable of binding to the analyte and an alkaline phosphatase label, thereby obtaining an alkaline phosphatase-labeled immune complex;

[0126] Mix the alkaline phosphatase-labeled immune complex with the chemiluminescent substrate solution;

[0127] Measure the optical signal of the mixture, and obtain the analysis result of the sample to be tested based on the optical signal,

[0128] wherein the chemiluminescent substrate solution comprises a chemiluminescent substrate, a fluorescent agent, and a surfactant, and the ratio of the photon count per second of the upper detection limit to the lower detection limit of the chemiluminescent substrate solution is above 30,000, or the linear detection lower limit of the chemiluminescent substrate solution is less than or equal to 2000 photons per second, and the linear detection upper limit of the photometer is greater than or equal to 100 M photons per second.

[0129] The chemiluminescent detection method uses a chemiluminescent substrate solution with a wide linear range of luminescence values, and the ratio of the photon count per second of the upper detection limit to the lower detection limit is above 30,000, so that it can be used for immunoassays with a concentration span of the analyte in the sample up to 10 5 ~10 6 orders of magnitude.

[0130] In some embodiments, the ratio of the photon count per second of the linear detection upper limit to the linear detection lower limit of the chemiluminescent substrate solution is above 50,000, and even up to above 60,000. In some embodiments, the ratio of the photon count per second of the upper detection limit to the lower detection limit of the chemiluminescent substrate solution is below 1,000,000, such as below 800,000, below 600,000, below 500,000, and even below 400,000.

[0131] Alternatively, the lower limit of linear detection of the chemiluminescent substrate solution is 1,000 to 3,000 photons per second, preferably 1,000 to 2,000 photons per second; the upper limit of linear detection of the chemiluminescent substrate solution is 100M to 200M photons per second, preferably 120M to 200M photons per second.

[0132] In some embodiments, the light signal of the assay mixture includes measuring the light signal of the mixture with a photometer, the lower detection limit of the photometer is less than or equal to 2,000 photons per second, and the upper detection limit of the photometer is greater than or equal to 100M photons per second.

[0133] Using the photometer can better match the wide range of luminescence values of the chemiluminescent substrate solution of the present disclosure, further simplify the detection steps, and improve the detection efficiency and accuracy.

[0134] It should be understood that, as described above, the chemiluminescence detection method of the present disclosure can also be used with a detection device equipped with a photometer having other detection ranges for wide linear range detection.

[0135] In some embodiments, the chemiluminescent substrate is a dioxetane compound, preferably a chlorinated dioxetane compound with a spiro - adamantane substituent, the fluorescent agent is selected from fluorescein and / or carboxyl - substituted fluorescein, and the surfactant is selected from water - soluble polymeric quaternary ammonium salts.

[0136] As described above, preferably, the chlorinated dioxetane compound with a spiro - adamantane substituent is ADP - STAR and CDP - STAR; the carboxyl - substituted fluorescein is 5 - carboxyfluorescein, 6 - carboxyfluorescein, and 5(6) - carboxyfluorescein.

[0137] Preferably, the water - soluble polymeric quaternary ammonium salt is a quaternary ammonium salt containing a vinyl group, preferably at least one of polyvinylbenzyl - trimethylammonium chloride, polyvinylbenzyl - benzyldimethylammonium chloride, and polyvinylbenzyl - tributylammonium chloride.

[0138] The contents of the chemiluminescent substrate, light agent, and water - soluble polymeric quaternary ammonium salt in the chemiluminescent substrate solution are as described above and will not be elaborated here.

[0139] In some embodiments, in the detection methods of the third and fourth aspects of the present disclosure, the analyte is human chorionic gonadotropin HCG.

[0140] In some embodiments, the concentration range of HCG in the sample to be tested is 1 to 200,000 mIU / mL. According to specific embodiments, the method is a double antibody sandwich method. According to specific embodiments, the capture reagent is superparamagnetic microparticles coated with HCG antibody. According to more specific embodiments, the HCG antibody is a monoclonal antibody.

[0141] The sample to be tested is as defined above.

[0142] Example

[0143] The following specific examples are used to illustrate various embodiments and advantages of the present disclosure, but the scope of the present disclosure is not limited by these.

[0144] Example 1: Linear performance using different chemiluminescent substrates

[0145] Chemiluminescent substrate solution formulation:

[0146] Chemiluminescent substrate: 200 mg

[0147] 5(6)-Carboxyfluorescein: 200 mg

[0148] Polyvinylbenzyltrimethylammonium chloride: 5 g

[0149] AMP-HCl: 50 mM

[0150] Magnesium chloride: 200 mg

[0151] Sodium azide: 1 g

[0152] Water: Add to a total volume of 1 L

[0153] According to the above formula, where the chemiluminescent substrates are shown in Table 1 below, a series of chemiluminescent substrate solutions 1-1 to 1-4 are prepared.

[0154] Table 1

[0155] Chemiluminescent substrate solution Chemiluminescent substrate 1-1 AMPPD 1-2 CSPD 1-3 ADP-STAR 1-4 CDP-STAR

[0156] Using the above chemiluminescent substrate solutions 1-1 to 1-4, tests are performed on the Mindray CL-6000i fully automatic chemiluminescent immunoassay analyzer. Different concentration gradients of alkaline phosphatase (AP enzyme) solutions are mixed with each chemiluminescent substrate solution respectively. After incubation for a period of time, light signals are collected, and the background signal values (i.e., the alkaline phosphatase concentration is 0) and luminescence signal values at a 2-minute light collection time are obtained respectively. The concentration of alkaline phosphatase in the system and the detection signal values are shown in Table 2 below.

[0157] Table 2

[0158]

[0159]

[0160] The light signals (photons / second) measured for the chemiluminescent substrate solutions from 1-1 to 1-4 were plotted against the concentration of alkaline phosphatase in the system and linearly fitted, as Figures 1A to 1D shown.

[0161] According to the data in Table 2 and Figures 1A to 1D it can be seen that the chemiluminescent substrate solutions 1-2, 1-3 and 1-4 have low background signals, while the chemiluminescent substrate solutions 1-3 and 1-4 simultaneously have the characteristics of high luminescence efficiency and have good linearity (R 2 ≥0.99) within the detected luminescence range, and ADP-STAR performs the best.

[0162] Example 2: Linear performance of ADP-STAR and different fluorescent agents

[0163] Chemiluminescent substrate solution formulation:

[0164] ADP-STAR: 200 mg

[0165] Fluorescent agent: 200 mg

[0166] Polyvinylbenzyltrimethylammonium chloride: 5 g

[0167] AMP-HCl: 50 mM

[0168] Magnesium chloride: 200 mg

[0169] Sodium azide: 1 g

[0170] Water: Added to a total volume of 1 L

[0171] According to the above formulation, with the fluorescent agents as shown in Table 3 below, a series of chemiluminescent substrate solutions 2-1 to 2-4 were prepared.

[0172] Table 3

[0173] Chemiluminescent substrate solution Fluorescent agent 2-1 Fluorescein 2-2 5-Carboxyfluorescein 2-3 6-Carboxyfluorescein 2-4 5(6)-Carboxyfluorescein

[0174] Using the above chemiluminescent substrate solutions 2-1 to 2-4, tests were carried out on the Mindray CL-6000i fully automatic chemiluminescent immunoassay analyzer. Alkaline phosphatase solutions with different concentration gradients were mixed with each chemiluminescent substrate solution respectively. After incubation for a period of time, light signals were collected, and the background signal values (i.e., the alkaline phosphatase concentration was 0) and luminescence signal values under a 2-minute light collection time were obtained respectively. The concentration of alkaline phosphatase in the system and the detected signal values are shown in Table 4 below.

[0175] Table 4

[0176]

[0177] The optical signals (photons / second) measured for the chemiluminescent substrate solutions 2-1 to 2-4 were plotted against the concentration of alkaline phosphatase in the system and linearly fitted, as Figures 2A to 2D shown.

[0178] According to the data in Table 4 and Figures 2A to 2D it can be seen that the chemiluminescent substrate solutions 2-1 to 2-4 all exhibit the characteristics of low background signal and high luminescence efficiency, and have good linearity (R 2 ≥0.99) within the detected luminescence range.

[0179] Example 3: Linear performance of CDP-STAR and different fluorescent agents

[0180] Prepare the chemiluminescent substrate solution according to the following formula:

[0181] CDP-STAR: 200 mg

[0182] Fluorescent agent: 200 mg

[0183] Polyvinylbenzyltrimethylammonium chloride: 5 g

[0184] AMP-HCl: 50 mM

[0185] Magnesium chloride: 200 mg

[0186] Sodium azide: 1 g

[0187] Water: Add to a total volume of 1 L

[0188] According to the above formula, with the fluorescent agents as shown in Table 5 below, a series of chemiluminescent substrate solutions 3-1 to 3-4 were prepared.

[0189] Table 5

[0190] Chemiluminescent substrate solution Fluorescent agent 3-1 Fluorescein 3-2 5-Carboxyfluorescein 3-3 6-Carboxyfluorescein 3-4 5(6)-Carboxyfluorescein

[0191] Using the above chemiluminescent substrate solutions 3-1 to 3-4, tests were carried out on the Mindray CL-6000i fully automatic chemiluminescent immunoassay analyzer. Alkaline phosphatase solutions with different concentration gradients were mixed with each chemiluminescent substrate solution respectively. After incubation for a period of time, the optical signals were collected, and the background signal values (i.e., the alkaline phosphatase concentration was 0) and luminescence signal values under a 2-min light collection time were obtained respectively. The concentration of alkaline phosphatase in the system and the detected signal values are shown in Table 6 below.

[0192] Table 6

[0193]

[0194] The optical signals (photons per second) measured for the chemiluminescent substrate solutions 3-1 to 3-4 were plotted against the concentration of alkaline phosphatase in the system and linearly fitted, as Figures 3A to 3D shown.

[0195] According to the data in Table 6 and Figures 3A to 3D it can be seen that for the chemiluminescent substrate solutions 3-1 to 3-4, they all exhibit the characteristics of low background signal and high luminescence efficiency, and have good linearity (R 2 ≥ 0.99) within the detected luminescence range.

[0196] Example 4: Linear performance chemiluminescent substrate solution formulation using ADP-STAR and different quaternary ammonium salt cationic surfactants:

[0197] ADP-STAR: 200 mg

[0198] Fluorescein: 200 mg

[0199] Quaternary ammonium salt surfactant: 5 g

[0200] AMP-HCl: 50 mM

[0201] Magnesium chloride: 200 mg

[0202] Sodium azide: 1 g

[0203] Water: Make up to a total volume of 1 L

[0204] According to the above formulation, where the quaternary ammonium salt cationic surfactants are as shown in Table 7 below, a series of chemiluminescent substrate solutions 4-1 to 4-4 were prepared.

[0205] Table 7

[0206]

[0207] Using the above chemiluminescent substrate solutions 4-1 to 4-4, tests were carried out on the Mindray CL-6000i fully automatic chemiluminescent immunoassay analyzer. Different concentration gradients of alkaline phosphatase solutions were mixed with each chemiluminescent substrate solution, and after incubation for a period of time, the optical signals were collected. The background signal values (i.e., the alkaline phosphatase concentration was 0) and the luminescence signal values under a 2-minute light collection time were obtained respectively. The concentration of alkaline phosphatase in the system and the detected signal values are as shown in Table 8 below.

[0208] Table 8

[0209]

[0210] The optical signals (photons per second) measured for the chemiluminescent substrate solutions 4-1 to 4-4 were plotted against the concentration of alkaline phosphatase in the system and linearly fitted, as Figures 4A to 4DAs shown

[0211] According to the data in Table 8 and Figures 4A to 4D it can be seen that the chemiluminescent substrate solutions 4-1 to 4-3 have low background signals and high luminescence efficiencies, and have good linearity (R 2 ≥0.99) within the detected luminescence range. The chemiluminescent substrate solution 4-4 has a high background signal and a low luminescence efficiency, and cannot maintain good linearity (R 2 <0.99) within the detected luminescence range, and the optical signal ratio between the upper detection limit and the lower detection limit is also small (less than 3000).

[0212] Example 5: Linear performance chemiluminescent substrate solution formulation using CDP-STAR and different quaternary ammonium salt cationic surfactants:

[0213] CDP-STAR: 200 mg

[0214] Fluorescein: 200 mg

[0215] Quaternary ammonium salt surfactant: 5 g

[0216] AMP-HCl: 50 mM

[0217] Magnesium chloride: 200 mg

[0218] Sodium azide: 1 g

[0219] Water: Add to a total volume of 1 L

[0220] According to the above formula, where the quaternary ammonium salt cationic surfactants are as shown in Table 9 below, a series of chemiluminescent substrate solutions 5-1 to 5-4 are prepared.

[0221] Table 9

[0222]

[0223] Using the above chemiluminescent substrate solutions 5-1 to 5-4, tests are carried out on the Mindray CL-6000i fully automatic chemiluminescent immunoassay analyzer. Different concentration gradients of alkaline phosphatase solutions are mixed with each chemiluminescent substrate solution respectively. After incubation for a period of time, optical signals are collected, and the background signal values (i.e., the alkaline phosphatase concentration is 0) and luminescence signal values under a 2-minute light collection time are obtained respectively. The concentration of alkaline phosphatase and the detection signal values in the system are as shown in Table 10 below.

[0224] Table 10

[0225]

[0226] The light signals (photons per second) measured for the chemiluminescent substrate solutions 5-1 to 5-4 were plotted against the concentration of alkaline phosphatase in the system, and linear fitting was performed, as Figures 5A to 5D shown.

[0227] According to the data in Table 10 and Figures 5A to 5D it can be seen that the chemiluminescent substrate solutions 5-1 to 5-3 have low background signals and high luminescence efficiencies, and have good linearity (R 2 ≥ 0.99) within the detected luminescence range. The chemiluminescent substrate solution 5-4 has a high background signal and low luminescence efficiency, and cannot maintain good linearity (R 2 < 0.99) within the detected luminescence range, and the ratio of the light signals at the upper detection limit and the lower detection limit is also small (less than 3000).

[0228] Example 6: Wide linear detection of HCG

[0229] Prepare the chemiluminescent substrate solution 6-1 according to the following formula:

[0230] ADP-STAR: 200 mg

[0231] Fluorescein: 200 mg

[0232] Polyvinylbenzyltrimethylammonium chloride: 5 g

[0233] AMP-HCl: 50 mM

[0234] Magnesium chloride: 200 mg

[0235] Sodium azide: 1 g

[0236] Water: Add to a total volume of 1 L

[0237] Prepare the chemiluminescent substrate solution 6-2 according to the following formula:

[0238] CDP-STAR: 200 mg

[0239] Fluorescein: 200 mg

[0240] Polyvinylbenzyltrimethylammonium chloride: 5 g

[0241] AMP-HCl: 50 mM

[0242] Magnesium chloride: 200 mg

[0243] Sodium azide: 1 g

[0244] Water: Add to a total volume of 1 L

[0245] Using the above chemiluminescent substrate solutions 6-1 to 6-2, serum samples with different HCG concentrations were tested on Mindray CL-6000i fully automatic chemiluminescent immunoassay analyzer. The sample concentration range was 1 to 200,000 mIU / mL, and the HCG detection kit was Mindray total β-human chorionic gonadotropin (TotalβHCG) assay kit (chemiluminescent immunoassay method). The number of photons per second of the optical signal collected when each sample was incubated with chemiluminescent substrate solutions 6-1 and 6-2 for 2 minutes was recorded, and the concentration of HCG in the sample was calculated based on the optical signal, denoted as the back-calculated concentration. The relative deviation of the back-calculated concentration was calculated relative to the nominal concentration of HCG in the sample, and the results are shown in Table 11 - Table 12 below.

[0246] Table 11

[0247]

[0248]

[0249] Table 12

[0250]

[0251] It can be seen from Table 11 and Table 12 that the chemiluminescent substrate solution of the present disclosure can be directly detected and analyzed without dilution within the concentration range of 1 to 200,000 mIU / mL of HCG in the sample during the detection of the HCG item, and the deviation of the detection result relative to the nominal concentration is within ±5%.

[0252] The above are only examples of some embodiments of the present disclosure, and do not limit the patent scope of the present disclosure. Any equivalent structural transformation made using the content of the specification and drawings of the present disclosure under the inventive concept of the present disclosure, or any direct / indirect application in other related technical fields, is included in the patent protection scope of the present disclosure.

Claims

1. A chemiluminescent substrate solution, characterized in that, It includes a chemiluminescent substrate, a fluorescein, and a water-soluble polymeric quaternary ammonium salt, wherein the chemiluminescent substrate is selected from chlorinated dioxetane compounds having a spiro-adamantyl substituent.

2. The chemiluminescent substrate solution according to claim 1, wherein The chlorinated dioxetane compounds having a spiro-adamantyl substituent are ADP-STAR and CDP-STAR.

3. The chemiluminescent substrate solution according to claim 1, characterized in that, The water-soluble polymeric quaternary ammonium salt is selected from quaternary ammonium salts containing polyvinyl groups, preferably at least one selected from polyvinylbenzyl-trimethylammonium chloride, polyvinylbenzyl-benzyldimethylammonium chloride, and polyvinylbenzyl-tributylammonium chloride.

4. The chemiluminescent substrate solution according to any one of claims 1-3, characterized in that, The ratio of the number of photons generated per second between the upper limit and the lower limit of the linear detection of the chemiluminescent substrate solution is above 30,000, preferably above 50,000.

5. The chemiluminescent substrate solution according to any one of claims 1 to 3, characterized in that The lower limit of the linear detection of the chemiluminescent substrate solution is less than or equal to 3000 photons / second, and the upper limit of the linear detection is greater than or equal to 100M photons / second.

6. The chemiluminescent substrate solution according to claim 5, wherein The lower limit of the linear detection of the chemiluminescent substrate solution is 1000 - 3000 photons / second, preferably 1000 - 2000 photons / second; the upper limit of the linear detection of the chemiluminescent substrate solution is 100M - 200M photons / second, preferably 120M - 200M photons / second.

7. The chemiluminescent substrate solution according to any one of claims 1-6, characterized in that, The chemiluminescent substrate solution includes 50 - 500 mg / L of the chemiluminescent substrate, 30 - 500 mg / L of the fluorescein, and 1 - 10 g / L of the water-soluble polymeric quaternary ammonium salt.

8. The chemiluminescent substrate solution according to claim 7, wherein The chemiluminescent substrate solution includes 150 - 250 mg / L of the chemiluminescent substrate, 150 - 250 mg / L of the fluorescein, and 3 - 7 g / L of the water-soluble polymeric quaternary ammonium salt.

9. The chemiluminescent substrate solution according to any one of claims 1-8, characterized in that, The chemiluminescent substrate solution further includes a buffer and a preservative.

10. The chemiluminescent substrate solution according to claim 9, wherein The buffer is selected from at least one of the Tris-HCl, AMP-HCl, AMPD-HCl, DEA-HCl, CHES-HCl, boric acid-NaOH, glycine-NaOH buffer systems. Preferably, the buffer is selected from the AMP-HCl buffer system.

11. The chemiluminescent substrate solution according to claim 9, wherein The preservative is selected from at least one of sodium azide, Proclin series, potassium sorbate, and sodium benzoate.

12. The chemiluminescent substrate solution according to any one of claims 1-11, characterized in that, The chemiluminescent substrate solution chemiluminesces through the catalysis of alkaline phosphatase.

13. The chemiluminescent substrate solution according to claim 12, wherein The alkaline phosphatase concentration is as low as 10 -19 mol / L.

14. The chemiluminescent substrate solution according to any one of claims 1-13, characterized in that, The chemiluminescent substrate solution is used for detecting human chorionic gonadotropin (HCG) in a test sample.

15. The chemiluminescent substrate solution according to claim 14, wherein The concentration range of the human chorionic gonadotropin (HCG) in the test sample is 1 - 200,000 mIU / mL. Preferably, the test sample is a blood sample.

16. The chemiluminescent substrate solution according to claim 14 or 15, wherein The chemiluminescent substrate solution is used for detecting human chorionic gonadotropin (HCG) in the test sample by the double antibody sandwich method.

17. The chemiluminescent substrate solution according to claim 16, wherein In the double antibody sandwich method, superparamagnetic particles coated with HCG antibody are used as the capture reagent, and an HCG antibody labeled with alkaline phosphatase is used as the label.

18. A chemiluminescence detection method, characterized in that, The chemiluminescent detection method includes: Mixing the test sample with a detection reagent, which includes a capture reagent that can bind to the analyte and an alkaline phosphatase label, to obtain an alkaline phosphatase-labeled immune complex; Mixing the alkaline phosphatase-labeled immune complex with the chemiluminescent substrate solution according to any one of claims 1 - 11 to obtain a mixture; Measure the optical signal of the mixture and obtain the analysis result of the analyte in the sample to be tested based on the optical signal.

19. A chemiluminescence detection method, characterized in that, The chemiluminescence detection method includes: Mix the sample to be tested with a detection reagent, where the detection reagent includes a capture reagent capable of binding to the analyte and an alkaline phosphatase label, so as to obtain an alkaline phosphatase-labeled immune complex; Mix the alkaline phosphatase-labeled immune complex with a chemiluminescent substrate solution to obtain a mixture; Measure the optical signal of the mixture and obtain the analysis result of the sample to be tested based on the optical signal, wherein the chemiluminescent substrate solution includes a chemiluminescent substrate, a fluorescent agent, and a surfactant, wherein the ratio of the number of photons generated per second between the upper linear detection limit and the lower linear detection limit of the chemiluminescent substrate solution is above 30,000, or the lower linear detection limit of the chemiluminescent substrate solution is less than or equal to 2000 photons per second, and the upper linear detection limit is greater than or equal to 100M photons per second.

20. The method according to claim 19, wherein The ratio of the number of photons generated per second between the upper linear detection limit and the lower linear detection limit of the chemiluminescent substrate solution is above 50,000, or the lower linear detection limit of the chemiluminescent substrate solution is 1000 - 3000 photons per second, preferably 1000 - 2000 photons per second; the upper linear detection limit of the chemiluminescent substrate solution is 100M - 200M photons per second, preferably 120M - 200M photons per second.

21. The method according to claim 19 or 20, characterized in that, Measuring the optical signal of the mixture includes measuring the optical signal of the mixture with a photometer, where the lower linear detection limit of the photometer is less than or equal to 2000 photons per second, and the upper linear detection limit of the photometer is greater than or equal to 100M photons per second.

22. The method according to any one of claims 19-21, characterized in that, The chemiluminescent substrate is a dioxetane compound, preferably selected from chloro-substituted dioxetane compounds with a spiro-adamantyl substituent, the fluorescent agent is selected from fluorescein and / or carboxyl-substituted fluorescein, and the surfactant is selected from water-soluble polymeric quaternary ammonium salt surfactants.

23. The method according to claim 22, wherein The chloro-substituted dioxetane compounds with a spiro-adamantyl substituent are ADP-STAR and CDP-STAR; the carboxyl-substituted fluoresceins are 5-carboxyfluorescein, 6-carboxyfluorescein, and 5(6)-carboxyfluorescein.

24. The method according to claim 22, wherein The water-soluble polymeric quaternary ammonium salt is a quaternary ammonium salt containing a vinyl group, preferably at least one of polyvinylbenzyl-trimethylammonium chloride, polyvinylbenzyl-benzyldimethylammonium chloride, and polyvinylbenzyl-tributylammonium chloride.

25. The method according to any one of claims 22-24, characterized in that, The chemiluminescent substrate solution includes 50 - 500 mg / L of the chemiluminescent substrate, 30 - 500 mg / L of the fluorescent agent, and 1 - 10 g / L of the water-soluble polymeric quaternary ammonium salt; preferably, the chemiluminescent substrate solution includes 150 - 250 mg / L of the chemiluminescent substrate, 150 - 250 mg / L of the fluorescent agent, and 3 - 7 g / L of the water-soluble polymeric quaternary ammonium salt.

26. The method according to any one of claims 18 - 25, characterized in that, The analyte is human chorionic gonadotropin (HCG). Preferably, the concentration range of HCG in the test sample is 1 to 200,000 mIU / mL. More preferably, the method is a double antibody sandwich method. More preferably, the capture reagent is superparamagnetic microparticles coated with HCG antibody.

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