Multi-stage determination type high-precision detection method and system, and kit

Abstract

Provided are a multi-stage determination type target object detection method and system, and a detection kit. The detection kit comprises a reagent with a structure linked to a solid-phase carrier and is used in conjunction with a detection method and detection system that have multi-stage determination steps and corresponding modules. On the basis of sample testing requirements, the reagent needs to be used in conjunction with the solid-phase carrier comprising a linking structure during the testing, offering flexibility and adaptability for different detection requirements. The detection method and system integrate the advantages of solid-phase washing detection technology and homogeneous detection technology, which not only avoids unnecessary washing when all samples are detected in a solid-phase mode, but also solves the problem of low accuracy caused by the presence of special samples when all samples are detected in a homogeneous mode.

Description

A multi-level judgment-based high-precision detection method, system, and reagent kit Technical Field

[0001] This invention relates to the field of in vitro diagnostics, specifically to immunoassay methods, systems, and reagent kits. Background Technology

[0002] Over the past 40 years, immunoassay technology has undergone a transformation from radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) to magnetic particle chemiluminescence (MPA). With continuously improving detection sensitivity, many clinically valuable biomarkers have been applied to clinical diagnosis. Luminescence detection methods involving solid-phase cleaning steps, such as MPA, have gradually replaced ELISA, capturing nearly 90% of the market share. While MPA is widely used clinically due to its sensitivity and specificity, it has also revealed some limitations. First, the magnetic cleaning process increases the complexity of the instrument's liquid path module and the instrument's cost. Second, the magnetic cleaning process limits the throughput of the detection instrument; laboratories with high sample volumes need to extend their working hours or add multiple devices to meet their testing needs. To avoid the complexity of solid-phase cleaning such as with magnetic beads, some homogeneous detection technologies, such as spatial proximity chemiluminescence, have gradually achieved technological breakthroughs.

[0003] In spatial proximity chemiluminescence (SPEC) technology, one antibody involved in the immunoreaction is labeled with horseradish peroxidase (HRP), and the other antibody is labeled with a luminescent compound, such as acridil (AE). In the presence of the target antigen, a sandwich immune complex is formed. The target antigen tightly binds the HRP and AE labeled on the two antibodies together, generating a light signal under the action of the substrate. The intensity of the light signal is directly proportional to the concentration of the target antigen. This technology has been disclosed in WO2010099486A1. Its advantages include: ① The formation of immune complexes based on antigen-antibody immunoreaction, with the amount of complexes monotonically correlated with the amount of analyte; the antigen-antibody immunoreaction ensures specificity of the detection. ② The immunoreaction occurs in a homogeneous system, ensuring high precision of the detection. ③ The signal is generated based on the proximity of the HRP and AE, therefore the instrument does not require a liquid path system for magnetic cleaning, resulting in lower cost, smaller size, and high throughput.

[0004] However, spatial proximity luminescence technology still has some limitations. For example, there is no magnetic cleaning process during the reaction, so it cannot remove interfering substances in the sample. The detection results are poor for highly interfering samples. For example, hemolysis and lipemia samples, which are common in some elderly people, can interfere with the immune response and thus affect the accuracy of the detection results. Compared with magnetic particle chemiluminescence, the detection sensitivity is lower.

[0005] In summary, existing detection technologies all have certain limitations. Clinical scenarios urgently need a fully automated, high-throughput detection technology with lower cost, less interference, and higher sensitivity to meet clinical needs. Summary of the Invention

[0006] The purpose of this invention is to provide a kit for detecting target analytes, as well as a testing method and system thereof. This kit and its testing method can reduce the number of unnecessary sample cleanings, reduce the frequency of solid-phase material detection methods, significantly reduce costs, increase detection throughput, and avoid the problems of low sensitivity and accuracy caused by special samples.

[0007] Therefore, one aspect of the present invention is to provide a target analyte detection kit, the kit comprising reagent A and reagent B, wherein reagent A contains a first binding molecule and the first binding molecule is coupled with compound 1;

[0008] The reagent B contains a second binding molecule, which is coupled with compound 2.

[0009] The first and / or second binding molecules further include at least one linker structure I;

[0010] Wherein, compound 1 has the function of catalyzing compound 2 to generate a signal; the first binding molecule and the second binding molecule can specifically bind to the target in the sample to form a specific binding complex; the connecting structure I does not form a specific binding state.

[0011] The working concentration of the first binding molecular complex of compound 1 in reagent A is 0.1-3.0 μg / mL; the working concentration of the second binding molecular complex of the compound in reagent B is 0.1-3.0 μg / mL.

[0012] "Target substance" refers to one or more biochemical substances whose specific content or concentration is to be determined through detection methods.

[0013] "Catalysis" means changing the activation free energy required for a reaction through a catalyst, thereby changing the chemical reaction rate of the reactants. Compound 1 does not directly act on compound 2, but rather through "catalysis" causes compound 2 to react chemically with other substances.

[0014] Furthermore, the first and second binding molecules are not coupled to a solid-phase support.

[0015] Furthermore, the kit also includes reagent C, which contains compound 3. Compound 2 and compound 3 react to generate a detectable signal, such as a light signal. When the first binding molecule in the kit is coupled with compound 1, and the first binding molecule coupled with compound 1 and the second binding molecule coupled with compound 2 bind to the target analyte, the distance between compound 1 and compound 2 is sufficient for compound 1 to catalyze compound 2 to generate a detectable signal. At the same distance, phosphatase and compound 2 cannot generate a detectable signal. Therefore, compound 1 is not a phosphatase, such as alkaline phosphatase.

[0016] Furthermore, compounds 1 and 2 are coupled to the first and second binding molecules, respectively, via flexible connecting units.

[0017] The "flexible connecting unit" is a non-rigid material. When the first and second binding molecules of compounds 1 and 2 are coupled to form a complex with the target analyte, compounds 1 and 2 can move around the periphery of the complex based on the flexibility of the "flexible connecting unit," allowing the substances capable of generating luminescent signals to be spatially close enough to produce a detectable signal. The "flexible connecting unit" is selected from polyoxyethylene ethers, polyoxypropylene ethers, alkyl groups, polyethyleneimines, peptides, and block compounds composed of the above substances. To ensure luminescence efficiency, the flexible connecting unit should not be too long. Therefore, the total effective length of the flexible connecting unit linking compounds 1 and 2 is preferably less than or equal to 200 nm, more preferably less than 180 nm, and even more preferably less than 150 nm; at the same time, the total length should be greater than the diameter of the binding molecules; or the total degree of polymerization of the flexible connecting unit is less than 500.

[0018] "Polyoxyethylene ethers," also known as polyoxyethylene (PEO) or polyoxyethylene (POE) compounds, refer to polymers with a PEG backbone, preferably compounds with a PEG backbone and active groups at the chain ends that can be linked to biomolecules. Specific examples include NHS-(PEG)n and Pierce. TM The PEG-SPDP series.

[0019] "Alkyl" includes straight-chain alkyl, branched alkyl, or alkylene groups with active groups at the chain ends, which are used to couple the molecule with compound 1 and compound 2.

[0020] A polypeptide is a polymer that contains at least two amino acids.

[0021] "Polyethyleneimine compounds" are a class of polymers with a main chain of "-CH2CH2NH-", including substances with side chains and / or substitutions at both ends.

[0022] "Blocking material" refers to a substance that contains two or more flexible connecting unit basic building blocks, such as a block polymer formed by the polyoxyethylene ether compound building block -OCH2CH2- and the polyethyleneimine compound building block -CH2CH2NH-.

[0023] The "total degree of polymerization" is the sum of the degrees of polymerization of the two flexible connecting units on the first and second bound molecules.

[0024] The "solid support" is a material with at least one dimension greater than 0.1 micrometer or 0.2 micrometer, preferably greater than 0.3 micrometer or 0.5 micrometer, in three dimensions. The material has adsorption properties or the surface of the material has active functional groups that can couple with the bound molecules. The material can be in the following forms: particles, microparticles, metal colloids, fibers, paper, beads, membranes, filter paper, test tubes, microplates, chips, glass slides, and microarrays, etc.

[0025] Furthermore, the first binding molecule and / or the second binding molecule are antibodies, antigens, and / or antigen-binding fragments. For example, when the target detectable is an antigen, the first binding molecule and the second binding molecule may be optionally antibodies or antibody fragments that are specific to that antigen; for example, when the target detectable is an antibody, the first binding molecule and / or the second binding antibody may be optionally a secondary antibody against the target detection antibody or an antigen.

[0026] The antigen-binding fragment is selected from any one of the antibody's F(ab)2, F(ab')2, Fab', Fab, Fv, and scFv.

[0027] Further, compound 1 is a substance capable of catalyzing the generation of hydroxyl radicals from a substrate, including transition metal salts and / or complexes, and peroxidases, specifically such enzymes as lactoperoxidase, microperoxidase, myeloperoxidase, halogen peroxidase, vanadium bromide peroxidase, horseradish peroxidase, fungal peroxidase, lignin peroxidase, peroxidase derived from *Arthromyces ramosus*, Mn-dependent peroxidase produced by white-rot fungi, or soybean peroxidase, and transition metal complexes such as ferrous protoporphyrin, etc., which catalyze the chemiluminescent oxidation of the substrate. Correspondingly, compound 2 is selected from substances that generate photons under the action of the enzyme described in compound 1, such as luminol, isoluminol, profenine, acridine ester, luciferin, 9,10-dihydroacrylamide, and phthalic acid hydrazide.

[0028] Furthermore, compound 3 in reagent C is selected from peroxide compounds; for example, hydrogen peroxide, urea peroxide, peracetic acid, or perborate; the pH value of reagent C is 5.5-9.5, preferably 6.5-8.5.

[0029] It is understandable that, unlike compound 1 which catalyzes compound 2 to produce a signal, compound 3 reacts directly with compound 2 to produce a signal. Therefore, at the same concentration, the signal produced by the reaction of compound 3 with compound 2 is more significant than that produced by compound 1 with compound 2. Alternatively, it can be understood that compound 1 has the effect of enhancing the signal production of compound 3 and compound 2.

[0030] "Optical signal" refers to the photons generated by compounds through chemical reactions or by external environmental excitation (such as changes in temperature, light irradiation, changes in magnetic field, etc.). Photons serve as information carriers, and the expected information can be obtained by detecting photons, such as by detecting fluorescence, flashes, or glows.

[0031] Furthermore, the kit also includes reagent D, which contains a solid-phase carrier coupled to linker structure II, wherein linker structure I and linker structure II specifically bind to each other to form a specific binding system; the specific binding system includes: antibody-antigen, avidin-biotin, streptavidin-biotin, and tag-catcher.

[0032] The working solution concentration of the solid-phase support complex coupled with linker structure II in reagent D is 0.2-5.0 mg / mL.

[0033] When linking structures I and II are antibodies and antigens with specific binding properties, they differ in type from the target analyte and the first and / or second binding molecules. Furthermore, the antibodies and antigens described in linking structures I and II will not specifically bind to the target analyte and the first and / or second binding molecules. Without limitation, the antibodies and antigens described in linking structures I and II can be obtained by means known in the art. For example, if linking structure I is fluorescein isothiocyanate (FITC), then the antibody against FITC in linking structure II is preferably a monoclonal antibody.

[0034] The "Tag-Catcher" system consists of two parts: a protein and a polypeptide (short peptide). These parts spontaneously form heteropeptide bonds through residues that specifically and with high affinity for each other. This includes the SpyTag-SpyCatcher system, the SdyTag-SdyCatcher system, the SnoopTag-SnoopCatcher system, and the DogTag-DogCatcher system. The binding mechanisms of these different "Tag-Catcher" systems are essentially the same, with high affinity and rapid reactions, requiring no additional chemical reagents or catalysts. For example, in the earliest discovered SpyTag-SpyCatcher system, the carboxyl group of aspartic acid in the SpyTag (aspartic acid residue) specifically recognizes the primary amino group of lysine in the SpyCatcher (lysine residue), forming an amide bond through dehydration. For preparation and principles, please refer to references 1 and 2. The preparation and principles of SdyTag-SdyCatcher, SnoopTag-SnoopCatcher, and DogTag-DogCatcher can be found in references 3-5, respectively. For the connection of Tag-Catchers with magnetic beads, antibodies, proteins, and other substances, and the preparation methods, please refer to references 5-10.

[0035] The specific binding of the two parts in the “Tag-Catcher” system is a covalent bond, which makes the binding less susceptible to environmental changes and the connection more stable.

[0036] Furthermore, the kit also includes an alkaline solution with a pH value greater than or equal to 8, more preferably greater than or equal to 9, 10, or 11. The alkaline solution can be prepared using a suitable alkaline substance, such as sodium hydroxide. It is understood that alkaline substances are commonly used promoters in chemiluminescence, particularly suitable for enzyme-free systems. For example, adding hydrogen peroxide to cleaned magnetic beads (containing the analyte) followed by an alkaline solution can generate a detectable luminescent signal.

[0037] Furthermore, the kit also includes a selective signal inhibitor, which can be understood as an agent used to enhance the signal during the assay. When applied to the detection of the target analyte, the signal obtained from the assay reaction system exceeds the background signal by a significantly greater degree than would occur without the inhibitor. It is also important to note that the reagent is essentially in an open state during use, allowing the inhibitor to come into full contact with air. The inhibitor is susceptible to oxidation in the environment, making its stability extremely crucial.

[0038] The signal inhibitors described in this invention are selected from, for example, those listed in WO2010099486A1, including ascorbic acid or its salts, phenoloxazine, 2-aminophenol, 2-amino-p-chlorophenol, 2-amino-o-chlorophenol, 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid TROLOX™, etc. 3-Aminotyrosine or its salt, p-Hydroxy-2-chlorophenol, p-Hydroxy-2-methoxyphenol, o-hydroxy-m-chlorophenol, And at least one of the compounds with the structure shown in Formula 1.

[0039] In addition, WO2010099486A1 disclosed that some structures had poor signal inhibitory effects. However, the applicant unexpectedly discovered that the compound with the structure shown in Formula 1 has outstanding effects in suppressing background interference signals, and is particularly significant in terms of stability.

[0040] In this embodiment, any two or three substituents in the substituted R1-R6 are selected as -OH and / or -NH2, and the remaining substituents are -H, -F, -Cl and / or -Br, with two or three substituents being -F, -Cl and / or -Br. Preferably, two substituents in the substituted R1-R6 are -F, -Cl and / or -Br.

[0041] Therefore, another aspect of the present invention is the use of the signal inhibitor described herein in a test kit, specifically for reducing background interference signals.

[0042] The concentration of the signal inhibitor in the detection system described in this invention is 10. -4 mM-10mM, for example 10 -3 mM-1mM or 10 -2 mM-1mM.

[0043] Another aspect of the present invention provides a detection composition comprising: a bound molecule coupled to a substance capable of generating hydroxyl radicals or a substance that generates photons under the action of hydroxyl radicals via a flexible connecting unit; optionally, the flexible connecting unit is selected from polyoxyethylene ether compounds, polyoxypropylene ether compounds, alkyl groups, polyethyleneimine compounds, polypeptides, and block compounds of the above substances; the bound molecule further comprises at least one connecting structure, the bound molecule being a specifically bound substance capable of specifically binding to a target; and the connecting structure does not form a specific connection and the bound molecule is not coupled to a solid-phase support.

[0044] Furthermore, the binding molecule is an antibody, an antigen, and / or an antigen-binding fragment.

[0045] Furthermore, the connection structure is selected from any two parts of the following specific binding systems, including: antibody-antigen system, avidin-biotin system, streptavidin-biotin system, and Tag-Catcher system.

[0046] Antibody-antigen systems and tag-catcher systems have the functions and types described above.

[0047] Furthermore, substances capable of generating hydroxyl radicals include horseradish peroxidase, and the compounds that generate photons under the action of hydroxyl radicals include at least one of luminol, isoluminol, profen, acridine ester, glimepiride, 9,10-dihydroacrylidine, and phthalohydrazide and their derivatives.

[0048] Another aspect of the present invention is to provide the use of the above composition in the preparation of a detection product; said product is used for the detection of blood, urine, bone marrow, cerebrospinal fluid, pleural effusion, ascites, pericardial fluid, joint fluid, hydrocele, bile and / or tissue samples; preferably blood, plasma, serum and urine samples; further, said product is a kit.

[0049] Another aspect of the present invention is to provide a judgmental detection method, comprising the following steps:

[0050] S1: Perform interference identification on the sample to be tested to obtain the interference value;

[0051] S2: Compare the interference value with the preset threshold, and then select to execute either S3A-S4A or S3B-S4B based on the comparison result.

[0052] in,

[0053] The S3A step includes: selecting reagents and reacting the sample to be tested with a reagent that does not contain a solid support;

[0054] The S3B step includes: selecting reagents and reacting the sample to be tested with reagents containing a solid support, and separating impurities in the detection system;

[0055] Steps S4A and S4B include measuring the reaction system and reporting the results.

[0056] Understandably, due to differences in detection methods, the reagents used in S3A and S3B also differ. For example, the reagents in step S3A include reagent A, reagent B, and reagent C, while the reagents in step S3B include reagent A, reagent B, reagent C, reagent D, a cleaning agent, and an alkaline solution. Furthermore, both S3A and S3B reagents may optionally contain signal inhibitors. Under conditions where there is no mutual interference, these reagents can be stored individually or as a mixture.

[0057] Furthermore, before step S1, the following step S0 is also included: comparing and judging the test items of the sample to be tested with preset items; and then selecting to execute S1 and subsequent steps or S3B-S4B steps based on the comparison and judgment results (as shown in Figure 2).

[0058] Therefore, the present invention also relates to a detection method, the detection method comprising:

[0059] S0: Compare and judge the test items of the sample to be tested with the preset items; based on the comparison and judgment results, select to execute (i) or (ii) detection process;

[0060] The detection process (i) includes:

[0061] S1: Perform interference identification on the sample to be tested to obtain the interference value;

[0062] S2: Compare the interference value with a preset threshold, and based on the comparison result, select to execute either step S3A-S4A or step S3B-S4B; where...

[0063] The S3A step includes: selecting reagents and reacting the sample to be tested with a reagent that does not contain a solid support;

[0064] The S3B step includes: selecting reagents and reacting the sample to be tested with reagents containing a solid support, and separating impurities in the detection system;

[0065] Steps S4A and S4B include: measuring the reaction system and reporting the test results;

[0066] The detection process described in (ii) includes:

[0067] The S3B step includes: selecting reagents and reacting the sample to be tested with reagents containing a solid support, and separating impurities in the detection system;

[0068] The S4B step includes measuring the reaction system and reporting the results.

[0069] The reagents in step S3A include reagent A, reagent B, and reagent C; the reagents in step S3B include reagent A, reagent B, reagent C, reagent D, and cleaning solution; further, the reagents in step S3A and / or step S3B also include signal inhibitors, and the reagents A, B, C, D, and signal inhibitors have the selection types described herein.

[0070] The "analyte" refers to the test performed on the sample to obtain quantitative information on one or more biochemical substances. The "preset item" refers to a pre-set test item. The purpose of setting the "preset item" is to reduce interference and improve test accuracy. A "preset item" can be determined, for example, based on clinical studies or literature. If the content of the target analyte in the sample is extremely low or there is significant interference, making accurate quantitative analysis impossible, then such a test item can be considered a "preset item." When the "analyte" is a "preset item" or contains a "preset item," then interference-eliminating test steps are required, such as steps S3B-S4B. If the "analyte" does not include a "preset item," then steps S1 and subsequent steps are used. Alternatively, if the target analyte in the sample is extremely low or there is significant interference, making accurate quantitative analysis impossible, then test items not falling into this category are set as "preset items." If the "analyte" meets the "preset item" criteria, steps S1 and subsequent steps are used; otherwise, steps S3B-S4B are used. For example, if the concentration of AD markers (such as tau-181) in the blood is low, the detection item for tau-181 in the blood sample is set as a "preset item". When the "to be tested" item is tau-181 in the blood sample, it is determined to meet the "preset item" and the S3B-S4B steps are adopted.

[0071] Furthermore, the relationship between the interference value and the preset threshold is determined using one or more of the following methods:

[0072] 1) Illuminate the sample to be tested or a mixture of the sample to be tested and the reagent with a light source, detect the transmitted light and / or the scattered light signal, and make a judgment by comparing the transmitted light and / or the scattered light signal with a preset threshold;

[0073] 2) By calculating the difference in absorbance measured at two wavelengths after dilution, semi-quantitative values ​​of serum / plasma samples are given and compared with preset thresholds for determination; sample values ​​include, for example, values ​​of lipemia, hemolysis and / or jaundice in the sample.

[0074] 3) The color features of the sample image are compared with a preset threshold to determine the result.

[0075] The "mixed solution of sample and reagent" refers to the solution obtained by mixing the original sample with a reagent that can enhance the detection effect.

[0076] Without limitation by examples, all methods for determining whether a sample has interfering values ​​employ known techniques in the art, such as using the methods and modules described in CN105980833B and CN113592842B to detect lipemia, hemolysis, and jaundice in blood samples. Alternatively, methods and instruments described in literature or commercially available products such as CN109374551A, CN204101456U, CN210894370U, or the EXC400 automated biochemical analyzer (Zhongyuan Huiji Biotechnology Co., Ltd.) are used to detect the transmitted light and / or scattered light signals of the sample. A UV-4800 dual-beam spectrophotometer (Unico (Shanghai) Instruments Co., Ltd.) is used for dual-wavelength detection, and images are acquired using a regular camera or a miniature camera.

[0077] The "preset threshold" is a value set based on existing data and information, serving to distinguish between high-interference and low-interference samples. The data and information required to set the "preset threshold" can be obtained, for example, from clinical big data analysis of the differences between special samples and ordinary samples. For instance, hemolyzed blood samples (due to ruptured red blood cells) show significant differences in transmitted light compared to ordinary blood samples (relative to non-hemolyzed samples); hemolyzed samples have lower transmittance. Based on big data analysis of the transmittance range of ordinary blood samples, the "preset threshold" is set accordingly. The "preset threshold" can be appropriately and strictly set to ensure the accuracy of the test results. For example, to reduce interference from hemolyzed samples, the transmitted light "preset threshold" can be 5% or 10% lower than the transmittance of ordinary blood samples. When the transmittance of a sample is lower than the "preset threshold," it is determined to be a "high-interference" sample; when the transmittance of a sample is higher than the "preset threshold," it is determined to be a "low-interference" sample.

[0078] To further improve detection accuracy, preferably, multiple "preset thresholds" can exist in step S1, determined by different detection methods. A sample is considered low-interference only when the measured value of the sample meets the low-interference criteria for both "preset thresholds"; otherwise, it is considered high-interference. For example, two different "preset thresholds" can be determined using transmission method and dual-wavelength measurement. The sample is also tested using both transmission method and dual-wavelength measurement. When the difference between the transmittance and absorbance of the sample is higher than the "preset threshold" set for transmission method and lower than the "preset threshold" set for dual-wavelength measurement method, respectively, the sample is considered a low-interference sample.

[0079] The purpose of "impurity separation" or "impurity removal" described in this invention is to reduce the content of substances in the reaction system that interfere with the measurement results. Interference with the measurement results can occur in ways such as increasing background signals, suppressing the production of photons from luminescent compounds, or reducing the detection amount of the expected signal. Substances that produce these effects can be considered impurities. Methods for separating and removing impurities include washing, centrifugation, sedimentation, adsorption (such as HPLC, chromatography columns, etc.), and liquid-liquid separation. These examples are not limited to specific methods; any method that reduces the content of impurities in the measurement system or reduces their influence on the measurement system can be considered an effective method for "impurity separation."

[0080] Furthermore, based on preset items or interference judgments, multiple "impurity separation" steps can be performed, such as two or more; furthermore, cleaning with a solid carrier is the preferred impurity separation method.

[0081] Furthermore, the reagents used in the detection method of the present invention have the composition of the reagent kit described above, or the reagent kit of the present invention is used in conjunction with the detection method of the present invention for the detection of the target analyte.

[0082] Another aspect of the present invention is to provide a multi-level judgment-based detection system, the detection system including a detection item comparison module, an interference identification and analysis module, an impurity separation module, a reagent aspiration module, an optical detection module, a signal processing module, etc.

[0083] The comparison module is used to compare the information of the item to be tested with that of the preset item.

[0084] The interference identification and analysis module is used to analyze sample interference information;

[0085] The impurity separation module is used to separate impurities and reduce substances in the detection system that affect the detection results. Furthermore, the impurity separation module includes a cleaning submodule, which uses the difference between reagents bound to a solid support and reagents not bound to a solid support to separate impurities. The difference is manifested in the following ways: difference in magnetic field, difference under centrifugal force, difference under gravity, difference under buoyancy, difference in solubility, or difference in retention time in the chromatography column.

[0086] The optical testing module is used to detect the signals generated by the reaction system;

[0087] The signal processing module is used to process signals and convert them into visual information, such as figures or numbers.

[0088] The detection system can have at least two different detection channels to detect high-interference samples and low-interference samples respectively. Therefore, based on this, the detection system also includes a sample transfer module, which transfers the samples to different detection channels based on the interference identification results.

[0089] Alternatively, the detection system may have only one detection channel, where high-interference samples and low-interference samples are detected by different reagents drawn by the reagent aspiration module within the same detection channel.

[0090] Furthermore, whether to invoke the impurity separation module depends on the analysis results of the interference identification and analysis module.

[0091] In addition, based on the need for impurity separation, the detection system also includes a module for transferring samples before and after impurity separation. This module can be the aforementioned sample transfer module or a separate module.

[0092] In this invention, biological samples containing the target analyte can exist in any form. The technical solution described in this invention is particularly suitable for samples that are prone to generating high interference values, such as blood, plasma, serum, and urine.

[0093] The "kit" of this invention is based on reagents A and B, and may optionally contain reagents C, D and / or signal inhibitors, as well as other reagents, depending on the detection requirements.

[0094] In this invention, a "reagent kit" refers to a detection system composed of detection reagents. Different detection reagents can be stored in the same packaging container, or stored separately in containers, or some reagents can be mixed in one container while others are stored separately. Containers containing different reagents can be connected together, partially connected, or not connected at all. For example, depending on factors such as system control and equipment, the reagent detection system may include reagent A, reagent B, and reagent D, which are stored separately and used as needed during detection; or, for example, the reagent detection system may include reagent A, reagent B, reagent C, and reagent D, with reagent A and reagent C stored in one container and reagent B and reagent D stored separately, used as needed during detection.

[0095] In this invention, "specific binding" has the meaning known in the art, and refers to a targeted biological binding process in which a certain ligand interacts with a specific structural site in vitro or in vivo, which can be competitively blocked by the corresponding substance, such as the binding between antigen and antibody or between receptor and ligand, or the binding between biotin and avidin.

[0096] In this invention, the “coupling” method includes two parts of the substance being directly linked through active functional groups (direct coupling), or indirectly linked through one or more substances (indirect coupling). Direct coupling is, for example, the connection between a solid-phase carrier with a carboxyl group on its surface and the amino part of an antibody, thereby directly coupling the solid-phase carrier and the antibody. Indirect coupling is, for example, the two parts are linked through a secondary antibody or through avidin-biotin.

[0097] The kit and its detection composition described in this invention are applicable to the detection of both highly interfering samples and ordinary samples. Depending on the sample, different reagents in the kit can be selected to detect the sample, thereby meeting both the needs of high-throughput detection and the accuracy of the results.

[0098] For example, in one embodiment of the present invention, the kit includes:

[0099] 1) Reagent A: Contains the first bound molecule of compound 1 coupled together;

[0100] 2) Reagent B: Contains a second bound molecule coupled with compound 2;

[0101] 3) Reagent D: Contains a solid support coupled to linker structure II;

[0102] The first or second binding molecule includes at least one linker structure I, and linker structures I and II have specific binding effects. The detection principle is as follows: The sample interference value is determined. If the sample has low interference, the target analyte (such as an antigen) is captured directly using the first binding molecule in reagent A and the second binding molecule in reagent B. Compound 2 generates a light signal under the catalytic action of compound 1, and the detection is completed. If the sample has high interference, the first and second binding molecules that have captured the target analyte are separated from the reaction system using the solid-phase support in reagent D, and then subsequent detection is performed, thereby reducing interference and improving detection accuracy. The first binding molecule, the second binding molecule, compound 1, compound 2, and linker structures I and II are consistent with the previous description.

[0103] For example, in another embodiment of the present invention, the kit includes:

[0104] 1) Reagent A: Contains the first binding molecule;

[0105] 2) Reagent B: Contains a second bound molecule coupled with compound 2;

[0106] 3) Reagent C: Contains compound 3;

[0107] 4) Reagent D: Contains a solid support coupled to linker structure II;

[0108] The first and / or second binding molecules include at least one linker structure I, which has specific binding activity. The detection principle is as follows: The sample interference value is assessed. If the sample has low interference, the target analyte (e.g., antigen) is captured directly using the first binding molecule in reagent A and the second binding molecule in reagent B. Compound 2 reacts with compound 3 in reagent C to generate a light signal, completing the detection. If the sample has high interference, the first and second binding molecules, which have captured the target analyte, are separated from the reaction system using the solid-phase support in reagent D before subsequent detection, thereby reducing interference and improving detection accuracy. The first binding molecule, second binding molecule, compound 2, compound 3, and linker structures I and II are consistent with the previous description.

[0109] For example, in another embodiment of the present invention, the kit includes:

[0110] 1) Reagent A: Contains the first bound molecule of compound 1 coupled together;

[0111] 2) Reagent B: Contains a second bound molecule coupled with compound 2;

[0112] 3) Reagent C: Contains compound 3;

[0113] 4) Reagent D: Contains a solid support coupled to linker structure II;

[0114] The first and / or second binding molecules include at least one linker structure I, which has specific binding activity. The detection principle is as follows: The sample interference value is assessed. If the sample has low interference, the target analyte (e.g., antigen) is captured directly using the first binding molecule in reagent A and the second binding molecule in reagent B. Compound 1 catalyzes the reaction of compound 2 with compound 3 in reagent C to generate a light signal, thus completing the detection. If the sample has high interference, the first and second binding molecules, which have captured the target analyte, are separated from the reaction system using the solid-phase support in reagent D before subsequent detection, thereby reducing interference and improving detection accuracy. The first binding molecule, second binding molecule, compound 1, compound 2, compound 3, and linker structures I and II are consistent with the previous description.

[0115] Optionally, the above-described embodiments also include signal inhibitors, and when reagents with solid-phase supports are used, a cleaning solution is also included.

[0116] Referring to Figure 1, which is a schematic diagram of a detection implementation method of the present invention, the composition of the reagent kit varies depending on whether the sample is high-interference or low-interference.

[0117] Referring to Figure 2, which is a flowchart illustrating an embodiment of the detection method of the present invention, preset items are first set in the detection system. When step S0 judges the preset items and the items to be tested, and the items to be tested do not conform to the preset items, the interference value of the sample is identified in step S1 to obtain the interference. In step S2, the interference value is compared with a preset threshold. If the comparison result is low interference, the sample is reacted in step S3A using a reagent without a solid phase. In step S4A, the signal generated during the reaction process is detected and a corresponding result report is output. If the comparison result is high interference, the sample is reacted in step S3B using a reagent containing a solid phase and impurities are removed and separated. In step S4B, the signal generated during the reaction process is detected and a corresponding result report is output.

[0118] When the detection item does not meet the preset content, in step S3B, a reagent containing a solid phase is used to carry out the reaction and impurity removal and separation. In step S4B, the signal generated during the reaction process is detected and the corresponding result report is output.

[0119] Referring to Figure 3, which is a schematic diagram of an embodiment of the detection system of the present invention, the system includes a comparison module S10, an interference identification and analysis module S20, an impurity separation module S30, a reagent aspiration module S40, an optical detection module S50, and a signal processing module S60.

[0120] Based on the technical solution described in this invention, the inventors believe that the beneficial effects of this invention are at least as follows:

[0121] 1) A novel test reagent with a structure that connects to a solid support is proposed. The test reagent is then used in conjunction with the solid support containing the connecting structure during the test process, depending on the sample test requirements. This makes it flexible and can be matched with different detection needs.

[0122] 2) The kit described in this invention contains a variety of reagents, and different types of reagents can be formulated according to different sample detection needs. It integrates the advantages of solid-phase cleaning detection technology and homogeneous detection technology, which avoids unnecessary cleaning when the whole sample is detected in solid-phase mode, and also avoids the problem of low accuracy caused by special samples when the whole sample is detected in homogeneous mode.

[0123] 3) The reagent kit described in this invention has a wider range of applications and can be used to detect various types of samples, meeting the needs of complex application scenarios and significantly improving the reliability of the reagent kit.

[0124] 4) The detection method and detection system described in this invention have multi-level judgment steps and corresponding modules. Different modules are selected according to different judgment results, making the detection method more flexible and more suitable for the detection of sample groups with complex situations. The detection time is shorter than that of solid phase detection under high-throughput detection, and the detection accuracy is higher than that of all-homogeneous phase detection.

[0125] 5) A new class of signal inhibitors suitable for homogeneous reactions is proposed, which have higher stability in practical applications.

[0126] References:

[0127] Document 1: Zakeri, B. & Howarth, M. Spontaneous Intermolecular Amide Bond Formation between Side Chains for Irreversible Peptide Targeting. J. Am. Chem. Soc. 132, 4526–4527 (2010);

[0128] Document 2: Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc. Natl. Acad. Sci. USA 2012, 109, E690–E697;

[0129] Document 3: Tan LL, et al.Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly.PLoS ONE 11(10):e0165074;

[0130] Document 4: Veggiani G, et al, Programmable polyproteams built using twin peptide superglues. Proc Natl Acad Sci US A. 2016 Feb 2; 113(5): 1202-7;

[0131] Document 5: Anthony H. Keeble, et al, DogCatcher allows loop-friendly protein-protein ligation, Cell Chem Bio, 29(2), P339-350.E10, DOI: https: / / doi.org / 10.1016 / j.chembiol.2021.07.005;

[0132] Document 6: X.Pei, et al, Putting precision and elegance in enzyme immobilization with bio-orthogonal chemistry, Chem.Soc.Rev., 2022, 51, 7281, DOI: 10.1039 / D1CS01004B;

[0133] Reference 7: CN117723749A;

[0134] Reference 8: CN114990146A;

[0135] Reference 9: CN117362447A;

[0136] Reference 10: CN116444623B.

[0137] Instruction manual illustrations

[0138] Figure 1 is a schematic diagram of a detection method according to the present invention.

[0139] Figure 2 is a schematic diagram of a detection method used in this invention.

[0140] Figure 3 is a schematic diagram of a detection system used in this invention. Detailed Implementation

[0141] The present invention will be further explained with reference to specific embodiments. Where specific techniques or conditions are not specified in this embodiment, operations should be performed according to conventional techniques and instrument manuals in the art. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially or through self-development, and the relevant antibodies are all self-developed by Chongqing Aisens Biotechnology Co., Ltd.

[0142] Example 1: Preparation of raw materials

[0143] Reagent A:

[0144] Weigh 5 mg HRP and dissolve it in 1 ml of distilled water; add 0.2 ml of freshly prepared 0.1 M NaIO4 solution and stir for 20 minutes at room temperature in the dark; transfer the above solution to a dialysis bag and dialyze against 1 mM pH 4.4 sodium acetate buffer overnight at 4°C; add 20 μl of 0.2 M pH 9.5 carbonate buffer to raise the pH of the aldehyde-modified HRP to 9.3, then immediately add 10 mg IgG (thyroid-stimulating hormone (TSH) monoclonal antibody) to 1 ml of 0.01 M carbonate buffer and gently stir for 2 hours at room temperature in the dark; add 0.1 ml of freshly prepared 4 mg / ml NaBH4 solution, mix well, and let stand at 4°C for 2 hours; transfer the above solution to a dialysis bag and dialyze against 0.15 M sodium acetate buffer. Dialyze to pH 7.4 PBS to remove ammonium ions (detected with Nessler's reagent), centrifuge at 10000 rpm for 30 min to remove precipitate, and the supernatant is the enzyme conjugate. After determining the concentration, add an equal volume of 60% glycerol and aliquot, then store at 4°C. Before aliquoting, add BSA at a ratio of 10 mg / ml as a stabilizing agent.

[0145] Reagent B-1:

[0146] Weigh 5 mg of TSH antibody, desalt it using a PD-10 desalting column, and elute with 2.5 mL of biotin-conjugated buffer (50 mM triethanolamine, pH 8.4). Then dilute the antibody to 1 mg / mL with biotin buffer A. Weigh 10 mg of each antibody separately. NHS-Biotin and NHS-acridine were dissolved thoroughly in DMSO to a concentration of 3.0 mg / ml. The NHS-Biotin and NHS-acridine solutions were added to the antibody solution at a molar ratio of antibody:NHS-Biotin:NHS-acridine = 1:20:20. The mixture was incubated at 25°C (±2°C) for 180 minutes. The resulting solution was desalted using a PD-10 desalting column to remove unreacted molecules, eluted with 2.5 ml of biotin-conjugated buffer B, and the concentration was determined using a UV spectrophotometer before storage at 4°C. BSA was added at a concentration of 10 mg / ml as a stabilizing agent before aliquoting.

[0147] Reagent B-2:

[0148] Measure 5 mg of TSH antibody, desalt it using a PD-10 desalting column, and elute with 2.5 mL of biotin-conjugated buffer A. Then dilute the antibody to 1 mg / mL with biotin-buffered saline (50 mM triethanolamine, pH 8.4). Weigh 10 mg each of NHS-Biotin and NHS-(PEG). 24-Acridine compounds, fully dissolved in DMSO to a concentration of 3.0 mg / ml, were prepared according to the antibody: NHS-Biotin: NHS-(PEG) ratio. 24 -Acridine compounds in a molar ratio of 1:20:20, measuring NHS-Biotin and NHS-(PEG). 24 The acridine compound solution was added to the antibody solution above and incubated at 25°C (with an allowable error range of ±2°C) for 180 minutes. The resulting solution was desalted using a PD-10 desalting column to remove unreacted molecules, eluted with 2.5 ml of biotin-conjugated buffer (50 mM triethanolamine, pH 8.4), and the concentration was determined using a UV spectrophotometer before freezing at 4°C or -20°C. BSA was added at a ratio of 10 mg / ml as a stabilizing agent before aliquoting.

[0149] Reagent B-3:

[0150] Measure 5 mg of TSH antibody, desalt it using a PD-10 desalting column, and elute with 2.5 mL of biotin-conjugated buffer A. Then dilute the antibody to 1 mg / mL with biotin-buffered saline (50 mM triethanolamine, pH 8.4). Weigh 10 mg each of NHS-Biotin and NHS-(L-Lysine). 11 -Acridine compounds, fully dissolved in DMSO to a concentration of 3.0 mg / ml, were prepared according to the antibody: NHS-Biotin: NHS-(L-Lysine) formula. 11 The acridine compound was prepared in a molar ratio of 1:20:22, and the amounts of NHS-Biotin and NHS-(L-Lysine) were measured. 11 The acridine compound solution was added to the antibody solution above and incubated at 28°C (with an allowable error range of ±2°C) for 150 minutes. The resulting solution was desalted using a PD-10 desalting column to remove unreacted molecules, eluted with 2.5 ml of biotin-conjugated buffer (50 mM triethanolamine, pH 8.4), and the concentration was determined using a UV spectrophotometer before freezing at 4°C or -20°C. BSA was added at a ratio of 10 mg / ml as a stabilizing agent before aliquoting.

[0151] Reagent C:

[0152] Hydrogen peroxide was dissolved in TBS buffer (pH≈8), with a final concentration of 0.1M for hydrogen peroxide and a final concentration of 10mM for TBS.

[0153] Reagent D:

[0154] The magnetic beads coated with streptavidin, purchased from Thermo, with a diameter of 2.8 μm, were washed and diluted to 1 mg / mL for later use.

[0155] Alkaline solution:

[0156] A solution with a pH of 12 was prepared using sodium hydroxide.

[0157] Washing solution: 500mM PBS buffer, 10% sodium ions, 0.7% EDTA-2Na, 0.5% SDS, 1% Tween 20, 0.1% sodium azide, 0.1% defoamer, pH around 7.

[0158] Example 2: Evaluation of Signal Inhibitors

[0159] The effectiveness of the signal inhibitor was evaluated from two aspects: stability and elimination of background interference. The prepared signal inhibitor solution was placed in test tubes and exposed to air for 0 min, 5 min, and 20 min before luminescence signal testing. The signal-to-noise ratio (S / N) (positive sample signal / negative sample signal) was used as the indicator; a higher ratio indicated better effectiveness.

[0160] Test Procedure: First, prepare a positive serum sample with a TSH antigen concentration of 5.0 μIU / mL. Add 60 μL each of reagent A and reagent B-1 from Example 1 to 20 μL of the positive serum sample, mix well, and react at 37°C for 10 min. Then, add 85 μL of reagent C and 6 μL of a 0.1 mM signal inhibitor. The generated light signal is captured by a photomultiplier tube, and the intensity of the light signal is quantified. The antigen concentration of the negative serum sample is 0, and other reagents are kept consistent. Test information and results are shown in Table 1.

[0161] Table 1 Test results for different substances

[0162] As shown in Table 1, the effectiveness of signal inhibitors such as vitamin C is consistent with that of WO2010099486A1. However, the influence of air on signal inhibitors is unavoidable. Considering storage and practical use, signal inhibitors can be selected as substances in which two or three of the R1-R6 substituents of the benzene ring are selected as -OH and / or -NH2, and the remaining substituents are -H, -F, -Cl and / or -Br, with two or three substituents being -F, -Cl and / or -Br. Preferably, two of the substituents in the substituted R1-R6 are -F, -Cl and / or -Br, and more preferably, the substituent is Cl.

[0163] Example 3: Effect of reagent composition on signal

[0164] To demonstrate the differences in detection results between reagents with different compositions and structures, the performance of reagents with different compositions and structures in terms of signal output can be intuitively understood by measuring the detection signal and signal-to-noise ratio.

[0165] The reagents prepared in Step 1 were used to prepare kits and their performance was tested. Kit 1 included reagent A, reagent B-2, reagent C, and signal inhibitor 14 from Table 1 (exposed to air for 20 min); Kit 2 included reagent A, reagent B-3, and signal inhibitor 14 from Table 1 (exposed to air for 20 min), and reagent C; Kit 3 included reagent A, reagent B-3, reagent C, reagent D, signal inhibitor 14 from Table 1 (exposed to air for 20 min), alkaline solution, and washing solution. The concentrations of reagents A, B, and C were kept consistent in Kits 1-3. Samples with different TSH concentrations were prepared by adding TSH to the buffer, and clinical serum samples with low, medium, and high concentrations were collected. The antigen concentrations of each sample are shown in Table 2. The performance of Kits 1-3 was analyzed using the following reaction modes.

[0166] Reaction modes of kits 1 and 2: Take 20 μl of each sample, add 60 μl of reagent A and 60 μl of reagent B to each sample, mix well, react at 37 degrees Celsius for 10 min, then add 85 μl of reagent C and 6 μl of signal inhibitor with a concentration of 0.1 mM. The generated light signal is captured by a photomultiplier tube, and then the intensity of the light signal is quantified.

[0167] Reaction mode of kit 3: Take 20 μl of each sample, add 60 μl each of reagent A and reagent B, and 30 μl of reagent D to each sample, mix well, and react at 37 degrees Celsius for 10 min. After 3-step magnetic separation and cleaning, add 85 μl of reagent C, 6 μl of 0.1 mM signal inhibitor and an appropriate amount of alkaline solution. The generated light signal is captured by a photomultiplier tube, and the intensity of the light signal is then quantified.

[0168] Table 2. Detection results of reagent kits with different compositions *The concentration of antigens in serum samples was determined using a commercially available kit (electrochemiluminescence immunoassay, Roche Diagnostics GmbH).

[0169] As shown in Table 2, the luminescent substrates in Kits 1 and 2 have lower steric hindrance due to the flexible unit connections, resulting in higher signals (not listed) and a higher signal-to-noise ratio compared to the reagents without flexible connecting units in Example 2. Since there is no cleaning step, the signal values ​​of Kits 1 and 2 are higher than those of Kit 3. Although the signal value of Kit 3 is slightly lower, it has a higher signal-to-noise ratio, mainly due to the lower background value after cleaning. These verifications demonstrate that flexible unit connections help improve the luminescent signal and signal-to-noise ratio, and the cleaning step significantly improves the signal-to-noise ratio.

[0170] Example 4: Effects of Reagent Composition and Detection Strategy Selection

[0171] To further demonstrate the beneficial effects of the multi-level judgment-based detection method described in this invention, the detection time (efficiency) and detection accuracy of different detection strategies were measured.

[0172] (1) Sample setup for time detection

[0173] First, a baseline sample with a TSH concentration of 0.05 μIU / mL was prepared by adding TSH to the buffer solution. Two 100 mL aliquots of the baseline sample were taken, and 1 mL of TBS buffer solution (pH 8.0) and 1 mL of red dye solution prepared with the buffer solution were added to each aliquot. These were designated as the experimental group and the interference group, respectively. The experimental group and the interference group formed the detection sample clusters. By controlling the number of interference group samples in the detection sample count, different sample clusters were formed. Different detection strategies were then used to detect different sample clusters, and the time differences under different strategies were determined. The results are shown in Table 4.

[0174] (2) Sample setup for accuracy testing

[0175] Samples containing different concentrations were prepared by adding TSH to the buffer solution (sample preparation), and clinical serum samples with low, medium, and high antigen concentrations were collected. Four 200 μL aliquots of each antigen concentration were taken. One sample was designated as the experimental group, and hemoglobin (final concentration 10 mg / dL), bilirubin (final concentration 5 mg / dL), and visceral lipid (final concentration 1000 mg / dL) were added to the remaining three samples, respectively, and designated as the hemolysis group, jaundice group, and lipemia group. The information of each sample is shown in Table 3, and the test results are shown in Table 5.

[0176] Table 3. Sample information used for accuracy analysis *Antigen concentrations in serum samples were determined using a commercially available kit (electrochemiluminescence immunoassay, Roche Diagnostics GmbH); **Intralipid, provided by Sigma

[0177] (3) Construction of solid-phase method for sample determination

[0178] The process includes the following steps: Take 20 μl of sample, add 60 μl each of reagent A and reagent B-1 from Example 1 and 30 μl of reagent D to the sample, mix well and react at 37°C for 10 min, perform 3-step magnetic separation and cleaning, then add 85 μl of reagent C and an appropriate amount of alkaline solution. The generated light signal is captured by a photomultiplier tube, and the light value signal is converted into a concentration value through a calibration curve.

[0179] (4) Constructing a homogeneous detection method

[0180] The procedure includes the following steps: Take 20 μl of the test sample, add 60 μl each of reagent A and reagent B-1 from Example 1 to the sample, mix well, and react at 37°C for 10 min. Then add 85 μl of reagent C and 6 μl of 0.05 mM analyte 10 from Table 1. The generated light signal is captured by a photomultiplier tube, and the light value signal is converted into a concentration value through a calibration curve.

[0181] (5) The technical testing method described in this invention

[0182] Includes the following steps:

[0183] A. The preset item is set to "NT-proBNP". After comparison, the test item "TSH" does not meet the preset item.

[0184] B. Measure the transmittance of three normal samples (serum samples with antigen of 2.44 μIU / mL) at a wavelength of 500 nm, and take 95% of the average transmittance as the "preset threshold";

[0185] C. Measure the transmittance of samples that do not meet the "preset items" and judge the interference value. Samples with transmittance higher than or equal to the "preset threshold" are judged as low interference samples, and samples with transmittance lower than the "preset threshold" are judged as high interference samples.

[0186] D. For samples with high interference, the above-mentioned "solid phase method" is used for determination; for samples with low interference, the above-mentioned "homogeneous phase detection" is used for detection.

[0187] (6) Results

[0188] As shown in Table 4, the detection time for a single sample clearly demonstrates that, compared to homogeneous detection, solid-phase detection significantly increases the detection time due to the added cleaning step. Taking a medium-sized hospital with a daily sample processing capacity of 800 samples as an example, the detection time using the solid-phase detection mode is more than twice that of the homogeneous detection mode. Even with normal interference samples present (10%), the detection time using the method described in this invention is increased by 18% and decreased by 44% compared to the completely homogeneous mode and the completely solid-phase mode, respectively. Even with a large number of abnormal samples appearing (25%), the detection time is increased by approximately 43% and decreased by approximately 33% compared to the completely homogeneous mode and the completely solid-phase mode, respectively.

[0189] It is evident that the detection method described in this invention has higher detection efficiency compared to a purely solid-phase detection mode, reduces unnecessary sample cleaning in the cleaning process, and significantly saves on detection costs, outputting more accurate detection results at a lower cost. Those skilled in the art should understand that, based on the above verification process, the advantages of the detection method described in this invention in terms of efficiency and cost will become even more pronounced as the sample volume increases.

[0190] Table 4 Detection efficiency of different detection strategies

[0191] As can be seen from the results in Table 5, the multi-level judgment detection method of the present invention is basically equivalent to the detection method using solid phase in terms of detection accuracy. However, since the detection method of the present invention has the ability to identify interfering samples, its detection accuracy is significantly improved compared with the homogeneous mode.

[0192] Based on the results shown in Tables 4 and 5, the multi-level judgment detection method of the present invention has the ability to accurately identify interference samples, and has higher detection efficiency, lower detection cost and higher accuracy. The detection method of the present invention is particularly suitable for high-throughput detection needs.

[0193] Table 5. Detection accuracy of different detection strategies

[0194] Example 5: Determination of NT-proBNP

[0195] NT-proBNP is the best laboratory indicator for evaluating heart failure, but its content in blood samples is extremely low. The detection of this type of substance has very high requirements for both the detection method and the detection reagent. Taking NT-proBNP detection as an example, this invention demonstrates the universality, sensitivity and accuracy of the detection method and reagent described in this invention.

[0196] (1) Raw material preparation:

[0197] Reagent A:

[0198] Weigh 5 mg HRP and dissolve it in 1 ml of distilled water; add 0.2 ml of freshly prepared 0.1 M NaIO4 solution and stir for 20 minutes at room temperature in the dark; transfer the above solution to a dialysis bag and dialyze against 1 mM pH 4.4 sodium acetate buffer overnight at 4°C; add 20 μl of 0.2 M pH 9.5 carbonate buffer to raise the pH of the aldehyde-modified HRP to 9.3, then immediately add 10 mg IgG (NT-proBNP monoclonal antibody) to 1 ml of 0.01 M carbonate buffer and gently stir for 2 hours at room temperature in the dark; add 0.1 ml of freshly prepared 4 mg / ml NaBH4 solution, mix well, and incubate at 4°C for another 2 hours; transfer the above solution to a dialysis bag and dialyze against 0.15 M sodium acetate buffer overnight at 4°C. Dialyze to pH 7.4 PBS to remove ammonium ions (detected with Nessler's reagent), centrifuge at 10000 rpm for 30 min to remove precipitate, and the supernatant is the enzyme conjugate. After determining the concentration, add an equal volume of 60% glycerol and aliquot, then store at 4°C. Before aliquoting, add BSA at a ratio of 10 mg / ml as a stabilizing agent.

[0199] Reagent B:

[0200] Measure 5 mg of NT-proBNP antibody (NT-proBNP monoclonal antibody, Chongqing Aisens Biotechnology Co., Ltd.), desalt it using a PD-10 desalting column, and elute with 2.5 ml of biotin-conjugated buffer A. Then dilute the antibody to 1 mg / mL with biotin-conjugated buffer A. Weigh 10 mg of each... NHS-Biotin and NHS-(PEI) 20 -Acridine compounds, fully dissolved in DMSO to a concentration of 3.0 mg / ml, were prepared according to the antibody:NHS-Biotin:NHS-(PEI) ratio. 20 -Acridine compounds in a 1:20:20 molar ratio, measuring NHS-Biotin and NHS-(PEI). 20 The acridine compound solution was added to the antibody solution above and incubated at 25°C (with an allowable error range of ±2°C) for 180 minutes. The resulting solution was desalted using a PD-10 desalting column to remove unreacted molecules, eluted with 2.5 ml of biotin-conjugated buffer B, and the concentration was determined using a UV spectrophotometer before storage at 4°C. Before aliquoting, BSA was added at a ratio of 10 mg / ml as a stabilizing agent.

[0201] Reagents C and D, alkaline solution and cleaning solution are the same as in Example 1.

[0202] (2) Sample Construction

[0203] Samples with different concentrations were prepared by adding NT-proBNP to the buffer solution (sample preparation), and two concentrations of clinical serum samples were collected. Four 200 μL aliquots of each concentration were taken. One sample was designated as the experimental group, and hemoglobin (final concentration 10 mg / dL), bilirubin (final concentration 5 mg / dL), and visceral lipids (final concentration 1000 mg / dL) were added to the remaining three samples, respectively, and designated as the hemolysis group, jaundice group, and lipemia group. The information for each sample is shown in Table 6.

[0204] Table 6 NT-proBNP Detection Sample Information *The antigen concentration in serum samples was determined using a kit (Elecsys proBNP II STAT, Roche Diagnostics GmbH).

[0205] (3) Construction of solid-phase method for sample determination

[0206] The process includes the following steps: Take 20 μl of sample, add 60 μl each of reagent A and reagent B prepared in Example 5 and 30 μl of reagent D to the sample, mix well and react at 37°C for 10 min, perform 3-step magnetic separation and cleaning, then add 85 μl of reagent C and an appropriate amount of alkaline solution, the generated light signal is captured by a photomultiplier tube, and the light value signal is converted into a concentration value through a calibration curve.

[0207] (4) Construction of homogeneous sample determination method

[0208] The process includes the following steps: Take 20 μl of sample, add 60 μl each of reagent A and reagent B prepared in Example 5 to the sample, mix well and react at 37°C for 10 min, then add 85 μl of reagent C and 5 μl of 0.05 mM vitamin C. The generated light signal is captured by a photomultiplier tube, and the light value signal is converted into a concentration value through a calibration curve.

[0209] (5) Construction of the technical detection method described in this invention

[0210] Includes the following steps:

[0211] A. The preset item is set to "NT-proBNP". After comparison, the test item "NT-proBNP" is found to be compatible with the preset item.

[0212] B. All samples were measured using the "solid phase method".

[0213] (6) Effect

[0214] Table 7. Detection results of NT-proBNP in different samples

[0215] As can be seen from the test results in Table 7, for ultra-low concentration test items, a more accurate test method was adopted after comparing the preset items with the test items. This avoids the problem of inaccurate test results caused by directly using homogeneous detection methods, and also avoids the influence of interfering samples on the test results. Therefore, the present invention has higher accuracy than homogeneous detection and is not affected by interfering samples.

[0216] It should also be noted that the exemplary embodiments mentioned in this invention describe methods or systems based on a series of steps or apparatus. However, this invention is not limited to the order of the steps described above; that is, the steps can be performed in the order mentioned in the embodiments, or in a different order, or several steps can be performed simultaneously.

[0217] In this invention, features described and / or illustrated for one embodiment may be used in the same or similar manner in one or more other embodiments, and / or combined with or in place of features of other embodiments.

[0218] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, various modifications and variations can be made to the embodiments of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A target analyte detection kit, characterized in that, The kit includes reagent A and reagent B; reagent A contains a first binding molecule, which is coupled with compound 1; reagent B contains a second binding molecule, which is coupled with compound 2; the first binding molecule or the second binding molecule further includes at least one linker structure I. Wherein, compound 1 has the function of catalyzing compound 2 to generate a signal, the first binding molecule and the second binding molecule can specifically bind to the target in the sample to form a specific binding complex, and the first binding molecule and the second binding molecule are not coupled to a solid support.

2. The kit according to claim 1, characterized in that, The kit also includes reagent C, which contains compound 3; Wherein, the reaction of compound 2 and compound 3 produces a detectable signal; Preferably, compound 3 is selected from peroxides.

3. The kit according to claim 1 or 2, characterized in that, Compound 1 is a substance that can catalyze the generation of hydroxyl radicals from a substrate, including at least one of transition metal salts and / or complexes and peroxidases; preferably a peroxidase.

4. The kit according to any one of claims 1-3, characterized in that, The compound 2 is at least one of the compounds that generate photons under the action of hydroxyl radicals; preferably, it includes at least one of luminol, isoluminol, profen base, acridine ester, glimepiride, 9,10-dihydroacrylidine and phthalic acid hydrazide.

5. The kit according to any one of claims 1-4, characterized in that, Compound 1 and Compound 2 are coupled to the first binding molecule and the second binding molecule respectively via flexible connecting units; preferably, the flexible connecting units are selected from polyoxyethylene ethers, polyoxypropylene ethers, alkyl groups, polyethyleneimines, polypeptides, or blocks composed of the above substances.

6. The kit according to any one of claims 1-5, characterized in that, The kit also includes an alkaline solution with a pH value greater than or equal to 8.

7. The kit according to any one of claims 1-6, characterized in that, The kit further includes reagent D, which contains a solid-phase carrier coupled to linker structure II, wherein linker structure I and linker structure II specifically bind to each other to form a specific binding system; preferably, the specific binding system includes an antigen-antibody specific affinity system, an avidin-biotin specific binding system, a streptavidin-biotin specific binding system, and a Tag-Catcher specific binding system.

8. The kit according to any one of claims 1-7, characterized in that, The kit also includes signal inhibitors, including ascorbic acid or its salts, phenoloxazine, 2-aminophenol, 2-amino-p-chlorophenol, 2-amino-o-chlorophenol, 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid TROLOX™. 3-Aminotyrosine or its salt, p-Hydroxy-2-chlorophenol, p-Hydroxy-2-methoxyphenol, o-hydroxy-m-chlorophenol, and at least one of the compounds with the structure shown in Formula 1, In this group, any two or three of the substituents in the substituted R1-R6 are selected as -OH and / or -NH2, and the remaining substituents are -H, -F, -Cl and / or -Br, and two or three of the substituents are -F, -Cl and / or -Br.

9. The kit according to any one of claims 1-8, characterized in that, The kit also includes a cleaning solution.

10. A detection composition, characterized in that, The composition contains a binding molecule, which is coupled via a flexible linker to a substance that can catalyze the generation of hydroxyl radicals from a substrate or a substance that generates photons under the action of hydroxyl radicals. The binding molecule can specifically bind to the target analyte; The binding molecule further contains at least one linking structure, and the binding molecule is not coupled to a solid-phase support, wherein the linking structure is selected from specific binding substances.

11. The composition according to claim 10, characterized in that, The binding molecule is an antibody, an antigen, and / or an antigen-binding fragment.

12. The composition according to claim 10 or 11, characterized in that, The specific binding substance is selected from any two parts of the following specific binding systems, including: antibody-antigen specific binding system, avidin-biotin specific binding system, streptavidin-biotin specific binding system, and tag-catcher specific binding system.

13. The composition according to any one of claims 9-12, characterized in that, The compounds that generate photons under the action of hydroxyl radicals include at least one of luminol, isoluminol, profen base, acridine ester, glimepiride, 9,10-dihydroacrylidine, phthaloyl hydrazide, and derivatives thereof.

14. Use of the composition according to any one of claims 9-13 in the preparation of a test product.

15. The application as described in claim 14, characterized in that, The product is used for the detection of blood, urine, bone marrow, cerebrospinal fluid, pleural effusion, ascites, pericardial fluid, joint fluid, hydrocele, bile and / or tissue samples; preferably blood, plasma, serum and urine samples.

16. The application as described in claim 14 or 15, characterized in that, The product in question is a reagent kit.

17. A judgment-based detection method, characterized in that, The detection method includes the following steps: S1: Perform interference identification on the sample to be tested to obtain the interference value; S2: Compare the interference value with the preset threshold, and then select to execute either S3A-S4A or S3B-S4B based on the comparison result. in, The S3A step includes: selecting reagents and reacting the sample to be tested with a reagent that does not contain a solid support; The S3B step includes: selecting reagents and reacting the sample to be tested with reagents containing a solid support, and separating impurities in the detection system; Steps S4A and S4B include measuring the reaction system and reporting the results.

18. A judgment-based detection method, characterized in that, The detection method includes: S0: Compare and judge the test items of the sample to be tested with the preset items; based on the comparison and judgment results, select to execute (i) or (ii) detection process; The detection process (i) includes: S1: Perform interference identification on the sample to be tested to obtain the interference value; S2: Compare the interference value with a preset threshold, and based on the comparison result, select to execute either step S3A-S4A or step S3B-S4B; where... The S3A step includes: selecting reagents and reacting the sample to be tested with a reagent that does not contain a solid support; The S3B step includes: selecting reagents and reacting the sample to be tested with reagents containing a solid support, and separating impurities in the detection system; Steps S4A and S4B include: measuring the reaction system and reporting the test results; The detection process described in (ii) includes: The S3B step includes: selecting reagents and reacting the sample to be tested with reagents containing a solid support, and separating impurities in the detection system; The S4B step includes measuring the reaction system and reporting the results.

19. The method according to claim 17 or 18, characterized in that, The interruption value is compared with a preset threshold using one or more of the following methods: 1) Illuminate the sample to be tested or a mixture of the sample to be tested and the reagent using a light source, detect the transmitted light and / or scattered light signals, and make a judgment by comparing the transmitted light and / or scattered light signals with a preset threshold; 2) By calculating the difference in absorbance measured under dual wavelengths after dilution, the values ​​of serum / plasma samples are given in a semi-quantitative manner and compared with the preset threshold for determination; 3) The color features of the sample image are compared with a preset threshold to determine the result.

20. The detection method according to any one of claims 17-19, characterized in that, Based on the comparison results with the preset threshold, after the sample to be tested reacts with the reagent containing the solid support, an impurity removal step is performed.

21. The detection method according to any one of claims 17-20, characterized in that, The reagents used in step S3A include reagent A, reagent B, and reagent C; the reagents used in step S3B include reagent A, reagent B, reagent C, reagent D, and cleaning solution. The reagent A contains a first binding molecule, which is coupled with compound 1; the reagent B contains a second binding molecule, which is coupled with compound 2; the first binding molecule or the second binding molecule further includes at least one linking structure I; Wherein, compound 1 has the function of catalyzing compound 2 to generate a signal, the first binding molecule and the second binding molecule can specifically bind to the target in the sample to form a specific binding complex, and the first binding molecule and the second binding molecule are not coupled to a solid support; reagent C contains compound 3, wherein compound 2 and compound 3 react to generate a detectable signal; reagent D contains a solid support coupled to linking structure II, and linking structure I and linking structure II specifically bind to each other to form a specific binding system.

22. The detection method as described in claim 21, characterized in that, The reagents in step S3A and / or step S3B further include signal inhibitors, such as ascorbic acid or its salts, phenoloxazine, 2-aminophenol, 2-amino-p-chlorophenol, 2-amino-o-chlorophenol, 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid TROLOX™, etc. 3-Aminotyrosine or its salt, p-Hydroxy-2-chlorophenol, p-Hydroxy-2-methoxyphenol, o-hydroxy-m-chlorophenol, and at least one of the compounds with the structure shown in Formula 1, In this group, any two or three of the substituents in the substituted R1-R6 are selected as -OH and / or -NH2, and the remaining substituents are -H, -F, -Cl and / or -Br, and two or three of the substituents are -F, -Cl and / or -Br.

23. Application of the compound with the structure shown in Formula 1 in the preparation of detection kits. in, Two or three of the substituted R1-R6 groups are selected as -OH and / or -NH2, and the remaining substituents are -H, -F, -Cl and / or -Br, with two or three substituents being -F, -Cl and / or -Br.

24. The application as described in claim 23, characterized in that, The compound is used to improve the signal-to-noise ratio and / or reduce background interference.

25. A multi-level judgment-based detection system, characterized in that, The detection system includes: a comparison module, an interference identification and analysis module, an impurity separation module, a reagent absorption module, an optical detection module, and a signal processing module, wherein... The comparison module is used to compare the information of the item to be tested with that of the preset item. The interference identification and analysis module is used to analyze sample interference information; The impurity separation module is used to separate impurities and reduce substances in the detection system that affect the detection results; The optical testing module is used to detect the signals generated by the reaction system; The signal processing module is used to process signals and convert them into visual information.

26. The detection system as described in claim 25, characterized in that, The impurity separation module includes a cleaning submodule, which uses the difference between reagents bound to a solid support and reagents not bound to a solid support to separate impurities.

27. The detection system as described in claim 25 or 26, characterized in that, The detection system has at least two different detection channels, which are used to detect high-interference samples and low-interference samples respectively.

28. The detection system as described in claim 27, characterized in that, The detection system also includes a sample transfer module, the function of which includes: transferring samples to different detection channels based on interference identification results.

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