A multi-level judgment type high-precision detection method, system and kit

Abstract

The present invention discloses a multi-level judgment-based target detection method, system and detection kit. The detection kit includes reagents having a connection structure with a solid-phase carrier, and is used in cooperation with a detection method and a detection system having multi-level judgment steps and corresponding modules. According to the sample test requirements, the reagents are used in cooperation with the solid-phase carrier containing the connection structure during the test process, which is flexible in use and can match different detection requirements. Therefore, the detection method and system described in the present invention integrate the advantages of solid-phase washing detection technology and homogeneous detection technology, avoiding both unnecessary washing when the whole sample is detected in the solid-phase mode and the problem of low accuracy caused by special samples when the whole sample is detected in the homogeneous detection mode.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostics, and particularly to immunoassay methods, systems and kits. Background Art

[0002] In the past nearly 40 years, immunoassay technology has undergone a transformation from radioimmunoassay technology, enzyme-linked immunosorbent assay technology to magnetic particle chemiluminescence technology. With the continuous improvement of detection sensitivity, many biomarkers with great clinical value have been applied to clinical diagnosis. Luminescence detection methods containing a solid-phase washing step, such as magnetic particle chemiluminescence technology, have gradually replaced enzyme-linked immunosorbent assay technology and occupied nearly 90% of the market share. Although magnetic particle chemiluminescence technology has been widely used clinically due to its sensitivity and specificity, some limitations have also been exposed. First, the magnetic washing action increases the difficulty of the liquid path module of the instrument and the instrument cost; second, due to the magnetic washing action, the detection throughput of the detection instrument is limited. Inspection departments with a large sample detection volume need to extend working hours or add multiple devices to meet the detection requirements. To avoid the complexity brought by solid-phase washing such as magnetic beads, some homogeneous detection technologies, such as proximity ligation assay, have gradually achieved technological breakthroughs.

[0003] In proximity ligation assay, one antibody participating in the immune reaction is labeled with horseradish peroxidase (HRP), and another antibody participating in the immune reaction is labeled with a luminescent compound, such as acridinium ester (AE). In the presence of the target antigen, a sandwich immune complex can be formed, and the target antigen can tightly connect the HRP and AE labeled on the two antibodies. Under the action of the substrate, a light signal is generated. The intensity of the light signal is proportional to the concentration of the target antigen, and the related technology has been disclosed in WO2010099486A1. Its technical advantages are as follows: ① Based on the antigen-antibody immune reaction, an immune complex is formed, and the amount of the complex is monotonically related to the amount of the analyte; the antigen-antibody immune reaction ensures the specificity of the detection. ② The immune reaction occurs in a homogeneous system, and the homogeneous reaction ensures relatively high detection precision; ③ A signal is generated based on the proximity of HRP and AE, so the instrument does not require a liquid path system to achieve magnetic washing, the instrument cost is low, the volume is small, and high throughput can be achieved.

[0004] However, proximity ligation assay still has some limitations. For example, there is no magnetic washing process during the reaction, and interfering substances in the sample cannot be removed, resulting in poor detection results for samples with high interference. For example, hemolytic and lipemic samples that often appear in some elderly populations will interfere with the immune reaction, thereby affecting the accuracy of the detection results; compared with magnetic particle chemiluminescence, the detection sensitivity is relatively low.

[0005] In summary, existing detection technologies all have certain limitations. There is an urgent need in the clinical scenario for a fully automated, high-throughput detection technology with lower costs, lower interference, and higher sensitivity to meet clinical needs. Summary of the Invention

[0006] The object of the present invention is to provide a kit for detecting a target analyte, its testing method, and system. The kit and its detection method can reduce the number of washings of unnecessary samples, reduce the frequency of use of solid-phase material detection methods, significantly reduce the cost, improve the detection throughput, and also avoid the problems of low sensitivity and accuracy caused by special samples.

[0007] Therefore, in one aspect of the present invention, there is provided a kit for detecting a target substance, the kit comprising reagent A and reagent B, wherein reagent A contains a first binding molecule, and the first binding molecule is conjugated to compound 1;

[0008] Reagent B contains a second binding molecule, and the second binding molecule is conjugated to compound 2;

[0009] At least one linking structure I is further included on the first binding molecule and / or the second binding molecule;

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

[0011] Among them, the working concentration of the first binding molecule complex conjugated with compound 1 in reagent A is 0.1 - 3.0 μg / mL; the working concentration of the second binding molecule complex conjugated with the compound in reagent B is 0.1 - 3.0 μg / mL.

[0012] "Target substance" refers to a certain or certain biochemical substances whose specific content or concentration is desired to be obtained through detection means.

[0013] "Catalyze" means to change the activation free energy required for a reaction through a catalyst, change the chemical reaction rate of the reactants. Compound 1 does not directly act on compound 2, but through the "catalytic" effect, it enables compound 2 to undergo a chemical reaction with other substances.

[0014] Furthermore, the first binding molecule and the second binding molecule are not conjugated to a solid-phase carrier.

[0015] Furthermore, the kit further includes reagent C, and reagent C contains compound 3. Among them, a detectable signal, such as an optical signal, is generated after the reaction of compound 2 and compound 3; when the first binding molecule in the kit is conjugated with compound 1, when the first binding molecule conjugated with compound 1, the second binding molecule conjugated with compound 2, and the target to be detected bind together, the distance between compound 1 and compound 2 is sufficient for compound 1 to catalyze compound 2 to generate a detectable signal, while a detectable signal cannot be generated between phosphatase and compound 2 at the same distance. Therefore, further, compound 1 is not a phosphatase, such as alkaline phosphatase.

[0016] Furthermore, compound 1 and compound 2 are conjugated to the first binding molecule and the second binding molecule respectively through a flexible linking unit.

[0017] The "flexible linking unit" is a non-rigid substance. When the first binding molecule and the second binding molecule conjugated with compound 1 and compound 2 form a complex with the target to be detected, compound 1 and compound 2 can move around the periphery of the complex based on the flexibility of the "flexible linking unit" so that the substances capable of generating a luminescence signal are close enough in space to generate a detectable signal. The "flexible linking unit" is selected from polyoxyethylene ether substances, polyoxypropylene ether substances, alkyl groups, polyethyleneimine substances, polypeptides, and block copolymers composed of the above substance units. In order to ensure the luminescence efficiency, the flexible linking unit should not be too long. Therefore, preferably, the total effective length of the flexible linking unit linking compound 1 and compound 2 is less than or equal to 200 nm, preferably less than 180 nm, more preferably less than 150 nm; at the same time, the total length should be greater than the diameter of the binding molecule; or the total degree of polymerization of the flexible linking unit is less than 500.

[0018] "Polyoxyethylene ether substances" are also called poly(ethylene oxide) (PEO) or poly(ethylene glycol) (POE) substances, which refer to polymers with a PEG main chain. Preferably, they are a class of compounds with a PEG main chain and active groups capable of being linked to biomolecules at the chain ends. Specifically, for example, NHS-(PEG)n, Pierce TM PEG-SPDP series in

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

[0020] "Polypeptides" are polymers including at least two amino acids.

[0021] "Polyethyleneimine substances" are a class of polymers with a main chain of "-CH 2 CH 2 NH-", including substances with substitutions on the side chains and / or both ends.

[0022] "Block substance" refers to a substance containing two or more basic constituent units of flexible connecting unit substances. For example, the constituent unit of polyoxyethylene ether compounds -OCH 2 CH 2 - and the constituent unit of polyethyleneimine compounds -CH 2 CH 2 NH- form a block polymer.

[0023] "Total degree of polymerization" is the sum of the degrees of polymerization of the two parts of flexible connecting units on the first binding molecule and the second binding molecule.

[0024] "Solid-phase carrier" is a material with at least one dimension in three-dimensional dimensions greater than 0.1 μm or 0.2 μm, preferably greater than 0.3 μm or 0.5 μm. The material has an adsorption effect or the surface of the material has reactive functional groups that can be coupled with the binding molecule. The material can have the following forms: particles, microparticles, metal colloids, fibers, papers, beads, membranes, filter papers, 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 analyte is an antigen, the first binding molecule and the second binding molecule are optionally antibodies or antibody fragments specific to the antigen; for example, when the target analyte is an antibody, the first binding molecule and / or the second binding antibody are optionally antigens or secondary antibodies against the target analyte antibody.

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

[0027] Furthermore, Compound 1 is a substance that can catalyze the substrate to generate hydroxyl radicals, including transition metal salts and / or complexes and peroxidases. Specific enzymes such as lactoperoxidase, microperoxidase, myeloperoxidase, haloperoxidase, vanadium bromoperoxidase, horseradish peroxidase, fungal peroxidase, lignin peroxidase, peroxidase from Arthromyces ramosus, Mn-dependent peroxidase produced by white rot fungi, or soybean peroxidase, and transition metal complexes such as ferroprotoporphyrin, etc., are substances that can catalyze the chemiluminescent oxidation of the substrate. Correspondingly, Compound 2 is a substance that generates photons under the action of the enzyme of Compound 1, such as luminol, isoluminol, lucigenin, acridinium ester, lucigenin, 9,10-dihydroacridine, and phthalhydrazide.

[0028] Further, 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 can be understood that, different from the case where compound 1 catalyzes compound 2 to generate a signal, compound 3 directly reacts with compound 2 to generate a signal. Therefore, at the same concentration, the signal effect generated by the reaction of compound 3 and compound 2 is more obvious than that of the interaction between compound 1 and compound 2. Or it can be understood that compound 1 has the effect of enhancing the signal generation of compound 3 and compound 2.

[0030] "Optical signal" means that a compound generates photons through a chemical reaction or is excited by an external environment (such as temperature change, light irradiation, magnetic field change, etc.). The photons serve as information carriers, and the expected information is obtained by detecting the photons. For example, relevant information is obtained by detecting fluorescence, flash, or glow.

[0031] Further, the kit further includes reagent D, which contains a solid-phase carrier coupled with linking structure II. Linking structure I and linking 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 carrier complex coupled with linking structure II in reagent D is 0.2 - 5.0 mg / mL.

[0033] When linking structures I and II are an antibody and an antigen with specific binding properties, their types are different from those of the detection target and the first binding molecule and / or the second binding molecule. The antibody and antigen of linking structures I and II will not specifically bind to the detection target and the first binding molecule and / or the second binding molecule either. Without being limited by examples, the antibody and antigen of 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 linking structure II is an anti - FITC antibody, preferably a monoclonal antibody.

[0034] The "Tag-Catcher" system consists of two parts: protein and polypeptide (short peptide). An isopeptide bond is spontaneously formed through residues that can specifically recognize each other with high affinity between the two parts. It includes the SpyTag-SpyCatcher system, SdyTag-SdyCatcher system, SnoopTag-SnoopCatcher system, and DogTag-DogCatcher system. The binding modes of different "Tag-Catcher" systems are basically the same. The binding between the two in the system is very high, the reaction is rapid, and no additional chemical reagents or catalysts are required. For example, in the earliest discovered SpyTag-SpyCatcher system, the carboxyl group of aspartic acid (aspartic acid residue) in SpyTag specifically recognizes the primary amino group of lysine (lysine residue) in SpyCatcher, and an amide bond is formed by dehydration. The preparation and principle can be found in References 1 and 2. The preparation and principle of SdyTag-SdyCatcher, SnoopTag-SnoopCatcher, and DogTag-DogCatcher can be found in References 3-5 respectively. The connection and preparation methods of Tag-Catcher with substances such as magnetic beads, antibodies, and proteins can be found in References 5-10.

[0035] The specific binding between the two parts in the "Tag-Catcher" system is a covalent bond binding. Therefore, after binding, it is less susceptible to environmental changes and the connection is more stable.

[0036] Furthermore, the kit further 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 with corresponding alkaline substances, such as sodium hydroxide. It can be understood that alkaline substances are commonly used promoters for chemiluminescence, especially suitable for enzyme-free chemiluminescence systems. For example, after adding hydrogen peroxide to the magnetic beads (capturing the analyte) after washing and then adding the alkaline solution, a detectable luminescence signal can be generated.

[0037] Furthermore, the kit also includes a signal inhibitor (Selective Signal Inhibiting Agent). The signal inhibitor can be understood as being used to increase the signal in the measurement process. When it is applied to the detection of the target, the degree to which the signal obtained from the measurement reaction system exceeds the background signal is significantly greater than that without the signal inhibitor. At the same time, it should be noted that during the use of the reagent, it is basically in an open state, and the signal inhibitor will be in full contact with the air. The signal inhibitor is easily oxidized in the environment. Therefore, the stability of the signal inhibitor itself is extremely important.

[0038] The signal inhibitors described in the present invention are selected from the types listed in WO2010099486A1, and include, for example, ascorbic acid or its salts, phenoxazine, 2-aminophenol, 2-amino-p-chlorophenol, 2-amino-o-chlorophenol, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid TROLOXTM, 3-aminotyrosine or its salts, p-hydroxy-2-chlorophenol, p-hydroxy-2-methoxyphenol, o-hydroxy-m-chlorophenol, and at least one of the compounds having the structure shown in Formula 1.

[0039] In addition, WO2010099486A1 discloses that the signal inhibitors of some structures have poor effects. However, the applicant unexpectedly found that the compounds having the structure shown in Formula 1 have outstanding effects in suppressing background interference signals, and are particularly remarkable in terms of stability.

[0040]

[0041] Among them, any two or three substituents in the substituted R 1 -R 6 are respectively -OH and / or -NH 2 , and the remaining substituents are respectively -H, -F, -Cl and / or -Br, and two or three substituents are respectively -F, -Cl and / or -Br. Preferably, there are two substituents in the substituted R 1 -R 6 which are respectively -F, -Cl and / or -Br.

[0042] Therefore, another aspect of the present invention lies in the use of the signal inhibitors described in the present invention in test kits, specifically for reducing background interference signals.

[0043] The concentration of the signal inhibitors described in the present invention in the detection system is 10 -4 mM - 10 mM, for example 10 -3 mM - 1 mM or 10 -2 mM - 1 mM.

[0044] Another aspect of the present invention is to provide a detection composition, which contains: a binding molecule in which a substance capable of generating hydroxyl radicals or a substance generating photons under the action of hydroxyl radicals is coupled through a flexible linking unit; optionally, the flexible linking unit is selected from polyoxyethylene ether compounds, polyoxypropylene ether compounds, alkyl groups, polyethyleneimine compounds, polypeptides, and block copolymers of the above-mentioned substance units; the binding molecule further contains at least one linking structure, the binding molecule is a specifically binding substance and can specifically bind to a target; and the linking structure does not form a specific linkage and the binding molecule is not coupled to a solid phase carrier.

[0045] Further, the binding molecule is an antibody, an antigen, and / or an antigen-binding fragment.

[0046] Further, the linking structure is selected from any one of two parts in the following specific binding systems, including: antibody-antigen system, avidin-biotin system, streptavidin-biotin system, and Tag-Catcher system.

[0047] The antibody-antigen system and the Tag-Catcher system have the functions and species selections as described above.

[0048] Further, the substance capable of generating hydroxyl radicals includes horseradish peroxidase, and the compound generating photons under the action of hydroxyl radicals includes at least one of luminol, isoluminol, lucigenin, acridinium ester, lucigenin, 9,10-dihydroacridine, and phthalhydrazide and derivatives of the above substances.

[0049] Another aspect of the present invention is to provide the application of the above composition in the preparation of a detection product; the product is used for the detection of blood, urine, bone marrow, cerebrospinal fluid, pleural effusion, ascites, pericardial fluid, synovial fluid, hydrocele, bile, and / or tissue samples; preferably blood, plasma, serum, and urine samples; further, the product is a kit.

[0050] Another aspect of the present invention is to provide a judgment-based detection method, including the following steps:

[0051] S1: Perform interference recognition on the sample to be tested to obtain an interference value;

[0052] S2: Compare the interference value with a preset threshold, and then select to execute steps S3A-S4A or steps S3B-S4B according to the comparison result;

[0053] Among them,

[0054] Step S3A includes: selecting a reagent and reacting the sample to be tested with a reagent not containing a solid phase carrier;

[0055] Step S3B includes: selecting reagents, reacting the sample to be tested with the reagents containing a solid phase carrier, and separating impurities in the detection system;

[0056] Steps S4A and S4B include: measuring the reaction system and reporting the detection result.

[0057] It can be understood that due to the differences in detection methods, the reagents selected in S3A and S3B are also different. For example, the reagents in step S3A include reagent A, reagent B, and reagent C, and the reagents in step S3B include reagent A, reagent B, reagent C, reagent D, a cleaning agent, and an alkaline solution; meanwhile, the reagents in S3A and S3B optionally contain a signal inhibitor. In the absence of mutual influence, the above reagents can be stored separately or together as a mixture.

[0058] Further, before step S1, the following step S0 is also included: comparing and judging the item to be tested of the sample to be tested with a preset item; and then selecting to execute S1 and subsequent steps or steps S3B - S4B according to the comparison and judgment results (as Figure 2 shown).

[0059] Therefore, the present invention also relates to a detection method, and the detection method includes:

[0060] S0: comparing and judging the item to be tested of the sample to be tested with a preset item; and then selecting to execute the detection process of (i) or (ii) according to the comparison and judgment results;

[0061] The detection process of (i) includes:

[0062] S1: identifying interference of the sample to be tested to obtain an interference value;

[0063] S2: comparing the interference value with a preset threshold, and then selecting to execute steps S3A - S4A or steps S3B - S4B according to the comparison result; wherein,

[0064] Step S3A includes: selecting reagents and reacting the sample to be tested with the reagents without a solid phase carrier;

[0065] Step S3B includes: selecting reagents, reacting the sample to be tested with the reagents containing a solid phase carrier, and separating impurities in the detection system;

[0066] Steps S4A and S4B include: measuring the reaction system and reporting the detection result;

[0067] The detection process of (ii) includes:

[0068] Step S3B includes: selecting reagents, reacting the sample to be tested with the reagents containing a solid phase carrier, and separating impurities in the detection system;

[0069] Step S4B includes: measuring the reaction system and reporting the detection result.

[0070] The reagents for step S3A include reagent A, reagent B, and reagent C; the reagents for step S3B include reagent A, reagent B, reagent C, reagent D, and a cleaning solution; further, signal inhibitors are also included in the reagents for step S3A and / or step S3B, and the reagents reagent A, reagent B, reagent C, reagent D, and the signal inhibitor have the selected types described herein.

[0071] The "item to be measured" is the item for which a test sample is to be tested, and the purpose is to obtain quantitative information about a certain biochemical substance or certain biochemical substances. The "preset item" is a test item set in advance. The purpose of setting the "preset item" is to reduce interference and provide detection accuracy. The "preset item" can be determined, for example, based on clinical research or literature. Since the content of the target substance to be detected in the sample is extremely low or there is extremely high interference and accurate quantitative analysis cannot be performed, such test items can be regarded as "preset items". When the "item to be measured" is a "preset item" or includes a "preset item", detection steps for eliminating interference need to be adopted, such as steps S3B - S4B; when the "item to be measured" does not include a "preset item", otherwise steps S1 and subsequent steps are adopted; or since the content of the target substance to be detected in the sample is extremely low or there is extremely high interference and accurate quantitative analysis cannot be performed, test items that are not such cases are set as "preset items". When the "item to be measured" meets the "preset item", steps S1 and subsequent steps are adopted, otherwise steps S3B - S4B are adopted. Specifically, for example, the content of the AD biomarker (such as tau - 181) in blood is low, and the test item for detecting tau - 181 in a blood sample is set as a "preset item". When the "item to be measured" is to detect tau - 181 in a blood sample, it is determined to meet the "preset item", and steps S3B - S4B are adopted.

[0072] Further, the relationship between the interference value and the preset threshold is judged by one or more of the following methods:

[0073] 1) Irradiate the test sample or the mixed solution of the test sample and the reagent with a light source, detect the transmitted light and / or scattered light signals, and judge by comparing the transmitted light and / or scattered light signals with the preset threshold;

[0074] 2) By calculating the difference in absorbance measured at two wavelengths of the diluted test sample, semi - quantitatively give the value of the serum / plasma sample, and compare it with the preset threshold for judgment; the sample value is, for example, the value of lipemia, hemolysis, and / or jaundice in the sample;

[0075] 3) Judge by comparing the color characteristics of the sample picture with the preset threshold.

[0076] The "mixed solution of the sample to be tested and the reagent" refers to the solution obtained by mixing the original sample with a reagent that can enhance the detection effect.

[0077] Without being limited by examples, the above methods for judging whether there are interference values in the sample all adopt means known in the art. For example, the methods and modules described in CN105980833B and CN113592842B are used to detect lipemic, hemolytic and icteric blood samples. Another example is to use the methods and instruments described in documents such as CN109374551A, CN204101456U, CN210894370U, or the EXC400 automatic biochemical analyzer (Zhongyuan Huiji Biotechnology Co., Ltd.) to detect the transmitted light and / or scattered light signals of the sample, and use a UV-4800 double-beam spectrophotometer (Unico (Shanghai) Instruments Co., Ltd.) for dual-wavelength detection, and the pictures are obtained by means such as an ordinary camera or a micro camera.

[0078] The "preset threshold" is a value set based on existing data and information, which has the function of distinguishing high-interference samples and low-interference samples. The data and information required to set the "preset threshold" can be obtained, for example, from analyzing the differences between special samples and ordinary samples in clinical big data. For example, compared with ordinary blood samples (relative to non-hemolytic samples), there are significant differences in the transmitted light between hemolytic blood samples with ruptured red blood cells and ordinary blood samples. For example, the light transmittance of hemolytic samples is lower. Based on big data, the light transmittance value range of ordinary blood samples is obtained, and the "preset threshold" is set accordingly. And the "preset threshold" can be appropriately tightened to ensure the accuracy of the detection results. For example, to reduce the interference of hemolytic samples, the "preset threshold" of transmitted light can be 5% or 10% lower than the light transmittance value of ordinary blood samples. When the light transmittance of the detected sample is lower than the "preset threshold", it is determined as a "high-interference" sample; when the light transmittance of the detected sample is higher than the "preset threshold", it is determined as a "low-interference" sample.

[0079] To further improve the detection accuracy, preferably, there can be multiple "preset thresholds" in step S1, which are obtained by measurement under different detection methods. When the measured value of the detected sample meets the low-interference determination conditions when compared with two "preset thresholds", the sample is determined as a low-interference sample; otherwise, it is determined as a high-interference sample. For example, two different "preset thresholds" are determined by the transmission method and dual-wavelength measurement. The detected sample is also detected by the transmission method and dual-wavelength. When the transmittance and absorbance difference of the detected sample are respectively higher than the "preset threshold" set by the transmission method and lower than the "preset threshold" set by the dual-wavelength measurement method, the sample is determined as a low-interference sample.

[0080] In the present invention, the purpose of "separating impurities" or "removing impurities" is to reduce the content of substances in the reaction system that interfere with the measurement results. The ways of interfering with the measurement results include, for example, increasing the background signal, suppressing the photon output of the luminescent compound, reducing the detected amount of the expected signal, etc. Substances that can have the above effects can all be regarded as impurities. The ways of separating and removing impurities include, for example, cleaning, centrifugation, sedimentation, adsorption (such as HPLC, chromatography columns, etc.), liquid separation, etc. Without being limited by the examples, any way that can reduce the content of impurities in the measurement system and reduce its influence on the measurement system can be regarded as an effective way of "separating impurities".

[0081] Further, according to the preset items or interference judgment, the "impurity separation" step can be carried out multiple times, such as two or more times; further, the cleaning method with a solid-phase carrier is a preferred impurity separation method.

[0082] Further, the reagent used in the detection method described in the present invention has the composition of the reagent described in the aforementioned kit, or the kit described in the present invention is used in combination with the detection method described in the present invention for the detection of the target analyte.

[0083] Another aspect of the present invention is to provide a multi-level judgment detection system, which includes a detection item comparison module, an interference recognition and analysis module, an impurity separation module, a reagent aspiration module, an optical detection module, a signal processing module, etc.

[0084] The comparison module is used to compare the information of the item to be measured with the preset item information;

[0085] The interference recognition and analysis module is used to analyze the sample interference information;

[0086] The impurity separation module is used to separate impurities and reduce the substances in the detection system that affect the detection results; further, the impurity separation module includes a cleaning sub-module, and the cleaning sub-module uses the differences between the reagent combined with the solid-phase carrier and the reagent not combined with the solid-phase carrier to separate the impurity substances. The differences are manifested as: differences in the magnetic field, differences under the action of centrifugal force, differences under the action of gravity, differences under the action of buoyancy, differences in solubility, or differences in the retention time in the chromatography column.

[0087] The optical test module is used to detect the signal generated by the reaction system;

[0088] The signal processing module is used to process the signal and convert the signal into visual information, such as a graph or a number, etc.

[0089] The detection system can have at least two different detection channels for detecting high-interference samples and low-interference samples respectively. Therefore, based on this situation, the detection system further includes a sample transfer module, which transfers samples to different detection channels based on the interference recognition result.

[0090] Alternatively, the detection system can have only one detection channel, and high-interference samples and low-interference samples are detected in the same detection channel by sucking different reagents through a reagent sucking module.

[0091] Furthermore, whether to call the impurity separation module is determined according to the analysis result of the interference recognition and analysis module.

[0092] In addition, based on the need for impurity separation, the detection system further includes a module for transferring samples before and after impurity separation. This module can be the aforementioned sample transfer module or a separate module.

[0093] In the present invention, the biological sample containing the target analyte can exist in any form, and the technical solution of the present invention is particularly applicable to samples prone to high interference values, such as blood, plasma, serum, and urine.

[0094] The "kit" of the present invention is based on reagent A and reagent B, and optionally further contains reagent C, reagent D, and / or signal inhibitor and other reagents according to the detection needs.

[0095] In the present invention, the "kit" refers to a detection system composed of detection reagents. Different detection reagents can be stored in the same packaging container, or separately in containers, or some reagents are mixed and stored in a container while other reagents are stored separately in a container. The containers storing different reagents can be connected together, or partially connected together, or not connected together at all. For example, depending on factors such as system control and equipment, the reagent detection system includes reagent A, reagent B, and reagent D, and reagent A, reagent B, and reagent D are stored separately and used as needed during detection; or for example, the reagent detection system includes reagent A, reagent B, reagent C, and reagent D, reagent A and reagent C are stored in one container, reagent B and reagent D are stored separately, and used as needed during detection.

[0096] In the present invention, "specific binding" has the meaning well-known in the art and is a biological binding process in which a ligand with a specific orientation and capable of being competitively blocked by a corresponding substance interacts with a specific structural site in vitro or in vivo, such as the binding between an antigen and an antibody, or between a receptor and a ligand, or the binding between biotin and avidin, etc.

[0097] In the present invention, the "coupling" method includes the direct connection of two substances through active functional groups (direct coupling), or the indirect connection through one or more substances (indirect coupling). For example, the direct coupling method is the connection between a solid-phase carrier with a carboxyl group on its surface and the amino part of an antibody, so that the solid-phase carrier is directly coupled with the antibody. The indirect coupling method is, for example, the connection between two parts through a secondary antibody or through avidin-biotin.

[0098] The kit and the detection composition thereof according to the present invention can be simultaneously applicable to the detection of highly interfering samples and ordinary samples. According to the different samples, different reagents in the kit are selected to detect the samples, so as to meet the needs of high-throughput detection and the accuracy of the results.

[0099] For example, in one embodiment of the present invention, the kit includes:

[0100] 1) Reagent A: containing a first binding molecule conjugated with compound 1;

[0101] 2) Reagent B: containing a second binding molecule conjugated with compound 2;

[0102] 3) Reagent D: containing a solid-phase carrier conjugated with linking structure II;

[0103] The first binding molecule or the second binding molecule includes at least one linking structure I, and the linking structures I and II have specific binding effects. The detection principle is as follows: judge the sample interference value. If it is a low-interference sample, directly use the first binding molecule in reagent A and the second binding molecule in reagent B to capture the target to be detected (such as an antigen). Compound 2 generates a light signal under the catalytic action of compound 1, and the detection is completed. If it is a high-interference sample, use the solid-phase carrier in reagent D to separate the first binding molecule and the second binding molecule that have captured the target analyte from the reaction system, and then perform subsequent detection, so as to reduce interference and improve the detection accuracy. The first binding molecule, the second binding molecule, compound 1, compound 2, linking structure I and II are the same as those described above.

[0104] For example, in another embodiment of the present invention, the kit includes:

[0105] 1) Reagent A: containing a first binding molecule;

[0106] 2) Reagent B: containing a second binding molecule conjugated with compound 2;

[0107] 3) Reagent C: containing compound 3;

[0108] 4) Reagent D: containing a solid-phase carrier conjugated with linking structure II;

[0109] The first binding molecule and / or the second binding molecule comprise at least one linking structure I, and the linking structures I and II have specific binding effects. The detection principle is as follows: judge the sample interference value. If it is a low-interference sample, directly use the first binding molecule in reagent A and the second binding molecule in reagent B to capture the target to be detected (such as an antigen). Compound 2 reacts with compound 3 in reagent C to generate a light signal, and the detection is completed. If it is a high-interference sample, use the solid-phase carrier in reagent D to separate the first binding molecule and the second binding molecule that have captured the target analyte from the reaction system, and then perform subsequent detection, thereby reducing interference and improving the detection accuracy. The first binding molecule, the second binding molecule, compound 2, compound 3, linking structures I and II are the same as those described above.

[0110] For example, in another embodiment of the present invention, the kit comprises:

[0111] 1) Reagent A: containing a first binding molecule conjugated with compound 1;

[0112] 2) Reagent B: containing a second binding molecule conjugated with compound 2;

[0113] 3) Reagent C: containing compound 3;

[0114] 4) Reagent D: containing a solid-phase carrier conjugated with the linking structure II;

[0115] The first binding molecule and / or the second binding molecule comprise at least one linking structure I, and the linking structures I and II have specific binding effects. The detection principle is as follows: judge the sample interference value. If it is a low-interference sample, directly use the first binding molecule in reagent A and the second binding molecule in reagent B to capture the target to be detected (such as an antigen). Compound 1 catalyzes the reaction between compound 2 and compound 3 in reagent C to generate a light signal, and the detection is completed. If it is a high-interference sample, use the solid-phase carrier in reagent D to separate the first binding molecule and the second binding molecule that have captured the target analyte from the reaction system, and then perform subsequent detection, thereby reducing interference and improving the detection accuracy. The first binding molecule, the second binding molecule, compound 1, compound 2, compound 3, linking structures I and II are the same as those described above.

[0116] Optionally, a signal inhibitor is further included in the above-listed embodiments, and a cleaning solution is also included when using a reagent with a solid-phase carrier.

[0117] Reference Figure 1 , Figure 1 is a schematic diagram of a detection embodiment described in the present invention. According to the differences between high-interference samples and low-interference samples, the composition of the kit is different.

[0118] Reference Figure 2 , Figure 2It is a schematic flowchart of an implementation mode of a detection method according to the present invention. First, preset items are set in the detection system. When it is judged in step S0 that the preset items and the items to be detected do not match, the sample interference value is identified in step S1 to obtain the interference value. In step S2, the interference value is compared with a preset threshold. Samples with a low interference comparison result are reacted with a reagent without a solid phase in step S3A, and the signals generated during the reaction are detected in step S4A and a report of the corresponding results is output; for samples with a high interference comparison result, a reagent containing a solid phase is used for reaction and impurity removal and separation in step S3B, and the signals generated during the reaction are detected in step S4B and a report of the corresponding results is output.

[0119] When the item to be detected does not match the preset item content, a reagent containing a solid phase is used for reaction and impurity removal and separation in step S3B, and the signals generated during the reaction are detected in step S4B and a report of the corresponding results is output.

[0120] Reference Figure 3 , Figure 3 It is a schematic diagram of an implementation mode of a detection system according to the present invention, including 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.

[0121] Based on the technical solution of the present invention, the inventor believes that the beneficial effects of the present invention are at least as follows:

[0122] 1) A test reagent with a completely new structure is proposed. The test reagent contains a structure connected to a solid-phase carrier, and the test reagent is used in combination with the solid-phase carrier with a connection structure during the test according to the sample test requirements, which is flexible to use and can match different detection needs;

[0123] 2) The kit according to the present invention contains a variety of reagents, and different reagent types can be proportioned according to different sample detection requirements, integrating the advantages of solid-phase cleaning detection technology and homogeneous detection technology, avoiding unnecessary cleaning when the whole sample is detected in the solid-phase mode, and avoiding the problem of low accuracy due to the existence of special samples when the whole sample is detected in the homogeneous mode.

[0124] 3) The kit according to the present invention has a wider application scenario, can be applied to the detection needs of various different samples, meets the requirements of complex application scenarios, and significantly improves the reliability of the kit.

[0125] 4) The detection method and detection system of the present invention have multiple judgment steps and corresponding modules. Different modules are selected according to different judgment results, and the detection method is more flexible, more suitable for the detection of sample groups with complex situations. Under high-throughput detection, the detection time is shorter than that of solid-phase detection, and the detection accuracy is higher than that of homogeneous detection.

[0126] 5) A new type of signal inhibitor applicable to homogeneous reactions is proposed, which has higher stability in actual application scenarios.

[0127] References:

[0128] Literature 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);

[0129] Literature 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;

[0130] Literature 3: Tan LL, et al. Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly. PLoS ONE 11(10): e0165074;

[0131] Literature 4: Veggiani G, et al, Programmable polyproteams built using twin peptide superglues. Proc Natl Acad Sci U S A. 2016 Feb 2; 113(5): 1202-7;

[0132] 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;

[0133] Document 6: X. Pei, et al, Putting precision and elegance in enzyme immobilisation with bio-orthogonal chemistry,Chem.Soc.Rev., 2022, 51, 7281, DOI: 10.1039 / D1CS01004B;

[0134] Document 7: CN117723749A;

[0135] Document 8: CN114990146A;

[0136] Document 9: CN117362447A;

[0137] Document 10: CN116444623B.

[0138] Attached drawings of the specification

[0139] Figure 1 A detection schematic diagram of the present invention.

[0140] Figure 2 A schematic diagram of the process flow of a detection method adopted by the present invention.

[0141] Figure 3 A schematic diagram of a detection system adopted by the present invention. Detailed implementation manners

[0142] The present invention will be further explained in combination with specific embodiments. For those not specifying specific technologies or conditions in this embodiment, operations shall be performed according to the conventional technical methods in the art and the content of the instrument instructions. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase or self-research. The relevant antibodies are all self-developed by Chongqing Aishengsi Biotechnology Co., Ltd.

[0143] Example 1: Preparation of raw materials

[0144] Reagent A:

[0145] Weigh 5 mg of HRP and dissolve it in 1 ml of distilled water; add 0.2 ml of freshly prepared 0.1 M NaIO4 solution and stir in the dark at room temperature for 20 minutes; put the above solution into a dialysis bag and dialyze it against 1 mM sodium acetate buffer at pH 4.4 overnight at 4 °C; add 20 μl of 0.2 M carbonate buffer at pH 9.5 to raise the pH of the above aldehyde-activated HRP to 9.3, then immediately add 10 mg of IgG (thyroid-stimulating hormone (TSH) monoclonal antibody), and gently stir in the dark at room temperature for 2 hours in 1 ml of 0.01 M carbonate buffer; add 0.1 ml of freshly prepared 4 mg / ml NaBH4 solution, mix well, and then let it stand at 4 °C for 2 hours; put the above solution into a dialysis bag and dialyze it against 0.15 M PBS at pH 7.4 to remove ammonium ions (detected with Nessler's reagent), centrifuge at 10000 r / min for 30 min to remove the precipitate, and the supernatant is the enzyme conjugate. After measuring the concentration, add an equal volume of 60% glycerol and dispense it, and store it at 4 °C. Before dispensing, add BSA at a ratio of 10 mg / ml as its stabilizing and protecting agent.

[0146] Reagent B-1:

[0147] Measure 5 mg of TSH antibody, desalt it using a PD-10 desalting column, and elute it with 2.5 ml of biotin conjugation buffer (50 mM triethanolamine, pH 8.4), then dilute the antibody to 1 mg / mL with biotin buffer A. Weigh 10 mg each of NHS-Biotin and NHS-acridine compound, dissolve them thoroughly with DMSO to 3.0 mg / ml, and according to the ratio of antibody:NHS-Biotin:NHS-acridine compound = 1:20:20 (molar ratio), measure the NHS-Biotin and NHS-acridine compound solutions and add them to the above antibody solution, and let it stand and react at 25 °C (allowable error range is ±2 °C) for 180 minutes. The reacted solution is desalted using a PD-10 desalting column to remove unreacted molecules, eluted with 2.5 ml of biotin conjugation buffer B, and stored at 4 °C after measuring the concentration with an ultraviolet spectrophotometer. Before dispensing, add BSA at a ratio of 10 mg / ml as its stabilizing and protecting agent.

[0148] Reagent B-2:

[0149] Measure 5 mg of TSH antibody, desalt it using a PD-10 desalting column, and elute it with 2.5 ml of biotin conjugation buffer A, then dilute the antibody to 1 mg / mL with biotin buffer (50 mM triethanolamine, pH 8.4). Weigh 10 mg each of NHS-Biotin and NHS-(PEG) 24- Acridine compound, fully dissolved in DMSO to 3.0 mg / ml, according to antibody:NHS - Biotin:NHS-(PEG) 24 - Acridine compound = 1:20:20 (molar ratio), measure NHS - Biotin and NHS-(PEG) 24 - Acridine compound solution, then add it to the above antibody solution, place it at 25 °C (allowable error range is ±2 °C) and let it stand for reaction for 180 minutes. The reacted solution is desalted with a PD - 10 desalting column to remove unreacted molecules, eluted with 2.5 ml of biotin coupling buffer (50 mM triethanolamine, pH 8.4), and the concentration is measured with an ultraviolet spectrophotometer and then stored frozen at 4 °C or -20 °C. Before aliquoting, add BSA as its stabilizing protectant at a ratio of 10 mg / ml.

[0150] Reagent B - 3:

[0151] Measure 5 mg of TSH antibody, desalt it with a PD - 10 desalting column, elute it with 2.5 ml of biotin coupling buffer A, and then dilute the antibody to 1 mg / mL with biotin buffer (50 mM triethanolamine, pH 8.4). Weigh 10 mg of NHS - Biotin and NHS-(L - Lysine) 11 - Acridine compound, fully dissolved in DMSO to 3.0 mg / ml, according to antibody:NHS - Biotin:NHS-(L - Lysine) 11 - Acridine compound = 1:20:22 (molar ratio), measure NHS - Biotin and NHS-(L - Lysine) 11 - Acridine compound solution, then add it to the above antibody solution, place it at 28 °C (allowable error range is ±2 °C) and let it stand for reaction for 150 minutes. The reacted solution is desalted with a PD - 10 desalting column to remove unreacted molecules, eluted with 2.5 ml of biotin coupling buffer (50 mM triethanolamine, pH 8.4), and the concentration is measured with an ultraviolet spectrophotometer and then stored frozen at 4 °C or -20 °C. Before aliquoting, add BSA as its stabilizing protectant at a ratio of 10 mg / ml.

[0152] Reagent C:

[0153] Dissolve hydrogen peroxide in TBS buffer (pH≈8), where the final concentration of hydrogen peroxide is 0.1 M and the final concentration of TBS is 10 mM.

[0154] Reagent D:

[0155] The streptavidin - coated magnetic beads purchased from Thermo, with a diameter of 2.8 μm, after being washed, are diluted to 1 mg / mL for standby.

[0156] Alkaline solution:

[0157] Prepare a solution with a pH of 12 by sodium hydroxide.

[0158] Washing solution: 500 mM PBS buffer, 10% sodium ions, 0.7% EDTA-2Na, 0.5% SDS, 1% Tween 20, 0.1% sodium azide, 0.1% antifoaming agent, with a pH of about 7.

[0159] Example 2: Evaluation of signal inhibitors

[0160] Evaluate the effect of signal inhibitors from two aspects: the stability of signal inhibitors and the elimination of background interference. Place the prepared signal inhibitor solution in a test tube and expose it to air for 0 min, 5 min, and 20 min, and then perform luminescence signal testing. Using the signal-to-noise ratio S / N (positive sample signal / negative sample signal) as an index, the higher the ratio, the better the effect.

[0161] Test procedure: First, prepare a test positive serum sample with a TSH antigen concentration of 5.0 μIU / mL. Add 60 μL of reagent A and reagent B-1 in Example 1 to 20 μL of the test positive serum sample respectively, mix well, react at 37 °C for 10 min, then add 85 μL of reagent C and 6 μL of a 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; the antigen concentration of the negative serum sample is 0, and other reagents remain the same. The test information and results are shown in Table 1.

[0162] Table 1 Test results of different substances

[0163]

[0164]

[0165] As can be seen from Table 1, the effectiveness of signal inhibitors such as vitamin C is consistent with WO2010099486A1. However, the influence of air on signal inhibitors is inevitable. Considering storage and actual use, the signal inhibitor can be selected as R in which the benzene ring is substituted 1 -R 6 Any two or three of the substituents are -OH and / or -NH 2 of the substance, and the remaining substituents are -H, -F, -Cl, and / or -Br respectively, and two or three of the substituents are -F, -Cl, and / or -Br respectively. Preferably, the substituted R 1 -R 6 has two substituents which are -F, -Cl, and / or -Br respectively, and more preferably the substituent is Cl.

[0166] Example 3: Influence of Reagent Composition on Signal

[0167] To prove the differences in reagents with different compositions and structures in the test results, the performance of reagents with different compositions and structures in signal output can be intuitively understood by detecting signals and signal-to-noise ratios.

[0168] The reagent prepared in Example 1 was made into a kit and its performance was tested. Kit 1 includes Reagent A, Reagent B-2, Reagent C, and Signal Inhibitor 14 in Table 1 (in contact with air for 20 min); Kit 2 includes Reagent A, Reagent B-3, Signal Inhibitor 14 in Table 1 (in contact with air for 20 min), and Reagent C; Kit 3 includes Reagent A, Reagent B-3, Reagent C, Reagent D, Signal Inhibitor 14 in Table 1 (in contact with air for 20 min), an alkaline solution, and a cleaning solution. The concentrations of Reagents A, B, and C in Kits 1-3 are kept consistent. 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 following reaction modes were used to analyze the performance of Kits 1-3.

[0169] Reaction mode of Kits 1 and 2: Take 20 μl of each sample, add 60 μl of Reagent A and Reagent B to each sample respectively, mix well, react at 37 °C for 10 min, then add 85 μl of Reagent C and 6 μl of a signal inhibitor with a concentration of 0.1 mM. The generated optical signal is captured by a photomultiplier tube, and then the intensity of the optical signal is quantified.

[0170] Reaction mode of Kit 3: Take 20 μl of each sample, add 60 μl of Reagent A and Reagent B, and 30 μl of Reagent D to each sample respectively, mix well, react at 37 °C for 10 min, undergo 3-step magnetic separation and cleaning, then add 85 μl of Reagent C, 6 μl of a signal inhibitor with a concentration of 0.1 mM, and an appropriate amount of alkaline solution. The generated optical signal is captured by a photomultiplier tube, and then the intensity of the optical signal is quantified.

[0171] Table 2 Test Results of Kits with Different Compositions

[0172]

[0173] * The antigen concentration in the serum sample was measured by a commercially available kit (electrochemiluminescence method, Roche Diagnostics GmbH)

[0174] As can be seen from Table 2, since the luminescent substrates in Kit 1 and Kit 2 are connected by a flexible unit, the steric hindrance is smaller, and the signal is higher than that of the reagent without a flexible connection unit in Example 2 (not listed). At the same time, the signal-to-noise ratio is also higher. Due to the absence of a washing step, the signal values of Kit 1 and Kit 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 because the background value is lower after washing. Through the above verification, it can be seen that the connection of the flexible unit helps to improve the luminescence signal and the signal-to-noise ratio, and the washing step can more significantly improve the signal-to-noise ratio.

[0175] Effect of Reagent Composition and Detection Strategy Selection in Example 4

[0176] To further prove the beneficial effects of the multi-level judgment detection method described in the present invention, the detection time (efficiency) and detection accuracy of different detection strategies were measured.

[0177] (1) Sample Setting for Time Detection

[0178] First, a basic sample with a TSH concentration of 0.05 μIU / mL was prepared by adding TSH to a buffer solution. Two 100 mL basic samples were taken, and 1 mL of TBS buffer (pH 8.0) and 1 mL of a red dye solution prepared with the buffer were added to the two basic samples, which were respectively recorded as the experimental group and the interference group. The experimental group and the interference group formed a detection sample group. By controlling the number of interference groups in the detection samples, sample groups in different situations were formed, and different detection strategies were successively used to detect different sample groups to determine the time differences under different strategies. The results are shown in Table 4.

[0179] (2) Sample Setting for Accuracy Detection

[0180] Samples with different concentrations (prepared samples) were prepared by adding TSH to a buffer solution, and clinical serum samples with low, medium, and high antigen concentrations were collected. Four samples of 200 μL each were taken for each antigen concentration sample. One sample was recorded as the experimental group, and hemoglobin (final concentration 10 mg / dL), bilirubin (final concentration 5 mg / dL), and lipoid (final concentration 1000 mg / dL) were added to the remaining three samples, which were respectively recorded as the hemolysis group, the jaundice group, and the lipemia group. The information of each sample is shown in Table 3, and the detection results are shown in Table 5.

[0181] Table 3 Sample Information for Accuracy Analysis

[0182]

[0183] *The antigen concentration in the serum sample was measured using a commercially available kit (electrochemiluminescence method, Roche Diagnostics GmbH); **Intralipid, provided by Sigma

[0184] (3) Construction of the method for measuring samples by the solid-phase method

[0185] It includes the following steps: Take 20 μl of the sample, add 60 μl each of reagent A and reagent B-1 in Example 1 and 30 μl of reagent D to the sample, mix well and react at 37 °C for 10 min. After three-step magnetic separation and washing, then add 85 μl of reagent C and an appropriate amount of alkaline solution. The generated optical signal is captured by a photomultiplier tube, and through the calibration curve, the optical value signal is converted into a concentration value.

[0186] (4) Construction using the homogeneous detection method

[0187] It includes the following steps: Take 20 μl of the test sample, add 60 μl each of reagent A and reagent B-1 in 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 in Table 1. The generated optical signal is captured by a photomultiplier tube, and through the calibration curve, the optical value signal is converted into a concentration value.

[0188] (5) The technical detection method described in the present invention

[0189] It includes the following steps:

[0190] A. Set the preset item as "NT-proBNP". After judgment by the comparison module, the test item "TSH" does not conform to the preset item;

[0191] B. Measure the transmittance of three normal samples (serum samples with an antigen of 2.44 μIU / mL) at a wavelength of 500 nm, and take 95% of the average value of the transmittance as the "preset threshold";

[0192] C. Measure the transmittance of the sample that does not conform to the "preset item" and judge the interference value. Samples with a transmittance higher than or equal to the "preset threshold" are determined to be low-interference samples, and samples with a transmittance lower than the "preset threshold" are determined to be high-interference samples;

[0193] D. For high-interference samples, use the above-mentioned "solid-phase method" for determination; for low-interference samples, use the above-mentioned "homogeneous detection" for detection.

[0194] (6) Results

[0195] As can be seen from Table 4, from the detection time of a single sample, it can be seen that compared with homogeneous detection, the detection time of the solid-phase detection method increases significantly due to the addition of a washing step. Taking the daily sample detection throughput of 800 samples in a medium-sized hospital as an example, the detection time of the completely solid-phase detection mode exceeds twice that of the homogeneous detection mode. In the presence of normal interfering samples (10%), the detection time of the method described in the present invention increases by 18% and decreases by 44% respectively compared with the completely homogeneous mode and the completely solid-phase mode. Even when a large number of abnormal samples appear (25%), the detection time increases by about 43% and decreases by 33% respectively compared with the completely homogeneous mode and the completely solid-phase mode.

[0196] It can be seen that the detection method described in the present invention has higher detection efficiency compared with the completely solid-phase detection mode, reduces the entry of unnecessary washed samples into the washing link, and will also significantly save the detection cost, outputting more accurate detection results at a lower cost. Those skilled in the art should understand that based on the above verification process, when the sample detection volume further increases, the advantages of the detection method described in the present invention in terms of efficiency and cost will become more obvious.

[0197] Table 4 Detection Efficiency of Different Detection Strategies

[0198]

[0199] From the results in Table 5, it can be seen that the multi-level judgment detection method described in the present invention is basically equivalent to the detection method completely using a solid phase in terms of detection accuracy. However, due to the ability of the detection described in the present invention to identify interfering samples, its detection accuracy is significantly improved compared with the completely homogeneous mode.

[0200] Combining the results shown in Table 4 and Table 5, the multi-level judgment detection method described in the present invention has an accurate ability to identify interfering samples, higher detection efficiency, lower detection cost, and higher accuracy. The detection method described in the present invention is particularly suitable for high-throughput detection requirements.

[0201] Table 5 Detection Accuracy of Different Detection Strategies

[0202]

[0203]

[0204] Example 5: Determination of NT-proBNP

[0205] NT-proBNP is the best laboratory test index for evaluating heart failure, but its content in blood samples is extremely low, and there are extremely high requirements for the detection methods and detection reagents of such substances. Further, taking the NT-proBNP detection as an example, the universality, sensitivity and accuracy of the detection method and detection reagent described in the present invention are demonstrated.

[0206] (1) Raw material preparation:

[0207] Reagent A:

[0208] Weigh 5 mg of HRP and dissolve it in 1 ml of distilled water; add 0.2 ml of freshly prepared 0.1 M NaIO4 solution and stir in the dark at room temperature for 20 minutes; put the above solution into a dialysis bag and dialyze it against 1 mM sodium acetate buffer solution with pH 4.4 overnight at 4 °C; add 20 μl of 0.2 M carbonate buffer solution with pH 9.5 to raise the pH of the above aldehyde HRP to 9.3, and then immediately add 10 mg of IgG (NT-proBNP monoclonal antibody), gently stir in the dark at room temperature for 2 hours in 1 ml of 0.01 M carbonate buffer solution; add 0.1 ml of freshly prepared 4 mg / ml NaBH4 solution, mix well, and then place it at 4 °C for 2 hours; put the above solution into a dialysis bag and dialyze it against 0.15 M PBS with pH 7.4 to remove ammonium ions (detected with Nessler's reagent), centrifuge at 10000 r / min for 30 min to remove the precipitate, and the supernatant is the enzyme conjugate. After measuring the concentration, add an equal volume of 60% glycerol for aliquoting and store at 4 °C. Before aliquoting, add BSA as its stabilizing protectant according to the ratio of 10 mg / ml.

[0209] Reagent B:

[0210] Measure 5 mg of NT-proBNP antibody (NT-proBNP monoclonal antibody, Chongqing Aishengsi Biotechnology Co., Ltd.), desalt it using a PD-10 desalting column, and elute it with 2.5 ml of biotin coupling buffer A, and then dilute the antibody to 1 mg / mL with biotin buffer A. Weigh 10 mg each of NHS-Biotin and NHS-(PEI) 20 -acridine compound, dissolve it thoroughly with DMSO to 3.0 mg / ml, and according to the ratio of antibody:NHS-Biotin:NHS-(PEI) 20 -acridine compound = 1:20:20 (molar ratio), measure NHS-Biotin and NHS-(PEI) 20The solution of the acridine compound was added to the above antibody solution and allowed to stand and react at 25 °C (with an allowable error range of ±2 °C) for 180 minutes. The reaction solution was desalted using a PD-10 desalting column to remove unreacted molecules, eluted with 2.5 ml of biotin conjugation buffer B, and stored at 4 °C after measuring the concentration using an ultraviolet spectrophotometer. Before aliquoting, BSA was added as a stabilizing protectant at a ratio of 10 mg / ml.

[0211] Reagent C, Reagent D, the alkaline solution, and the cleaning solution were the same as in Example 1.

[0212] (2) Sample construction

[0213] Samples containing different concentrations were prepared by adding NT-proBNP to the buffer (prepared samples), and two concentrations of clinical serum samples were collected. Four aliquots of 200 μL were taken for each concentration sample. One aliquot was designated as the experimental group, and hemoglobin (prepared to a final concentration of 10 mg / dL), bilirubin (prepared to a final concentration of 5 mg / dL), and lipid (prepared to a final concentration of 1000 mg / dL) were added to the remaining three samples, which were designated as the hemolysis group, jaundice group, and lipemia group, respectively. The information of each sample is shown in Table 6.

[0214] Table 6 Information of NT-proBNP detection samples

[0215]

[0216] * The antigen concentration in the serum sample was measured using a kit (Elecsys proBNP II STAT, Roche Diagnostics GmbH)

[0217] (3) Construction of the method for determining samples by the solid-phase method

[0218] It includes the following steps: Take 20 μl of the 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. After three-step magnetic separation and washing, then add 85 μl of Reagent C and an appropriate amount of alkali solution. The generated light signal was captured by a photomultiplier tube, and through the calibration curve, the light value signal was converted into a concentration value.

[0219] (4) Construction of the method for determining samples by the homogeneous method

[0220] It includes the following steps: Take 20 μl of the 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 was captured by a photomultiplier tube, and through the calibration curve, the light value signal was converted into a concentration value.

[0221] (5) Construction of the technical detection method of the present invention

[0222] It includes the following steps:

[0223] A. Set the preset item as "NT-proBNP". After judgment by the comparison module, the item to be measured "NT-proBNP" conforms to the preset item;

[0224] B. Use the "solid phase method" to measure all samples.

[0225] (6) Effect

[0226] Table 7 Detection results of NT-proBNP in different samples

[0227]

[0228] From the detection results in Table 7, for the detection items with ultra-low concentration, after the comparison and judgment between the preset item and the item to be measured, a more accurate detection method is adopted, avoiding the problem of inaccurate detection results caused by directly using the homogeneous detection method, and at the same time avoiding the influence of interfering samples on the detection results. Therefore, the present invention has higher accuracy than the homogeneous detection and is not affected by interfering samples.

[0229] It should also be noted that in the exemplary embodiments mentioned in the present invention, some methods or systems are described based on a series of steps or devices. However, the present invention is not limited to the order of the above steps, that is, the steps can be executed in the order mentioned in the embodiments, or different from the order in the embodiments, or several steps can be executed simultaneously.

[0230] In the present invention, the features described and / or illustrated for one embodiment can be used in the same way or in a similar way in one or more other embodiments, and / or combined with the features of other embodiments or replace the features of other embodiments.

[0231] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the embodiments of the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A target detection kit, characterized in that: The kit comprises reagent A and reagent B; the reagent A comprises a first binding molecule, and the first binding molecule is coupled to compound 1; the reagent B comprises a second binding molecule, and the second binding molecule is coupled to compound 2; the first binding molecule or the second binding molecule further comprises at least one connection structure I; Wherein, the compound 1 has the function of catalyzing the 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 phase carrier; The kit further comprises a reagent D, wherein the reagent D comprises a solid phase carrier coupled with a connecting structure II, and the connecting structure I and the connecting structure II are specifically bound to each other to form a specific binding system.

2. The kit according to claim 1, characterized in that The kit further comprises a reagent C, wherein the reagent C contains compound 3; Wherein, the compound 2 and the compound 3 generate a detectable signal after the reaction.

3. The kit according to claim 2, characterized in that The compound 3 is selected from peroxides.

4. The kit according to any one of claims 1 to 3, characterized in that The compound 1 is a substance that can catalyze a substrate to produce hydroxyl radicals, and includes at least one of a transition metal salt and / or a complex thereof and a peroxidase; the transition metal salt and / or a complex thereof includes ferrous protoporphyrin.

5. The kit according to claim 4, characterized in that The compound 1 is peroxidase.

6. The kit according to any one of claims 1 to 3, characterized in that The compound 2 is at least one of the compounds that generate photons under the action of hydroxyl radicals.

7. The kit according to claim 6, characterized in that The compound 2 is selected from at least one of luminol, isoluminol, rofenine, acridinium ester, lucigenin, 9,10-dihydroacridine and phthaloyl hydrazide.

8. The kit according to claim 4, characterized in that The compound 2 is at least one of the compounds that generate photons under the action of hydroxyl radicals.

9. The kit according to claim 8, characterized in that The compound 2 is selected from at least one of luminol, isoluminol, rofenine, acridinium ester, lucigenin, 9,10-dihydroacridine and phthaloyl hydrazide.

10. The kit according to any one of claims 1-3, 5, 7-9, characterized in that: Compound 1 and Compound 2 are coupled to the first binding molecule and the second binding molecule respectively through a flexible linker.

11. The kit according to claim 10, characterized in that The flexible connecting unit is selected from one or more of polyoxyethylene ether substances, polyoxypropylene ether substances, alkyl groups, polyethylene imine substances, polypeptides or blocks of the above substances.

12. The kit according to any one of claims 1-3, 5, 7-9 or 11, characterized in that The kit also includes an alkaline solution, and the pH value of the alkaline solution is greater than or equal to 8.

13. The kit according to claim 10, characterized in that The kit also includes an alkaline solution, and the pH value of the alkaline solution is greater than or equal to 8.

14. The kit according to claim 13, wherein The specific binding system comprises one of 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.

15. The kit according to claim 12, characterized in that The specific binding system comprises one of 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.

16. The kit according to any one of claims 1-3, 5, 7-9, 11 or 15, characterized in that The kit also includes a signal inhibitor, which includes ascorbic acid or its salt, phenoxazine, 2-aminophenol, 2-amino-p-chlorophenol, 2-amino-o-chlorophenol, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid TROLOXTM, 3-aminotyrosine or a salt thereof, 4-Hydroxy-2-chlorophenol, 4-Hydroxy-2-methoxyphenol, o-Hydroxy-m-chlorophenol, and at least one of the compounds of the structure shown in Formula 1, Among them, any two or three substituents among the substituted R1-R6 are -OH and / or -NH2, and the remaining substituents are -H, -F, -Cl and / or -Br, and two or three substituents are -F, -Cl and / or -Br.

17. The kit according to claim 12, characterized in that The kit also includes a signal inhibitor, which includes ascorbic acid or its salt, phenoxazine, 2-aminophenol, 2-amino-p-chlorophenol, 2-amino-o-chlorophenol, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid TROLOXTM, 3-aminotyrosine or a salt thereof, 4-Hydroxy-2-chlorophenol, 4-Hydroxy-2-methoxyphenol, o-Hydroxy-m-chlorophenol, and at least one of the compounds of the structure shown in Formula 1, Among them, any two or three substituents among the substituted R1-R6 are -OH and / or -NH2, and the remaining substituents are -H, -F, -Cl and / or -Br, and two or three substituents are -F, -Cl and / or -Br.

18. The kit according to any one of claims 1-3, 5, 7-9, 11, 15 or 17, characterized in that The kit also includes a cleaning solution.

19. The kit according to claim 16, wherein The kit also includes a cleaning solution.

20. A judgment-type luminescence detection method, characterized in that: The detection method comprises the following steps: S1: Perform interference identification on the sample to be tested and obtain the interference value; S2: Compare the interference value with a preset threshold value, and select to execute steps S3A and S4A or steps S3B and S4B according to the comparison result; in, Step S3A includes: selecting a reagent and reacting the sample to be tested with the reagent without a solid phase carrier; Step S3B includes: selecting reagents and reacting the sample to be tested with the reagent containing the solid phase carrier, and separating impurities in the detection system; Steps S4A and S4B include: measuring the reaction system and reporting the test results; The reagents selected in step S3A and step S3B include reagent A and reagent B; the reagent A contains a first binding molecule, and the first binding molecule is coupled to compound 1; the reagent B contains a second binding molecule, and the second binding molecule is coupled to compound 2; the first binding molecule or the second binding molecule also includes at least one connection structure I; Wherein, the 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 phase carrier.

21. A judgment-type luminescence detection method, characterized in that: The detection method comprises: S0: Compare and judge the test items of the sample to be tested with the preset items; and select to execute (i) or (ii) test process according to the comparison and judgment results; The (i) detection process comprises: S1: Perform interference identification on the sample to be tested and obtain the interference value; S2: Compare the interference value with the preset threshold, and select to execute steps S3A and S4A or steps S3B and S4B according to the comparison result; wherein, Step S3A includes: selecting a reagent and reacting the sample to be tested with the reagent without a solid phase carrier; Step S3B includes: selecting reagents and reacting the sample to be tested with the reagent containing the solid phase carrier, and separating impurities in the detection system; Steps S4A and S4B include: measuring the reaction system and reporting the test results; The (ii) detection process comprises: Step S3B includes: selecting reagents and reacting the sample to be tested with the reagent containing the solid phase carrier, and separating impurities in the detection system; The S4B step includes: measuring the reaction system and reporting the test results.

22. The method according to claim 21, characterized in that The reagents selected in step S3A and step S3B include reagent A and reagent B; the reagent A contains a first binding molecule, and the first binding molecule is coupled to compound 1; the reagent B contains a second binding molecule, and the second binding molecule is coupled to compound 2; the first binding molecule or the second binding molecule also includes at least one connection structure I; Wherein, the 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 phase carrier.

23. The method according to claim 20, 21 or 22, characterized in that The interference value is compared to a preset threshold value by one or more of the following methods: 1) irradiating the sample to be tested or the mixed solution of the sample to be tested and the reagent with a light source, detecting the transmitted light and / or scattered light signal, and making a judgment by comparing the transmitted light and / or scattered light signal with a preset threshold; 2) By calculating the difference in absorbance of the diluted sample under dual wavelengths, the value of the serum / plasma sample is given semi-quantitatively, and compared with the preset threshold value for determination; 3) Compare the color features of the sample image with the preset threshold.

24. The detection method according to claim 20, 21 or 22, characterized in that: According to the comparison result with the preset threshold value, after the step of reacting the sample to be tested with the reagent containing the solid phase carrier, the impurity removal step is performed.

25. The detection method according to claim 23, characterized in that: According to the comparison result with the preset threshold value, after the step of reacting the sample to be tested with the reagent containing the solid phase carrier, the impurity removal step is performed.

26. The detection method according to claim 24, characterized in that: The reagent used in step S3A also includes reagent C and / or the reagent selected in step S3B also includes reagent C, reagent D and cleaning solution; Among them, the reagent C contains compound 3, wherein the compound 2 and compound 3 generate a detectable signal after reaction; the reagent D contains a solid phase carrier coupled with a connecting structure II, and the connecting structure I and the connecting structure II specifically bind to each other to form a specific binding system.

27. The detection method according to claim 22 or 25, characterized in that: The reagent selected in step S3A also includes reagent C; and / or the reagent selected in step S3B also includes reagent D and a cleaning solution; Among them, the reagent C contains compound 3, wherein the compound 2 and compound 3 generate a detectable signal after reaction; the reagent D contains a solid phase carrier coupled with the connecting structure II, and the connecting structure I and the connecting structure II have specific binding to each other to form a specific binding system.

28. The detection method according to claim 27, characterized in that The reagents in step S3A and / or step S3B also include signal inhibitors, which include ascorbic acid or its salts, phenoxazine, 2-aminophenol, 2-amino-p-chlorophenol, 2-amino-o-chlorophenol, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid TROLOXTM, 3-aminotyrosine or a salt thereof, 4-Hydroxy-2-chlorophenol, 4-Hydroxy-2-methoxyphenol, o-Hydroxy-m-chlorophenol, and at least one of the compounds of the structure shown in Formula 1, Among them, any two or three substituents among the substituted R1-R6 are -OH and / or -NH2, and the remaining substituents are -H, -F, -Cl and / or -Br, and two or three substituents are -F, -Cl and / or -Br.

29. A multi-level judgment 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 item to be tested with the preset item information; 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 that affect the test results in the detection system; The optical testing module is used to detect the signal generated by the reaction system; The signal processing module is used to process signals and convert them into visual information.

30. The detection system according to claim 29, characterized in that The impurity separation module includes a cleaning submodule, and the cleaning submodule separates impurity substances by utilizing the difference between the reagent bound to the solid phase carrier and the reagent not bound to the solid phase carrier.

31. The detection system according to claim 29 or 30, characterized in that: The detection system has at least two different detection channels for detecting high-interference samples and low-interference samples respectively.

32. The detection system according to claim 31, characterized in that The detection system further includes a sample transfer module, and the function of the sample transfer module includes: transferring the sample to different detection channels based on the interference identification result.

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