Multifunctional detection kit

Abstract

The invention discloses a kit which comprises a binding molecule which is not connected with a solid phase and a signal inhibitor, different reagent types can be matched according to different sample detection requirements, and the kit integrates the advantages of a solid phase cleaning detection technology and a homogeneous phase detection technology. Not only is unnecessary cleaning avoided when a whole sample is detected in a solid phase mode, but also the problem of low accuracy caused by existence of a special sample when the whole sample is detected in a homogeneous phase mode is avoided. Meanwhile, by adding the signal inhibitor, the signal-to-noise ratio can be remarkably increased, the signal inhibitor with better stability is screened out, and the influence of environmental factors on the reagent in practice is reduced.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnosis, and in particular to a detection kit. Background Art

[0002] The spatial proximity chemiluminescence technology avoids the complexity brought by solid phase cleaning such as magnetic beads, and has become the representative of the new generation of luminescence detection technology. In the spatial proximity chemiluminescence, one antibody involved in the immune reaction is labeled with horseradish peroxidase (HRP), and the other antibody involved in the immune reaction is labeled with a luminescent compound, such as acridine ester (AE). In the presence of the target antigen, a sandwich immune complex can be formed. The target antigen can make the HRP and AE labeled on the two antibodies closely connected together, and generate a light signal under the action of the substrate. The intensity of the light signal is proportional to the concentration of the target antigen. The relevant technology has been disclosed in WO2010099486A1. Its technical advantages are: ① Based on the immune reaction of antigen and antibody, an immune complex is formed, and the amount of the complex is monotonically related to the amount of the analyte; the immune reaction of antigen and antibody ensures the specificity of the detection. ② The immune reaction occurs in a homogeneous system, and the homogeneous reaction ensures a high precision of the detection; ③ Based on the proximity of the positions of HRP and AE, the signal is generated, so the instrument does not need a liquid system to achieve magnetic cleaning, the instrument cost is low, the volume is small, and high throughput can be achieved.

[0003] However, spatial proximity luminescence technology still has some limitations. For example, there is no magnetic cleaning process in the reaction process, and the interference in the sample cannot be removed. The detection results for high-interference samples are poor. For example, hemolytic and lipemia samples that often appear in some elderly people will interfere with the immune response, thereby affecting the accuracy of the test results. Compared with magnetic particle chemiluminescence, the detection sensitivity is relatively low.

[0004] In summary, existing detection technologies all have certain limitations. Clinical scenarios urgently need a fully automatic, high-throughput detection technology with lower cost, lower interference and higher sensitivity to meet clinical needs. Summary of the invention

[0005] The purpose of the present invention is to provide a kit and a testing method and system for detecting target objects. The kit and the detection method can reduce the number of unnecessary sample cleanings, reduce the frequency of use of solid phase material detection methods, significantly reduce costs, and improve detection throughput. It can also avoid the problems of low sensitivity and accuracy caused by special samples.

[0006] Therefore, one aspect of the present invention is to provide a target detection kit, the kit comprising reagent A and reagent B, the reagent A containing a first binding molecule, the first binding molecule coupled to compound 1;

[0007] The reagent B contains a second binding molecule, and the second binding molecule is coupled to compound 2;

[0008] The first binding molecule and / or the second binding molecule further comprises at least one connecting structure I;

[0009] Among them, 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 connection structure I does not form a specific binding state.

[0010] The working concentration of the first binding molecule complex coupled with compound 1 in reagent A is 0.1-3.0 μg / mL; the working concentration of the second binding molecule complex coupled with the compound in reagent B is 0.1-3.0 μg / mL.

[0011] "Target" refers to a certain biochemical substance or substances whose specific content or concentration you want to know through detection means.

[0012] "Catalysis" means changing the activation free energy required for the reaction through a catalyst, thereby changing the chemical reaction rate of the reactants. Compound 1 does not act directly on compound 2, but rather causes compound 2 to react chemically with other substances through "catalysis".

[0013] Furthermore, the first binding molecule and the second binding molecule are not coupled to a solid phase carrier.

[0014] Further, the kit also includes reagent C, which contains compound 3. Wherein, 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, the first binding molecule coupled with compound 1 and the second binding molecule coupled with compound 2 are combined with the target to be detected, the distance between compound 1 and compound 2 is sufficient for compound 1 to catalyze compound 2 to generate a detectable signal, while phosphatase and compound 2 cannot generate a detectable signal at the same distance, so further, compound 1 is not a phosphatase, such as alkaline phosphatase.

[0015] Furthermore, compound 1 and compound 2 are coupled to the first binding molecule and the second binding molecule respectively through a flexible connection unit.

[0016] The "flexible linking unit" is a non-rigid substance. When the first binding molecule and the second binding molecule coupled 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 softness of the "flexible linking unit" so that the substance that can generate the luminescent signal is close enough in space to generate a detectable signal. The "flexible linking unit" is selected from polyoxyethylene ether substances, polyoxypropylene ether substances, alkyl, polyethyleneimine substances, polypeptides and blocks of the above substance composition units. In order to ensure the luminous efficiency, the flexible linking unit should not be too long. Therefore, it is preferred that the total effective length of the flexible linking unit linking compound 1 and compound 2 is less than or equal to 200nm, preferably less than 180nm, and more preferably less than 150nm; 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.

[0017] "Polyoxyethylene ether substances" are also called polyethylene oxide (PEO) or polyoxyethylene (POE) compounds, preferably compounds with a PEG main chain and an active group at the end of the chain that can be connected to a biological molecule, for example, NHS-(PEG)n, Pierce TM PEG-SPDP series.

[0018] "Alkyl" includes straight-chain alkyl and branched-chain alkyl groups having active groups at the chain ends, and the active groups are used to couple the binding molecules with Compound 1 and Compound 2.

[0019] A "polypeptide" is a polymer comprised of at least two amino acids.

[0020] The main chain of "polyethyleneimine" is "-CH 2 CH 2 NH-" polymers include those with substitutions on the side chains and / or at both ends.

[0021] "Block" refers to a substance containing two or more basic building blocks of flexible connecting units, such as the building blocks of polyoxyethylene ethers - OCH 2 CH 2 - and polyethyleneimine compounds form units -CH 2 CH 2 NH-block polymers formed.

[0022] The "total degree of polymerization" is the sum of the degrees of polymerization of the two flexible linker units on the first binding molecule and the second binding molecule.

[0023] A "solid phase carrier" is a material whose at least one dimension in the three-dimensional size is greater than 0.1 micron or 0.2 micron, preferably greater than 0.3 micron or 0.5 micron. The material has an adsorption effect or has active functional groups on the surface of the material that can be coupled with binding molecules. The material can be in the following forms: particles, microparticles, metal colloids, fibers, paper, beads, membranes, filter paper, test tubes, microplates, chips, slides, and microarrays, etc.

[0024] Further, the first binding molecule and / or the second binding molecule are antibodies, antigens and / or antigen binding fragments. For example, when the target detection object 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 detection object is an antibody, the first binding molecule and / or the second binding antibody are optionally secondary antibodies to the antigen or the anti-target detection antibody.

[0025] Further, the compound is a substance that can generate hydroxyl radicals and catalyze the chemiluminescence of the substrate, 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, transition metal complexes such as ferrous protoporphyrin, etc., which can catalyze the chemiluminescent oxidation of the substrate. Accordingly, compound 2 is selected from substances that generate photons under the action of the enzyme described in compound 1, such as luminol, isoluminol, rofenine, acridinium ester, lucigenin, 9,10-dihydroacridine and phthalic acid hydrazide.

[0026] Furthermore, the compound 3 in the reagent C is selected from a peroxide compound; for example, hydrogen peroxide, urea peroxide, peracetic acid or perborate; and the pH value of the reagent C is 5.5-9.5, preferably 6.5-8.5.

[0027] It can be understood that, unlike compound 1 catalyzing compound 2 to produce a signal, compound 3 directly reacts with compound 2 to produce a signal. Therefore, at the same concentration, the signal effect produced by the reaction of compound 3 and compound 2 is more obvious than the effect of compound 1 and compound 2. Alternatively, it can be understood that compound 1 has the effect of enhancing the signal production of compound 3 and compound 2.

[0028] "Optical signal" means that the compound generates photons through chemical reactions or is stimulated by the external environment (such as temperature changes, light irradiation, magnetic field changes, etc.). Photons serve as information carriers, and the expected information is obtained by detecting photons, for example, relevant information is obtained by detecting fluorescence, flash or glow.

[0029] Furthermore, the kit also includes reagent D, which contains a solid phase carrier coupled to 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; the specific binding system includes: antibody-antigen, avidin-biotin, streptavidin-biotin and Tag-Catcher.

[0030] The working solution concentration of the solid phase carrier complex coupled to the connecting structure II in the reagent D is 0.2-5.0 mg / mL.

[0031] When the connecting structures I and II are antibodies and antigens with characteristic affinity, they are different from the types of the detection target and the first binding molecule and / or the second binding molecule, and the antibodies and antigens described in the connecting structures I and II will not produce specific binding with the detection target and the first binding molecule and / or the second binding molecule. Without being limited by the examples, the antibodies and antigens described in the connecting structures I and II can be obtained by means known in the art, for example, if the connecting structure I is fluorescein isothiocyanate (FITC), then the antibody of the connecting structure II against FITC is preferably a monoclonal antibody.

[0032] The "Tag-Catcher" system is composed of two parts, protein and polypeptide (short peptide), and spontaneously forms an isopeptide bond through the residues that can specifically recognize each other with high affinity. It includes the SpyTag-SpyCatcher system, the SdyTag-SdyCatcher system, the SnoopTag-SnoopCatcher system, and the DogTag-DogCatcher system. The binding modes of different "Tag-Catcher" systems are basically the same. The two systems have high affinity and react quickly, and no additional chemical reagents or catalysts are required. For example, the earliest discovered SpyTag-SpyCatcher system, the carboxyl group (aspartic acid residue) of aspartic acid in SpyTag and the primary amino group (lysine residue) of lysine in SpyCatcher specifically recognize each other, and the amide bond formed by dehydration, the preparation and principle of which can be found in references 1 and 2. The preparation and principle of SdyTag-SdyCatcher, SnoopTag-SnoopCatcher and DogTag-DogCatcher are respectively referred to references 3-5. The connection and preparation of Tag-Catcher with magnetic beads, antibodies, proteins and other substances are referred to references 5-10.

[0033] The specific binding of the two parts in the "Tag-Catcher" system is a covalent bond, so after binding, it is less susceptible to environmental changes and the connection is more stable.

[0034] Further, the kit also includes an alkaline solution, the pH value of the alkaline solution is greater than or equal to 8, more preferably greater than or equal to 9, 10, or 11. The alkaline solution can be prepared by a corresponding alkaline substance, such as sodium hydroxide. It is understood that alkaline substances are commonly used promoters for chemiluminescence, and are particularly suitable for chemiluminescence in enzyme-free systems. For example, after adding hydrogen peroxide to the washed magnetic beads (capturing the analyte), a detectable luminescent signal can be generated after adding an alkaline solution.

[0035] Furthermore, the kit also includes a signal inhibitor (Selective Signal Inhibiting Agent), which can be understood as a signal used to increase the signal of the measurement process. When it is applied to target detection, the degree to which the signal obtained from the measurement reaction system exceeds the background signal is significantly greater than the degree of excess that occurs when there is no signal inhibitor. At the same time, it is necessary to pay attention to that the reagent is basically in an open state during use, and the signal inhibitor will be fully in contact with the air. The signal inhibitor is easily oxidized in the environment, so the stability of the signal inhibitor itself is extremely important.

[0036] The signal inhibitor of the present invention is selected from the types listed in WO2010099486A1, for example, including 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, At least one of them.

[0037] In addition, WO2010099486A1 disclosed that the signal inhibitors of some structures were less effective. However, the applicant unexpectedly discovered that the compound of the structure shown in Formula 1 has an outstanding effect in suppressing background interference signals, and is particularly remarkable in terms of stability.

[0038]

[0039] Among them, the substituted R 1 -R 6 The optional two or three substituents are -OH and / or -NH 2 The remaining substituents are -H, -F, -Cl and / or -Br, and two or three substituents are -F, -Cl and / or -Br, preferably, two substituents are -OH and / or -NH 2; Preferably, substituted R 1 -R 6 There are two substituents, namely -F, -Cl and / or -Br, more preferably there are two substituents, namely -F or -Cl.

[0040] The concentration of the signal inhibitor in the detection system of the present invention is 10 -4 mM-10mM, for example 10 -3 mM-5mM or 10 -2 mM-1mM.

[0041] Therefore, another aspect of the present invention is the use of the signal inhibitor of the present invention in a test kit, specifically for reducing background interference signals and / or improving the signal-to-noise ratio.

[0042] Another aspect of the present invention is to provide a judgment-type detection method, comprising the following steps:

[0043] S1: Perform interference identification on the sample to be tested and obtain the interference value;

[0044] S2: Compare the interference value with a preset threshold value, and select to execute steps S3A-S4A or S3B-S4B according to the comparison result;

[0045] in,

[0046] Step S3A includes: selecting a reagent and reacting the sample to be tested with the reagent without a solid phase carrier;

[0047] 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;

[0048] Steps S4A and S4B include: measuring the reaction system and reporting the test results.

[0049] It is understandable that due to the differences in detection methods, the reagents used 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, cleaning solution, and alkaline solution; at the same time, the reagents in S3A and S3B optionally contain signal inhibitors. In the absence of mutual influence, the above reagents can be stored separately or as a mixture.

[0050] Furthermore, before step S1, the following step S0 is also included: matching and judging the test item of the test sample with the preset item; and selecting to execute S1 and subsequent steps or steps S3B-S4B (such as Figure 2 shown).

[0051] The "items to be tested" are items to be tested for the sample to be tested, the purpose of which is to obtain quantitative information of a certain or certain biochemical substances, and the "preset items" are pre-set test items. The purpose of setting the "preset items" is to reduce interference and provide detection accuracy. "Preset items" can be determined, for example, based on clinical research or literature. Since the content of the target to be detected in the sample is extremely low or there is great interference and it is impossible to accurately quantify the analysis, this type of test item can be regarded as a "preset item". When the "items to be tested" are "preset items" or include "preset items", it is necessary to adopt a detection step to eliminate interference, such as using S3B-S4B steps; when the "items to be tested" do not include "preset items", otherwise S1 and subsequent steps are adopted; or because the content of the target to be detected in the sample is extremely low or there is great interference and it is impossible to accurately quantify the analysis, the test items that are not such cases are set as "preset items", and when the "items to be tested" meet the "preset items", S1 and subsequent steps are adopted, otherwise S3B-S4B steps are adopted. For example, the content of AD markers (such as tau-181) in the blood is low, and the detection item for tau-181 in the blood sample is set to a "preset item". When the "item to be tested" is to detect tau-181 in the blood sample, it is judged to meet the "preset item" and the S3B-S4B steps are used.

[0052] Furthermore, the relationship between the interference value and the preset threshold is determined by one or more of the following methods:

[0053] 1) Using a light source to illuminate the sample to be tested or a mixed solution of the sample to be tested and a reagent, 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;

[0054] 2) Semi-quantitatively give the serum / plasma sample value by calculating the difference in absorbance of the diluted sample under dual wavelengths, and compare and determine with the preset threshold; the sample value is, for example, the value of lipemia, hemolysis and / or icterus in the sample;

[0055] 3) Compare the color features of the sample image with the preset threshold.

[0056] The "mixed solution of the sample to be tested and the reagent" refers to a solution obtained by mixing the original sample with the reagent that can enhance the detection effect.

[0057] Without limitation to the examples, the above methods for determining whether a sample has an interference value all adopt means known in the art, such as using the methods and modules described in CN105980833B and CN113592842B to detect lipemia, hemolysis and icteric blood samples, and for example, using the methods and instruments described in literature or commercial products such as CN109374551A, CN204101456U, CN210894370U, or EXC400 automatic biochemical analyzer (Zhongyuan Huiji Biotechnology Co., Ltd.) to detect sample transmitted light and / or scattered light signals, using UV-4800 dual-beam spectrophotometer (Unico (Shanghai) Instrument Co., Ltd.) for dual-wavelength detection, and obtaining images by means of ordinary cameras or miniature cameras.

[0058] The "preset threshold" is a value set based on existing information and data, which has the function of distinguishing high-interference samples from low-interference samples. The information and data required to set the "preset threshold" can be obtained from, for example, the difference between special samples and ordinary samples analyzed by clinical big data. For example, due to the hemolysis of red blood cells, there is a significant difference in the transmitted light between the two blood samples (relative to non-hemolysis samples), such as the lower transmittance of hemolysis samples. Based on the big data, the transmittance value range of ordinary blood samples is obtained, and the "preset threshold" is set accordingly. The "preset threshold" can be appropriately and strictly provided to ensure the accuracy of the test results. For example, in order to reduce the interference of hemolysis samples, the "preset threshold" of transmitted light can be 5% or 10% lower than the transmittance value of ordinary blood samples. When the transmittance of the test sample is lower than the "preset threshold", it is judged as a "high-interference" sample; when the transmittance of the test sample is higher than the "preset threshold", it is judged as a "low-interference" sample.

[0059] To further improve the detection accuracy, preferably, there may be multiple "preset thresholds" in step S1, which are obtained by measuring under different detection methods. When the measured value of the test sample meets the low interference identification conditions compared with the two "preset thresholds", the sample will be identified as low interference, otherwise it will be identified as high interference. For example, two different "preset thresholds" are determined by transmission method and dual-wavelength measurement, and the detection sample also adopts transmission method and dual-wavelength detection sample. When the difference between the transmittance and absorbance of the test sample is 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 identified as a low-interference sample.

[0060] In the present invention, the biological sample containing the target detection object can exist in any form, such as body fluids (blood, serum, plasma, tears, sweat, urine, tissue secretions, cerebrospinal fluid, saliva), tissues (such as tissue sections, tissue extracts) or other samples from organisms. The technical solution of the present invention is particularly suitable for samples that are prone to high interference values, such as blood, plasma, serum and urine.

[0061] The "kit" of the present invention is based on reagent A and reagent B, and optionally contains reagent C, reagent D and / or signal inhibitor and other reagents according to detection requirements.

[0062] In the present invention, "test 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 and stored in containers, and other reagents can be stored separately in containers. The containers storing different reagents can be connected together, or partially connected together, or not connected together at all. For example, according to 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 taken as needed during detection; or for example, the reagent detection system includes reagent A, reagent B, reagent C and reagent D, and reagent A and reagent C are stored in one container, and reagent B and reagent D are stored separately, and are taken as needed during detection.

[0063] In the present invention, "specific binding" has the commonly known meaning in the art, which is a biological binding process in which a certain ligand that can be competitively blocked by corresponding substances interacts with a specific structural site in vitro or in vivo, such as the binding between antigen and antibody or receptor and ligand, the binding between biotin and avidin, etc.

[0064] In the present invention, the "coupling" method includes two parts of the substance being directly connected through an active functional group (direct coupling), or being indirectly connected through one or two or more substances (indirect coupling). A direct coupling method, for example, is the connection between a solid phase carrier with a carboxyl group on the surface and the amino portion of an antibody, thereby directly coupling the solid phase carrier to the antibody. An indirect coupling method, for example, is the connection of the two parts through a secondary antibody or through avidin-biotin.

[0065] refer to Figure 1 , Figure 1 This is a schematic diagram of a detection implementation method of the present invention. The composition of the kit is different depending on the high-interference sample and the low-interference sample.

[0066] refer to Figure 2 , Figure 2This is a flow chart of an implementation method of a detection method described in the present invention. Preset items are first set in the detection system. When the preset items and the items to be tested are judged in step S0 and the items to be tested do not meet the content of the preset items, the sample interference value is identified in step S1 to obtain the interference value. In step S2, the interference value is compared with the preset threshold value. For samples with low interference as a comparison result, a reagent without a solid phase is used and reacted in step S3A, and a signal generated in the reaction process is detected in step S4A and a report on the corresponding results is output; for samples with high interference as a comparison result, a reagent containing a solid phase is used and reacted as well as impurity removal and separation is performed in step S3B, and a signal generated in the reaction process is detected in step S4B and a report on the corresponding results is output.

[0067] When the detection item does not meet the preset item content, a reagent containing a solid phase is used in step S3B to carry out a reaction and remove impurities, and a signal generated during the reaction process is detected in step S4B and a report of the corresponding result is output.

[0068] Based on the technical solution of the present invention, the inventors believe that the beneficial effects of the present invention are at least:

[0069] 1) A test reagent with a completely new structure is proposed. The test reagent contains a structure connected to a solid phase carrier. According to the sample test needs, the test reagent is used in combination with the solid phase carrier containing the connection structure during the test process. It is flexible to use and can meet different detection needs.

[0070] 2) The kit described in the present invention contains a variety of reagents, and different types of reagents can be mixed according to different sample detection requirements. It integrates the advantages of solid phase cleaning detection technology and homogeneous detection technology, which not only avoids unnecessary cleaning when the whole sample is detected in solid phase mode, but also avoids the problem of low accuracy due to the presence of special samples when the whole sample is detected in homogeneous mode.

[0071] 3) The kit of the present invention has a wider range of application scenarios and can be applied to the detection needs of various types of samples, meeting the requirements of complex application scenarios and significantly improving the reliability of the kit.

[0072] 4) A new class of signal inhibitors suitable for homogeneous reactions is proposed, which has higher stability in practical application scenarios.

[0073] References:

[0074] 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);

[0075] 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;

[0076] Literature 3: Tan LL, et al. Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly. PLoS ONE 11(10):e0165074;

[0077] 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;

[0078] Literature 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;

[0079] 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;

[0080] Document 7: CN117723749A;

[0081] Reference 8: CN114990146A;

[0082] Reference 9: CN117362447A;

[0083] Document 10: CN116444623B.

[0084] Instruction Manual

[0085] Figure 1 A detection schematic diagram of the present invention.

[0086] Figure 2 A schematic flow chart of a detection method adopted by the present invention. DETAILED DESCRIPTION

[0087] The present invention is further explained in conjunction with specific examples. If no specific technology or conditions are specified in this example, the operation is carried out according to the conventional technical methods and instrument instructions in the art. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased from the market or self-developed. The antibodies described in the present invention are self-developed by Chongqing Aishengsi Bioengineering Co., Ltd.

[0088] Example 1: Preparation of raw materials

[0089] Reagent A:

[0090] Weigh 5 mg HRP and dissolve it in 1 ml distilled water; add 0.2 ml of freshly prepared 0.1 M NaIO 4 solution, stir at room temperature in the dark for 20 minutes; put the above solution into a dialysis bag, dialyze against 1 mM sodium acetate buffer at pH 4.4 at 4°C overnight; add 20 μl of 0.2 M pH 9.5 carbonate buffer to raise the pH of the above aldehyded HRP to 9.3, and then immediately add 10 mg IgG (thyroid stimulating hormone (TSH) monoclonal antibody, Chongqing Aishengsi Bioengineering Co., Ltd.) in 1 ml 0.01 M carbonate buffer, gently stir at room temperature in the dark for 2 hours; add 0.1 ml of freshly prepared 4 mg / ml NaBH4 solution, mix well, and let stand at 4°C for 2 hours; put the above solution into a dialysis bag, dialyze against 0.15 M pH 7.4 After PBS dialyzing to remove ammonium ions (detected with Nessler's reagent), centrifuge at 10,000 r / min for 30 min to remove the precipitate. The supernatant is the enzyme conjugate. After the concentration is determined, add an equal amount of 60% glycerol to aliquot and store at 4°C. Before aliquoting, add BSA at a ratio of 10 mg / ml as a stabilizing protective agent.

[0091] Reagent B-1:

[0092] 5 mg of TSH antibody was weighed, desalted using a PD-10 desalting column, eluted with 2.5 ml of biotin coupling buffer (50 mM triethanolamine, pH 8.4), and then diluted to 1 mg / mL with biotin buffer A. 10 mg NHS-Biotin and NHS-acridine compounds were fully dissolved in DMSO to 3.0 mg / ml. According to the ratio of antibody: NHS-Biotin: NHS-acridine compound = 1:20:20 (molar ratio), NHS-Biotin and NHS-acridine compound solutions were added to the above antibody solution and placed at 25°C (the allowable error range is ±2°C) for 180 minutes. The solution after the reaction was desalted with a PD-10 desalting column to remove molecules that did not participate in the reaction, eluted with 2.5 ml of biotin coupling buffer B, and stored at 4°C after measuring the concentration using a UV spectrophotometer. BSA was added as a stabilizing protective agent at a ratio of 10 mg / ml before packaging.

[0093] Reagent B-2:

[0094] 5 mg of TSH antibody was weighed, desalted using a PD-10 desalting column, eluted with 2.5 ml of biotin coupling buffer A, and then diluted to 1 mg / mL with biotin buffer (50 mM triethanolamine, pH 8.4). 10 mg of NHS-Biotin and NHS-(PEG) were weighed separately. 24-Acridine compound, fully dissolved in DMSO to 3.0 mg / ml, according to the antibody: NHS-Biotin: NHS-(PEG) 24 -Acridine compound = 1:20:20 (molar ratio), weigh NHS-Biotin and NHS-(PEG) 24 -Acridine compound solution was added to the above antibody solution and placed at 25°C (allowable error range is ±2°C) for 180 minutes. The solution after the reaction was desalted with a PD-10 desalting column to remove molecules not involved in the reaction, and eluted with 2.5ml biotin coupling buffer (50mM triethanolamine, pH8.4)). The concentration was measured using an ultraviolet spectrophotometer and then frozen at 4°C or -20°C. BSA was added as a stabilizing protective agent at a ratio of 10mg / ml before packaging.

[0095] Reagent B-3:

[0096] 5 mg of TSH antibody was weighed, desalted using a PD-10 desalting column, and eluted with 2.5 ml of biotin coupling buffer A, and then the antibody was diluted to 1 mg / mL with biotin buffer (50 mM triethanolamine, pH 8.4). 10 mg of NHS-Biotin and NHS-(L-Lysine) were weighed separately. 11 -Acridine compound, fully dissolved in DMSO to 3.0 mg / ml, according to the antibody: NHS-Biotin: NHS-(L-Lysine) 11 -Acridine compound = 1:20:22 (molar ratio), weigh NHS-Biotin and NHS-(L-Lysine) 11 -Acridine compound solution was added to the above antibody solution and placed at 28°C (allowable error range is ±2°C) for 150 minutes. The solution after the reaction was desalted with a PD-10 desalting column to remove molecules not involved in the reaction, and eluted with 2.5ml biotin coupling buffer (50mM triethanolamine, pH8.4)). The concentration was measured using a UV spectrophotometer and then frozen at 4°C or -20°C. BSA was added as a stabilizing protective agent at a ratio of 10mg / ml before packaging.

[0097] Reagent C:

[0098] Hydrogen peroxide was dissolved in TBS buffer (pH ≈ 8), wherein the final concentration of hydrogen peroxide was 0.1 M and the final concentration of TBS was 10 mM.

[0099] Reagent D:

[0100] The streptavidin-coated magnetic beads with a diameter of 2.8 μm purchased from Thermo were washed and diluted to 1 mg / mL for use.

[0101] Alkaline solution:

[0102] The solution was adjusted to pH 12 using sodium hydroxide.

[0103] Washing solution: 500 mM PBS buffer, 10% sodium ion, 0.7% EDTA-2Na, 0.5% SDS, 1% Tween 20, 0.1% sodium azide, 0.1% defoaming agent, pH about 7.

[0104] Example 2: Evaluation of signaling inhibitors

[0105] The effect of the signal inhibitor was evaluated from two aspects: signal inhibitor stability and elimination of background interference. The prepared signal inhibitor solution was placed in a test tube and exposed to air for 0min, 5min and 20min before the luminescence signal test was performed. The signal-to-noise ratio S / N (positive sample signal / negative sample signal) was used as an indicator. The higher the ratio, the better the effect.

[0106] Test steps: 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, mix well and react at 37 degrees Celsius for 10 minutes, then add 85 μl of reagent C and 6 μl of a signal inhibitor with a concentration of 0.1 mM, and the light signal generated by the addition 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.

[0107] Table 1 Test results of different substances

[0108]

[0109]

[0110] As shown in Table 1, the effectiveness of signal inhibitors such as vitamin C is consistent with WO2010099486A1. However, the influence of air on signal inhibitors is unavoidable. Considering storage and practical use, signal inhibitors can be selected as R 1 -R 6 The optional two or three substituents are -OH and / or -NH 2 The remaining substituents are -H, -F, -Cl and / or -Br, and two or three substituents are -F, -Cl and / or -Br, preferably, the substituted R 1 -R 6 There are two substituents in it, namely -F, -Cl and / or -Br, and more preferably the substituent is Cl.

[0111] Example 3: Effect of reagent composition on signal

[0112] In order to prove the differences in the detection results of reagents with different compositions and structures, the performance of reagents with different compositions and structures in signal output can be intuitively understood by detecting the signal and signal-to-noise ratio.

[0113] The reagents prepared in Implementation 1 were prepared into a kit and their performance was tested. Kit 1 includes reagent A, reagent B-2, reagent C and the signal inhibitor 14 in Table 1 (contact with air for 20 minutes); Kit 2 includes reagent A, reagent B-3 and the signal inhibitor 14 in Table 1 (contact with air for 20 minutes), reagent C; Kit 3 includes reagent A, reagent B-3, reagent C, reagent D, the signal inhibitor 14 in Table 1 (contact with air for 20 minutes), alkaline solution and cleaning solution, and the concentrations of reagents A, B and C in kits 1-3 are kept consistent. Samples containing different TSH concentrations were prepared by adding TSH to the buffer and collecting clinical serum samples with three concentrations of low, medium and high. The antigen concentration of each sample is shown in Table 2. The performance of kits 1-3 was analyzed using the following reaction mode.

[0114] Reaction mode of kits 1 and 2: Take 20μl of each sample, add 60μl of reagent A and reagent B to each sample, mix well, react at 37 degrees Celsius for 10 minutes, then add 85μl of reagent C and 6μl of signal inhibitor with a concentration of 0.1mM. The generated light signal is captured by a photomultiplier tube, and the intensity of the light signal is quantified.

[0115] Reaction mode of kit 3: Take 20μl of each sample, add 60μl of reagent A and reagent B, and 30μl of reagent D to each sample, mix well, and react at 37 degrees Celsius for 10 minutes. After 3 steps of magnetic separation and washing, add 85ul of reagent C, 6μl of signal inhibitor with a concentration of 0.1mM 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 quantified.

[0116] Table 2 Test results of different kit compositions

[0117]

[0118]

[0119] * Antigen concentration in serum samples was determined using a commercially available kit (electrochemiluminescence method, Roche Diagnostics GmbH)

[0120] As can be seen from Table 2, since the luminescent substrates in kits 1 and 2 are connected by flexible units, the steric hindrance is smaller, and the signal is higher (not listed) than that of the reagent without flexible connection units in Example 2, and the signal-to-noise ratio is also higher. Due to the absence of a washing 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 because the background value is lower after washing. It can be seen from the above verification that the flexible unit connection helps to improve the luminescent signal and signal-to-noise ratio, and the washing step can significantly improve the signal-to-noise ratio.

[0121] Example 4 Effect of reagent composition and detection strategy selection

[0122] In order to further demonstrate the beneficial effects of the multi-stage judgment detection method of the present invention, the detection time (efficiency) and detection accuracy of different detection strategies were measured.

[0123] (1) Sample setting for time detection

[0124] First, TSH was added to the buffer to prepare a basic sample with a TSH concentration of 0.05 μIU / mL. Two 100 mL basic samples were taken, and 1 ml of TBS buffer (pH 8.0) and 1 ml of red dye solution prepared by the buffer were added to the two basic samples, respectively, and recorded as the experimental group and the interference group. The experimental group and the interference group formed a test sample group. By controlling the number of interference groups in the number of test samples, sample groups of different situations were formed, and different detection strategies were used to detect different sample groups in turn, and the time differences used under different strategies were determined. The results are shown in Table 4.

[0125] (2) Sample setting for accuracy test

[0126] TSH was added to the buffer to prepare samples with different concentrations (prepared samples) and clinical serum samples with three antigen concentrations of low, medium and high were collected. Four samples of each antigen concentration were taken, each 200 μL, one of which was recorded 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 recorded as the hemolysis group, jaundice group and lipemia group, respectively. The information of each sample is shown in Table 3, and the test results are shown in Table 5.

[0127] Table 3 Sample information for accuracy analysis

[0128]

[0129] *Antigen concentration in serum samples was determined by commercially available kit (electrochemiluminescence method, Roche Diagnostics GmbH); **Intralipid, provided by Sigma

[0130] (3) Construction of solid phase method for sample determination

[0131] The method comprises the following steps: taking 20 μl of a sample, adding 60 μl of each of the reagent A and reagent B-1 in Example 1 and 30 μl of the reagent D to the sample, mixing well and reacting at 37° C. for 10 minutes, performing 3 steps of magnetic separation and washing, and then adding 85 μl of the 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.

[0132] (4) Construction using homogeneous detection method

[0133] The method comprises the following steps: taking 20 μl of the test sample, adding 60 μl of reagent A and reagent B-1 in Example 1 to the sample, mixing well and reacting at 37° C. for 10 min, then adding 85 μl of reagent C and 6 μl of 0.05 mM test substance 10 in Table 1, and capturing the generated light signal by a photomultiplier tube, and converting the light value signal into a concentration value through a calibration curve.

[0134] (5) Technical detection method of the present invention

[0135] The following steps are involved:

[0136] A. Set the preset item to "NT-proBNP". After the comparison module determines, the test item "TSH" does not meet the preset item;

[0137] 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 transmittance value as the "preset threshold";

[0138] C. Measure the transmittance of samples that do not meet the "preset items" and make interference value judgments. If the transmittance is higher than or equal to the "preset threshold", it is judged as a low-interference sample; if the transmittance is lower than the "preset threshold", it is judged as a high-interference sample;

[0139] D. For high-interference samples, the above-mentioned "solid phase method" is used for detection; for low-interference samples, the above-mentioned "homogeneous detection" is used for detection.

[0140] (6) Results

[0141] According to Table 4, it can be seen from the detection time of a single sample that, compared with homogeneous detection, the detection time of the solid phase detection method is significantly increased due to the addition of the cleaning step. Taking the daily sample detection processing volume of 800 samples in a medium-sized hospital as an example, the detection time of the solid phase detection mode is more than 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 is increased by 18% and reduced by 44% respectively relative to the complete homogeneous mode and the complete solid phase mode. Even if abnormal samples appear on a large scale (25%), the detection time is increased by about 43% and reduced by 33% relative to the complete homogeneous mode and the complete solid phase mode.

[0142] It can be seen that the detection method of the present invention has a higher detection efficiency than that of a solid phase detection mode, reduces unnecessary cleaning of samples entering the cleaning stage, and will also significantly save detection costs, outputting more accurate detection results at a lower cost. It should be understood by those skilled in the art that based on the above-mentioned verification process, when the sample detection amount is further increased, the detection method of the present invention will have more obvious advantages in efficiency and cost.

[0143] Table 4 Detection efficiency of different detection strategies

[0144]

[0145] From the results in Table 5, it can be seen that the multi-stage judgment detection method of the present invention is basically equivalent to the detection method that completely adopts the 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 detection method that completely adopts the homogeneous mode.

[0146] Combined with the results shown in Table 4 and Table 5, the multi-level judgment detection method of the present invention has accurate recognition capability for interference samples, higher detection efficiency, lower detection cost and higher accuracy. The detection method of the present invention is particularly suitable for high-throughput detection needs.

[0147] Table 5 Detection accuracy of different detection strategies

[0148]

[0149]

[0150] It should also be noted that the exemplary embodiments mentioned in the present invention describe some methods or systems 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 performed in the order mentioned in the embodiments, or in a different order from the embodiments, or several steps can be performed simultaneously.

[0151] In the present 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 features of other embodiments or replace features of other embodiments.

[0152] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the embodiments of the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A multifunctional 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 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, two or three of the substituted R1-R6 are optionally -OH and / or -NH2, and the remaining substituents are -H, -F, -Cl and / or -Br, and two or three substituents are optionally -F, -Cl and / or -Br.

2. The kit according to claim 1, characterized in that The signal inhibitor is a structural compound described in Formula 1; preferably, two of the substituted R1-R6 are optionally substituted with -OH and / or -NH2, the remaining substituents are -H, -F, -Cl and / or -Br, and two or three substituents are -F, -Cl and / or -B; more preferably, two of the substituted R1-R6 are optionally substituted with -OH and / or -NH2, the remaining substituents are -H, -F, -Cl and / or -Br, and two substituents are -F, -Cl and / or -Br; most preferably, two substituents are -F or -Cl.

3. The kit according to claim 1 or 2, 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 and a peroxidase, preferably a peroxidase.

4. The kit according to any one of claims 1 to 3, characterized in that The compound 2 is selected from at least one of compounds that generate photons under the action of hydroxyl radicals; preferably includes at least one of luminol, isoluminol, rofenine, acridinium ester, lucigenin, 9,10-dihydroacridine and phthaloyl hydrazide.

5. The kit according to any one of claims 1 to 4, characterized in that The kit further comprises a reagent C, which contains a compound 3; wherein the compound 2 and the compound 3 react to generate a detectable signal; preferably, the compound 3 is a peroxide.

6. The kit according to any one of claims 1 to 5, characterized in that Compound 1 and compound 2 are coupled to the first binding molecule and the second binding molecule respectively through a flexible connection unit; the flexible connection unit is selected from polyoxyethylene ether substances, polyoxypropylene ether substances, alkyl, polyethyleneimine substances, polypeptides or blocks of the above substance constituent units.

7. The kit according to any one of claims 1 to 6, characterized in that The kit also includes an alkaline solution and a cleaning solution, and the pH value of the alkaline solution is greater than or equal to 8.

8. The kit according to any one of claims 1 to 7, characterized in that The kit further comprises a reagent D, wherein the reagent D comprises a solid phase carrier coupled to a connecting structure II, and the connecting structure I and the connecting structure II are specifically bound to each other and form a specific binding system; The specific binding system comprises an antigen-antibody specific binding system, an avidin-biotin specific binding system, a streptavidin-biotin specific binding system and a Tag-Catcher specific binding system.

9. Application of the compound of formula 1 in the preparation of a detection kit, in, Among the substituted R1-R6, two or three substituents are -OH and / or -NH2, respectively, and the remaining substituents are -H, -F, -Cl and / or -Br, respectively, and two or three substituents are -F, -Cl and / or -Br, respectively.

10. The use according to claim 9, characterized in that The compounds are used to improve the signal-to-noise ratio and / or reduce background interference.

Images