Kit for detecting granzyme B, preparation method and application

Abstract

The invention relates to a kit for detecting granzyme B as well as a preparation method and application of the kit. The kit for detecting granzyme B comprises a magnetic microsphere working solution, a detection antibody and streptavidin labeled phycoerythrin. The invention also provides a preparation method of the kit for detecting granzyme B. The preparation method comprises the following steps: coupling a granzyme B antibody by using a magnetic fluorescence coding microsphere to obtain a granzyme B antibody-coupled magnetic fluorescence coding microsphere; labeling the granzyme B detection antibody with biotin to obtain a detection antibody; the preparation method comprises the following steps: mixing granzyme B antibody coupled magnetic fluorescence coded microspheres, a PBS buffer solution, a stabilizer, a preservative, a surfactant and a blocking agent to obtain a magnetic microsphere working solution, and assembling the magnetic microsphere working solution with a detection antibody and streptavidin labeled phycoerythrin to obtain the kit. The invention also provides a kit for detecting serum granzyme B. The invention solves the problem of poor detection reliability caused by false positive results and cross reaction in the existing serum granzyme B detection method.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and particularly relates to a kit for detecting granzyme B, a preparation method and an application thereof. Background Art

[0002] Granzyme B is a key protease secreted by cytotoxic T lymphocytes (CTLs) and natural killer cells (NK cells), and is released into the target cell gap through exocytosis. Under the synergistic action of perforin, granzyme B can penetrate into the interior of the target cell and trigger apoptosis, which plays a crucial role in resisting viral infections, participating in autoimmune reactions, and anti-tumor immunity.

[0003] Granzyme B, consisting of 247 amino acids, is a serine protease with a molecular weight of approximately 32 kDa. Its active site consists of a catalytic triad, including histidine (His59), aspartic acid (Asp103), and serine (Ser198). This catalytic triad is the core of its catalytic activity and can specifically cleave peptide bonds in substrates. The active center of granzyme B is designed to recognize and cleave specific substrates, especially substrates containing acidic amino acids (such as aspartic acid). The structure of its substrate-binding pocket can accommodate up to eight substrate residues, among which arginine (Arg226) is the key residue that forms a salt bridge with the P1 site of the substrate (usually aspartic acid). This property enables granzyme B to play an important role in inducing apoptosis of target cells, especially when activating the caspase pathway.

[0004] The production of granzyme B is a key link in the immune response mediated by CTLs and NK cells. After recognizing specific antigens, these immune cells are activated and rapidly proliferate, differentiating into cells with effector functions. They then migrate to the site of infection or tumor occurrence, recognize and bind to MHC molecules on the surface of target cells through the T cell receptor (TCR). During the contact with the target cell, CTLs release cytotoxic granules containing perforin and granzyme B. Perforin forms pores in the target cell membrane, creating a channel for the entry of granzyme B. Once granzyme B enters the target cell, it triggers a series of cascade reactions, mainly inducing apoptosis by activating caspase family proteins. In particular, granzyme B can directly cleave apoptosis-related caspases such as caspase-3 and caspase-7, and initiate the mitochondrial pathway by cleaving the Bid protein, ultimately leading to the release of cytochrome C and activating the endogenous apoptosis pathway. This finely regulated process is not only crucial for clearing viral infections but also plays a core role in anti-tumor immunity.

[0005] The detection of granzyme B is of great significance for evaluating the status of immune response. The expression level of serum granzyme B shows significant clinical significance in inflammatory diseases. For example, in rheumatoid arthritis (RA), the serum granzyme B level is strongly positively correlated with the joint destruction score of RA patients, suggesting that serum granzyme B can be used as a surrogate marker for predicting the severity of joint damage. In addition, the detection of serum granzyme B also helps to monitor the early response of tumors to immunotherapy, which plays an important role in evaluating the treatment effect and adjusting the treatment plan.

[0006] Currently, the main method for detecting serum granzyme B is enzyme-linked immunosorbent assay (ELISA). The ELISA method captures granzyme B through specific antibodies and generates measurable signals through enzyme-labeled secondary antibodies, thereby achieving the quantitative detection of serum granzyme B. However, ELISA has disadvantages such as false positive results, cross-reactivity, and laborious operation, so its application in routine clinical quantitative detection is limited. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a kit, preparation method, and application for detecting granzyme B, so as to solve the problem that the existing detection methods of serum granzyme B have poor detection reliability due to false positive results, and can also solve the problem that the existing detection methods of serum granzyme B have poor detection reliability due to cross-reactivity, and can also solve the problems of large sample consumption and low detection efficiency in the existing detection methods of serum granzyme B.

[0008] To achieve the above purpose, the technical scheme adopted by the present invention is as follows:

[0009] A kit for detecting granzyme B, comprising: a magnetic microsphere working solution, a detection antibody, and streptavidin-labeled phycoerythrin (SA-PE);

[0010] The components of the magnetic microsphere working solution include magnetic fluorescent encoded microspheres conjugated with granzyme B antibodies;

[0011] The detection antibody is selected from granzyme B detection antibodies and biotin.

[0012] Among them, magnetic microspheres are used, and a magnetic plate can be used for adsorption during the washing process, which can effectively reduce the washing time and improve the operation simplicity.

[0013] In ELISA, enzyme-linked amplification generates signals through the reaction of enzymes with substrates, and this process may lead to false positive results due to non-specific binding or other factors. In this application, fluorescence intensity detection is used to directly measure the fluorescence signal after the binding of antibodies to target proteins. This method can provide more direct quantitative information and reduce the false positives that may be introduced during the enzyme-linked amplification process.

[0014] Preferably, the granzyme B antibody-conjugated magnetic fluorescently encoded microspheres are selected from magnetic fluorescently encoded microspheres coated with granzyme B capture antibodies on the surface.

[0015] Preferably, the detection antibody is selected from biotin-labeled anti-human granzyme B monoclonal antibody.

[0016] Preferably, the components of the magnetic microsphere working solution further include PBS buffer, stabilizer, preservative, surfactant, and blocker.

[0017] By adding a highly efficient blocker to the magnetic working solution, through its high affinity and specific binding, the cross-reaction in immunoassay is effectively reduced, and the accuracy of the detection result is improved.

[0018] Preferably, the granzyme B antibody is an IgG-type antibody.

[0019] Preferably, the kit further includes a lyophilized calibrator, calibrator diluent, and washing solution.

[0020] Preferably, the lyophilized calibrator is selected from recombinant human granzyme B protein.

[0021] Preferably, the calibrator diluent is selected from serum.

[0022] Preferably, the washing solution is selected from PBS buffer.

[0023] Preferably, the concentration of the PBS buffer is 10 mM and the pH value is 7.4.

[0024] PBS buffer is a widely used phosphate buffer solution that mimics the salt concentration and pH value under physiological conditions. PBS buffer is commonly used in biomedical experiments such as cell culture, tissue processing, immunohistochemistry, in situ hybridization, etc. Its basic formula includes: sodium chloride (NaCl): 8 g; potassium chloride (KCl): 0.2 g; sodium dihydrogen phosphate (NaH 2 PO 4 ·H 2 O): 0.2 g; disodium hydrogen phosphate (Na 2 HPO 4 ·7H 2 O): 1.44 g. Dissolve the above components in 1 liter of distilled water and adjust the pH to 7.2 - 7.4. Sometimes, calcium ions (CaCl 2 ), magnesium ions (MgCl 2 ) or other components are added according to experimental requirements. PBS buffer can be purchased in ready-made powder or liquid form, or prepared by oneself according to needs.

[0025] Preferably, the stabilizer is selected from at least one of bovine serum albumin, human serum albumin, ovalbumin, trehalose, sucrose and mannitol.

[0026] Preferably, the stabilizer is selected from 0.1% bovine serum albumin.

[0027] Preferably, the preservative is selected from one or both of NaN 3 and ProClin 300.

[0028] Preferably, the preservative is selected from 0.1% ProClin 300.

[0029] Preferably, the blocker is selected from the heterophilic antibody blocker HBR-5.

[0030] By adding the highly efficient heterophilic antibody blocker HBR-5 to the magnetic working fluid, through its high affinity and specific binding, the cross-reaction in immunoassay is effectively reduced, and the accuracy of the detection result is improved.

[0031] Preferably, the surfactant is selected from at least one of Tween-20, Tween-80 and Triton X-100.

[0032] Preferably, the surfactant is selected from 0.02% Tween-20.

[0033] The present invention also provides a method for preparing a kit for detecting granzyme B as described in the present invention, comprising the following steps:

[0034] Adding magnetic fluorescent encoded microspheres into MES buffer, resuspending, adding EDC solution and Sulfo-NHS solution, magnetically separating and discarding the supernatant; then adding granzyme B antibody, supplementing MES buffer, reacting under the first temperature condition, magnetically separating and discarding the supernatant; adding TBS-B buffer, performing a blocking reaction under the first temperature condition, magnetically separating and discarding the supernatant; adding PBS-TBN buffer to resuspend to obtain magnetic fluorescent encoded microspheres conjugated with granzyme B antibody;

[0035] Dissolving biotin in DMSO (dimethyl sulfoxide) solution to obtain a biotin solution, diluting the granzyme B detection antibody with PBS buffer to obtain a diluted granzyme B detection antibody; mixing and reacting the biotin solution and the diluted granzyme B detection antibody to obtain a detection antibody;

[0036] Mixing the magnetic fluorescent encoded microspheres conjugated with granzyme B antibody, PBS buffer, stabilizer, preservative, surfactant and blocker to obtain a magnetic microsphere working fluid, and then assembling it with the detection antibody and streptavidin-labeled phycoerythrin (SA-PE) to obtain the kit.

[0037] Preferably, the mass ratio of the magnetic fluorescent coding microspheres to the granzyme B antibody is 1.0 mg: 40 μg.

[0038] Preferably, the molar ratio of the biotin to the granzyme B detection antibody is 50:1.

[0039] Preferably, the EDC solution is an EDC + MES buffer solution with a concentration of 50 mg / mL and a pH value of 4.75.

[0040] The EDC solution generally refers to a solution containing 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. This compound is a commonly used coupling agent, widely applied in organic synthesis, peptide synthesis, and the field of biochemistry, for promoting the reaction between carboxylic acid and amine to form amide bonds.

[0041] The MES buffer solution is a buffer system with a pH range between 5.5 and 6.7, widely used in biochemistry and molecular biology experiments. It is an organic buffer, with the full name of 2-(N-morpholino)ethanesulfonic acid, having good buffering capacity and stability. The MES buffer solution is particularly suitable for experiments that need to be carried out under conditions close to physiological pH values, such as enzyme activity assays, electrophoretic separation of proteins and nucleic acids, etc.

[0042] Preferably, the Sulfo-NHS solution is a Sulfo-NHS + MES buffer solution with a concentration of 50 mg / mL and a pH value of 4.75.

[0043] The Sulfo-NHS solution is a chemical reagent, with the full name of N-hydroxysuccinimide sulfate solution. It is usually used in biochemistry experiments, especially as an activator in protein coupling and labeling reactions. Sulfo-NHS can react with the amino groups on proteins to form stable amide bonds, thereby coupling other molecules such as fluorescent dyes, biotin, or other proteins to the target protein. This solution usually needs to be stored under low temperature and light-proof conditions.

[0044] Preferably, the concentration of the MES buffer solution is 50 mM and the pH value is 4.75.

[0045] Preferably, the TBS-B buffer solution is a Tris buffer solution + 1% BSA, and the pH value of the Tris buffer solution is 8.2.

[0046] TBS-B buffer is a commonly used chemical buffer solution in laboratories. TBS stands for Tris-buffered saline, and "B" usually indicates that this buffer contains specific additives or components, such as Bovine Serum Albumin (BSA), to prevent proteins from adsorbing to the surface of containers. This buffer is often used to dilute antibodies or for the washing steps of samples during immunological experiments, such as Western blotting.

[0047] Preferably, the PBS-TBN buffer is PBS buffer + 0.1% BSA + 0.02% Tween-20 + 0.1% Proclin300 + HBR-5, the pH value of the PBS buffer is 7.4, and the concentration of HBR-5 is 50 μg / mL.

[0048] Among them, Proclin300 is a preservative, usually used as a component of laboratory buffers and other solutions to inhibit microbial growth.

[0049] Preferably, the concentration of the PBS buffer is 10 mM and the pH value is 7.4.

[0050] Preferably, the first temperature is 25 °C.

[0051] Preferably, the reaction time under the first temperature condition is 3 h.

[0052] Preferably, the blocking reaction time under the first temperature condition is 12 - 18 h.

[0053] Preferably, the temperature for mixing and reacting the biotin solution and the diluted granzyme B detection antibody is room temperature, and the mixing and reaction time is 2 h.

[0054] Preferably, the preparation method includes: subjecting 1.0 mg of magnetic fluorescent encoded microspheres (8×10 6 microspheres) to magnetic separation to discard the supernatant, then adding them to 128 μL of MES buffer, resuspending, adding 16 μL of EDC solution and 16 μL of Sulfo-NHS solution, mixing evenly, mixing and reacting at 25 °C for 30 minutes, subjecting to magnetic separation to discard the supernatant, washing once with 160 μL of MES buffer, and subjecting to magnetic separation again to discard the supernatant; adding 40 μg of granzyme B antibody, supplementing MES buffer to 160 μL, mixing evenly, mixing and reacting at 25 °C for 3 hours, subjecting to magnetic separation to discard the supernatant; adding 0.32 mL of TBS-B buffer, mixing and blocking at 25 °C for 12 - 18 h, subjecting to magnetic separation to discard the supernatant; adding 0.8 mL of PBS-TBN buffer to resuspend to obtain granzyme B antibody-conjugated magnetic fluorescent encoded microspheres, and storing them at 4 °C;

[0055] Dissolve biotin in DMSO solution to obtain a biotin solution with a concentration of 4 mg / mL, and dilute the granzyme B detection antibody with 10 mM PBS buffer at pH 7.4 to obtain a granzyme B detection antibody with a concentration of 1 mg / mL. According to the molar ratio of biotin to the granzyme B detection antibody of 50:1, add the biotin solution and the diluted granzyme B detection antibody to a reaction vessel and mix and react at room temperature for 2 h. Centrifuge the reaction solution at 2000 r / min for 5 min using a 50 KD ultrafiltration tube, and concentrate the liquid to about 50 μL. The concentrated solution obtained is the detection antibody.

[0056] Mix the magnetic fluorescence-encoded microspheres conjugated with granzyme B antibody, PBS buffer, stabilizer, preservative, surfactant, and blocker to obtain a working solution of magnetic microspheres. Then assemble it with the detection antibody, streptavidin-labeled phycoerythrin (SA-PE), lyophilized calibrator, calibrator diluent, and cleaning solution to obtain a kit.

[0057] The present invention also provides a kit prepared by the preparation method as described in the present invention for detecting serum granzyme B.

[0058] Preferably, the kit combines with a flow cytometer to detect serum granzyme B.

[0059] Preferably, the detection range of the kit is 0.436 - 2500 pg / ml.

[0060] The beneficial effects of the present invention:

[0061] The kit for detecting granzyme B of the present invention is applicable to flow cytometers configured with 2 lasers and 4 channels or more. Through its high-speed sample collection, simplified sample processing steps, automated and intelligent design, multi-parameter analysis ability, and fast data processing, the flow cytometer greatly saves experimental time and improves the convenience of operation. The quantitative detection of serum granzyme B can be completed in 3 - 4 hours, making the detection faster, more reliable, and more stable.

[0062] The kit for detecting granzyme B of the present invention quantifies the target protein according to the fluorescence intensity of the antibody, eliminating the false positives generated by enzyme-linked amplification in the ELISA reaction.

[0063] The kit for detecting granzyme B of the present invention is based on the flow cytometry-encoded microsphere chip technology, thus having the high sensitivity and high precision of chemiluminescence, and at the same time having a wider detection range and better repeatability. The flow cytometry-encoded microsphere chip technology enables the analysis sensitivity of protein quantification to reach the pg / ml level, and the detection range is 0.436 - 2500 pg / ml. This makes the flow cytometry-encoded microsphere chip technology perform excellently in detecting trace biomarkers, especially suitable for early disease screening.

[0064] The kit for detecting granzyme B of the present invention is based on the flow cytometry encoded microsphere chip technology and has the advantage of fewer operation steps. It mainly involves sample addition, incubation, and result reading, simplifies the experimental process, and reduces the result deviation caused by manual operation. ELISA requires multiple washing steps and incubation processes, with relatively complex operations, and each step may affect the accuracy of the final result.

[0065] The magnetic fluorescent encoded microspheres used in the kit for detecting granzyme B of the present invention can be highly purified separated within a short time through a simple magnetic separation device, providing convenience in operation and avoiding the steps of adjusting complex parameters such as centrifugation speed, time, and temperature during the separation of traditional polystyrene microspheres. Thereby reducing the sample loss caused by centrifugation and maintaining the integrity of the sample, which has the value of popularization and application in the field of immunoassay technology. Description of the Drawings

[0066] Figure 1 It is a relationship curve between the concentration of serum granzyme B detected by the serum granzyme B detection kit and the signal value when the concentration of serum granzyme B is 0.436 pg / mL to 2500 pg / mL;

[0067] Figure 2 It is the spectral effect diagram of GZMB fluorescent encoded microspheres. Detailed Embodiments

[0068] The following will describe the embodiments of the present invention with reference to the drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention and not for limiting the protection scope of the present invention.

[0069] It should be noted that the diagrams provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner. Therefore, only the components related to the present invention are shown in the diagrams, rather than being drawn according to the number, shape, and size of the components in actual implementation. The type, quantity, and ratio of each component in actual implementation can be arbitrarily changed, and the component layout type may also be more complex.

[0070] The present invention aims to publicly provide a kit, preparation method, and application for detecting granzyme B to solve the problems that the existing detection methods for serum granzyme B have false positive results and cross-reactions, resulting in poor detection reliability, and can also solve the problems that the existing detection methods for serum granzyme B have large sample consumption and low detection efficiency.

[0071] Among them, a kit for detecting granzyme B includes: a magnetic microsphere working solution, a detection antibody, and streptavidin-labeled phycoerythrin (SA-PE);

[0072] The components of the magnetic microsphere working solution include magnetic fluorescent encoded microspheres conjugated with granzyme B antibodies;

[0073] The detection antibody is selected from a granzyme B detection antibody and biotin.

[0074] Among them, magnetic microspheres are used, and a magnetic plate can be used for adsorption during the washing process, which can effectively reduce the washing time and improve the operation simplicity.

[0075] Exemplarily, the magnetic fluorescent encoded microspheres conjugated with granzyme B antibodies are selected from magnetic fluorescent encoded microspheres coated with granzyme B capture antibodies on the surface.

[0076] Exemplarily, the detection antibody is selected from biotin-labeled anti-human granzyme B monoclonal antibody.

[0077] In some embodiments, the components of the magnetic microsphere working solution further include PBS buffer, stabilizer, preservative, surfactant, and blocker.

[0078] Exemplarily, the granzyme B antibody is an IgG-type antibody.

[0079] In some embodiments, the kit further includes a freeze-dried calibrator, a calibrator diluent, and a washing solution.

[0080] Exemplarily, the freeze-dried calibrator is selected from recombinant human granzyme B protein;

[0081] Exemplarily, the calibrator diluent is selected from serum;

[0082] In some embodiments, the washing solution is selected from PBS buffer.

[0083] Exemplarily, the concentration of the PBS buffer is 10 mM and the pH value is 7.4.

[0084] In some embodiments, the stabilizer is selected from at least one of bovine serum albumin, human serum albumin, ovalbumin, trehalose, sucrose, and mannitol.

[0085] Exemplarily, the stabilizer is selected from 0.1% bovine serum albumin.

[0086] In some embodiments, the preservative is selected from one or both of NaN 3 and ProClin 300.

[0087] Exemplarily, the preservative is selected from 0.1% ProClin 300.

[0088] In some embodiments, the surfactant is selected from at least one of Tween-20, Tween-80, and Triton X-100.

[0089] Exemplarily, the surfactant is selected from 0.02% Tween-20.

[0090] Exemplarily, the blocker is selected from the heterophilic antibody blocker HBR-5.

[0091] In some embodiments, there is also provided a method for preparing a kit for detecting granzyme B as in any of the above embodiments, comprising the following steps:

[0092] Add magnetic fluorescent encoded microspheres to MES buffer, resuspend, add EDC solution and Sulfo-NHS solution, and discard the supernatant by magnetic separation; then add granzyme B antibody, supplement MES buffer, react under the first temperature condition, and discard the supernatant by magnetic separation; add TBS-B buffer, perform a blocking reaction under the first temperature condition, and discard the supernatant by magnetic separation; add PBS-TBN buffer to resuspend to obtain magnetic fluorescent encoded microspheres conjugated with granzyme B antibody;

[0093] Dissolve biotin in DMSO solution to obtain a biotin solution, dilute the granzyme B detection antibody with PBS buffer to obtain a diluted granzyme B detection antibody; mix and react the biotin solution and the diluted granzyme B detection antibody to obtain a detection antibody;

[0094] Assemble the magnetic fluorescent encoded microspheres conjugated with granzyme B antibody, the detection antibody, and streptavidin-labeled phycoerythrin (SA-PE) to obtain the kit.

[0095] Exemplarily, the mass ratio of the magnetic fluorescent encoded microspheres to the granzyme B antibody is 1.0 mg: 40 μg.

[0096] Exemplarily, the molar ratio of biotin to the granzyme B detection antibody is 50:1.

[0097] Exemplarily, the EDC solution is EDC + MES buffer, with a concentration of 50 mg / mL and a pH value of 4.75.

[0098] Exemplarily, the Sulfo-NHS solution is Sulfo-NHS + MES buffer, with a concentration of 50 mg / mL and a pH value of 4.75.

[0099] Exemplarily, the concentration of the MES buffer is 50 mM and the pH value is 4.75.

[0100] Exemplarily, the TBS-B buffer is Tris buffer + 1% BSA, and the pH value of the Tris buffer is 8.2.

[0101] Exemplarily, the PBS-TBN buffer is PBS buffer + 0.1% BSA + 0.02% Tween-20 + 0.1% Proclin300 + HBR-5. The pH value of the PBS buffer is 7.4, and the concentration of HBR-5 is 50 μg / mL.

[0102] Exemplarily, the concentration of the PBS buffer is 10 mM and the pH value is 7.4.

[0103] Exemplarily, the first temperature is 25 °C.

[0104] Exemplarily, the reaction time under the first temperature condition is 3 h.

[0105] In some embodiments, the blocking reaction time under the first temperature condition is 12 - 18 h.

[0106] Exemplarily, the temperature for mixing and reacting the biotin solution and the diluted granzyme B detection antibody is room temperature, and the mixing and reaction time is 2 h.

[0107] Exemplarily, the preparation method includes: subjecting 1.0 mg of magnetic fluorescent encoded microspheres (8×10 6 microspheres) to magnetic separation to discard the supernatant, then adding them to 128 μL of MES buffer, resuspending, adding 16 μL of EDC solution and 16 μL of Sulfo-NHS solution, mixing evenly, mixing and reacting at 25 °C for 30 minutes, subjecting to magnetic separation to discard the supernatant, washing once with 160 μL of MES buffer, and subjecting to magnetic separation again to discard the supernatant; adding 40 μg of granzyme B antibody, supplementing MES buffer to 160 μL, mixing evenly, mixing and reacting at 25 °C for 3 hours, subjecting to magnetic separation to discard the supernatant; adding 0.32 mL of TBS-B buffer, mixing and blocking at 25 °C for 12 - 18 h, subjecting to magnetic separation to discard the supernatant; adding 0.8 mL of PBS-TBN buffer to resuspend to obtain granzyme B antibody-conjugated magnetic fluorescent encoded microspheres, and storing at 4 °C;

[0108] Dissolve biotin in DMSO solution to obtain a 4 mg / mL biotin solution, dilute the granzyme B detection antibody with 10 mM PBS buffer at pH 7.4 to obtain a 1 mg / mL granzyme B detection antibody; according to the molar ratio of biotin to granzyme B detection antibody of 50:1, add the biotin solution and the diluted granzyme B detection antibody to the reaction vessel, and mix and react at room temperature for 2 h; centrifuge the reaction solution at 2000 r / min for 5 min using a 50 KD ultrafiltration tube to concentrate the liquid to about 50 μL, and the obtained concentrated solution is the detection antibody;

[0109] Mix the magnetic fluorescent encoded microspheres conjugated with granzyme B antibody, PBS buffer, stabilizer, preservative, surfactant and blocker to obtain the working solution of magnetic microspheres, and then assemble it with detection antibody, streptavidin-labeled phycoerythrin (SA-PE), freeze-dried calibrator, calibrator diluent and washing solution to obtain the kit.

[0110] In some embodiments, there is also provided a kit prepared by the preparation method in any of the above embodiments for detecting serum granzyme B.

[0111] In some embodiments, the kit combines with a flow cytometer to detect serum granzyme B.

[0112] Exemplarily, the detection range of the kit is 0.436 - 2500 pg / ml.

[0113] The detection process of the above kit for detecting granzyme B in serum includes the following steps:

[0114] 1) Preparation of calibrator

[0115] 1. Prepare 8 0.6 mL centrifuge tubes as calibrator gradient tubes, labeled S0 - S7; leave the S7 tube empty, and add 150 μL of calibrator diluent to the S0 - S6 tubes.

[0116] 2. Add 0.5 mL of calibrator diluent to the calibrator vial, fully dissolve and let it stand at room temperature for 15 min, mix well and transfer 200 μL to the S7 tube to obtain the highest concentration calibrator solution;

[0117] 3. Take 100 μL of the calibrator solution in the S7 tube and transfer it to the S6 tube to mix well, which is the 1:2.5 diluted calibrator; sequentially perform gradient dilution to obtain the calibrator solutions in the S6 - S1 tubes; the calibrator diluent in the S0 tube is used as the 0 concentration calibrator. As shown in Table 1.

[0118] Table 1 shows the calibrator solutions of S0 - S7

[0119]

[0120] 2) Add 50 μL of the working solution of magnetic microspheres (including magnetic fluorescent encoded microspheres conjugated with granzyme B antibody, PBS buffer (10 mM, pH 7.4), 0.1% bovine serum albumin, 0.1% proclin 300, 0.02% Tween - 20 and 50 μg / mL blocker HBR - 5) (vortex for more than 30 s before adding), 50 μL of serum calibrator to the reaction tubes, mix the samples well, and incubate at 25°C in the dark and mix for 60 minutes;

[0121] 2) Magnetic separation to remove the supernatant, wash once with 200 μL of magnetic bead washing solution (PBS buffer (10 mM, pH 7.4)); magnetic separation or centrifugation to remove the supernatant, add 100 μL of biotin-labeled anti-human granzyme B monoclonal antibody, mix well, and incubate for 60 minutes at 25°C in the dark with gentle mixing;

[0122] 3) Magnetic separation to remove the supernatant, wash once with 200 μL of magnetic bead washing solution, magnetic separation again to remove the supernatant, add 100 μL of streptavidin-labeled phycoerythrin (SA-PE), and incubate for 30 minutes at 25°C in the dark with gentle mixing;

[0123] 4) Magnetic separation to remove the supernatant, wash once with 200 μL of magnetic bead washing solution, magnetic separation again to remove the supernatant, resuspend the microspheres with 200 μL of magnetic bead washing solution, and detect by flow cytometry.

[0124] Among them, the detection principle of the kit for detecting granzyme B for serum granzyme B is: the principle of magnetic microsphere flow immunofluorescence technology using the double antibody sandwich method. Reagent component A contains fluorescence-encoded magnetic microspheres coated with granzyme B capture antibody on the surface; reagent component B contains biotin-labeled granzyme B detection antibody. During detection, components A / B and the antigen in the sample form a double antibody sandwich complex, and the fluorescence intensity of the PE channel of the encoded microspheres is related to the concentration of the corresponding antigen in the sample. By detecting the fluorescence intensity of the PE channel with a flow cytometer and referring to the standard curve, the concentration of granzyme B (Granzyme B) in the sample can be calculated.

[0125] In order to make the technical problems, technical solutions and beneficial effects solved by the present application clearer, the preparation method and application of the kit of the present invention will be further described in detail below with specific examples and drawings. Obviously, the specific examples described are only a part of the embodiments of the present application, rather than all embodiments. The description of at least one exemplary embodiment below is actually only illustrative and in no way limits the present application and its application. Based on the specific embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present application.

[0126] For those not specifying specific techniques or conditions in the specific examples, follow the techniques or conditions described in the literature in the field or according to the product instructions. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0127] Example 1

[0128] A preparation method of a kit for detecting granzyme B, comprising the following steps:

[0129] Coating scale: 1.0 mg of microspheres (8×10 6 / 40 μg IgG antibody per microsphere;

[0130] S1. Take 1.0 mg of magnetic fluorescent encoded microspheres (8×10 6 microspheres) for magnetic separation, discard the supernatant, then add them to 128 μL of MES buffer and resuspend to obtain a magnetic fluorescent encoded microsphere suspension. Sequentially add 16 μL of EDC solution and 16 μL of Sulfo-NHS solution to the magnetic fluorescent encoded microsphere suspension, mix well, react at 25 °C for 30 minutes with mixing. Perform magnetic separation on the microspheres, discard the supernatant, wash once with 160 μL of MES buffer, perform magnetic separation on the microspheres again, and discard the supernatant to obtain a precipitate. Add 40 μg of granzyme B antibody to the precipitate, supplement MES buffer to 160 μL, mix well, react at 25 °C for 3 hours with mixing. Perform magnetic separation on the microspheres, discard the supernatant to obtain a precipitate. Add 0.32 mL of TBS-B buffer to the precipitate, react at 25 °C for 16 h with mixing for blocking, perform magnetic separation on the microspheres, and discard the supernatant. Resuspend with 0.8 mL of PBS-TBN buffer to obtain granzyme B antibody-conjugated magnetic fluorescent encoded microspheres and store at 4 °C;

[0131] The magnetic fluorescent encoded microspheres are polystyrene microspheres with a core embedded with Fe 3 O 4 and the outer layer is encoded with different ratios of APC and APC-Cy7 dyes, and has carboxyl-modified surfaces.

[0132] Among them, MES buffer: 50 mM, pH 4.75;

[0133] PBS buffer: 10 mM, pH 7.4;

[0134] EDC solution: 50 mg / mL, EDC + MES buffer (pH 4.75);

[0135] Sulfo-NHS solution: 50 mg / mL, Sulfo-NHS + MES buffer (pH 4.75);

[0136] TBS-B buffer: Tris buffer (pH 8.2) + 1% BSA;

[0137] Tris buffer: 50 mM, pH 8.2;

[0138] PBS-TBN buffer: PBS buffer (pH 7.4) + 0.1% BSA + 0.02% Tween-20 + 0.1% Proclin300 + HBR-5 (50 μg / mL);

[0139] S2. Dissolve biotin in DMSO (dimethyl sulfoxide) solution to obtain a biotin solution with a concentration of 4 mg / mL. Dilute the granzyme B detection antibody with 10 mM PBS buffer at pH 7.4 to obtain a granzyme B detection antibody with a concentration of 1 mg / mL. According to the molar ratio of biotin to the granzyme B detection antibody of 50:1, add the biotin solution and the diluted granzyme B detection antibody into a reaction container and mix and react at room temperature for 2 h. Centrifuge the reaction solution at 2000 r / min for 5 min using a 50 KD ultrafiltration tube, and concentrate the liquid to about 50 μL. The concentrated solution obtained is the detection antibody.

[0140] S3. Mix the magnetic fluorescent encoded microspheres conjugated with granzyme B antibody, PBS buffer, stabilizer, preservative, surfactant and blocker to obtain a working solution of magnetic microspheres, and then assemble it with the detection antibody, streptavidin-labeled phycoerythrin (SA-PE), freeze-dried calibrator, calibrator diluent and washing solution to obtain a kit.

[0141] In the working solution of magnetic microspheres, the PBS buffer is 10 mM PBS buffer at pH 7.4, the stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% proclin 300, the surfactant is 0.02% Tween-20, and the blocker is HBR-5 (50 μg / mL).

[0142] The freeze-dried calibrator is recombinant human granzyme B protein.

[0143] The calibrator diluent is serum.

[0144] The washing solution is PBS buffer (10 mM, pH 7.4).

[0145] Example 2

[0146] A method for preparing a kit for detecting granzyme B, comprising the following steps:

[0147] Coating scale: 1.0 mg microspheres (8×10 6 microspheres) / 20 μg IgG type antibody;

[0148] S1. Take 1.0 mg of magnetic fluorescent encoded microspheres (8×10 6The microspheres were magnetically separated to discard the supernatant, and then added to 128 μL of MES buffer and resuspended to obtain a suspension of magnetically fluorescently encoded microspheres. To the suspension of magnetically fluorescently encoded microspheres, 16 μL of EDC solution and 16 μL of Sulfo-NHS solution were added in sequence, mixed well, and reacted at 25 °C for 30 minutes. The microspheres were magnetically separated to discard the supernatant, washed once with 160 μL of MES buffer, and magnetically separated again to discard the supernatant to obtain a precipitate. To the precipitate, 20 μg of granzyme B antibody was added, and MES buffer was supplemented to 160 μL, mixed well, and reacted at 25 °C for 3 hours. The microspheres were magnetically separated to discard the supernatant to obtain a precipitate. To the precipitate, 0.32 mL of TBS-B buffer was added, and the mixture was reacted at 25 °C for 16 h with gentle mixing. The microspheres were magnetically separated to discard the supernatant. The precipitate was resuspended with 0.8 mL of PBS-TBN buffer to obtain granzyme B antibody-conjugated magnetically fluorescently encoded microspheres, which were stored at 4 °C.

[0149] The magnetically fluorescently encoded microspheres have an Fe 3 O 4 core embedded therein and are encoded with different ratios of APC and APC-Cy7 dyes on the outer layer, and are polystyrene microparticles with carboxyl modification on the surface.

[0150] Among them, MES buffer: 50 mM, pH 4.75;

[0151] PBS buffer: 10 mM, pH 7.4;

[0152] EDC solution: 50 mg / mL, EDC + MES buffer (pH 4.75);

[0153] Sulfo-NHS solution: 50 mg / mL, Sulfo-NHS + MES buffer (pH 4.75);

[0154] TBS-B buffer: Tris buffer (pH 8.2) + 1% BSA;

[0155] Tris buffer: 50 mM, pH 8.2;

[0156] PBS-TBN buffer: PBS buffer (pH 7.4) + 0.1% BSA + 0.02% Tween-20 + 0.1% Proclin300 + HBR-5 (50 μg / mL);

[0157] S2. Dissolve biotin in DMSO (dimethyl sulfoxide) solution to obtain a biotin solution with a concentration of 4 mg / mL. Dilute the granzyme B detection antibody with 10 mM PBS buffer at pH 7.4 to obtain a granzyme B detection antibody with a concentration of 1 mg / mL. According to the molar ratio of biotin to the granzyme B detection antibody of 50:1, add the biotin solution and the diluted granzyme B detection antibody into a reaction container and mix and react at room temperature for 2 h. Centrifuge the reaction solution at 2000 r / min for 5 min using a 50 KD ultrafiltration tube, and concentrate the liquid to about 50 μL. The concentrated solution obtained is the detection antibody.

[0158] S3. Mix the magnetic fluorescent encoded microspheres conjugated with granzyme B antibody, PBS buffer, stabilizer, preservative, surfactant and blocker to obtain a working solution of magnetic microspheres, and then assemble it with the detection antibody, streptavidin-labeled phycoerythrin (SA-PE), freeze-dried calibrator, calibrator diluent and washing solution to obtain a kit.

[0159] In the working solution of magnetic microspheres, the PBS buffer is 10 mM PBS buffer at pH 7.4, the stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% proclin 300, the surfactant is 0.02% Tween-20, and the blocker is HBR-5 (50 μg / mL).

[0160] The freeze-dried calibrator is recombinant human granzyme B protein.

[0161] The calibrator diluent is serum.

[0162] The washing solution is PBS buffer (10 mM, pH 7.4).

[0163] Example 3

[0164] A preparation method of a kit for detecting granzyme B, comprising the following steps:

[0165] Coating scale: 1.0 mg microspheres (8×10 6 microspheres) / 10 μg IgG-type antibody;

[0166] S1. Take 1.0 mg of magnetic fluorescent encoded microspheres (8×10 6The microspheres were magnetically separated to discard the supernatant, and then added to 128 μL of MES buffer and resuspended to obtain a suspension of magnetically fluorescent encoded microspheres. To the suspension of magnetically fluorescent encoded microspheres, 16 μL of EDC solution and 16 μL of Sulfo-NHS solution were added successively, mixed well, and reacted at 25 °C for 30 minutes. The microspheres were magnetically separated to discard the supernatant, washed once with 160 μL of MES buffer, and magnetically separated again to discard the supernatant to obtain a precipitate. To the precipitate, 10 μg of granzyme B antibody was added, and MES buffer was supplemented to 160 μL, mixed well, and reacted at 25 °C for 3 hours. The microspheres were magnetically separated to discard the supernatant to obtain a precipitate. To the precipitate, 0.32 mL of TBS-B buffer was added, and the reaction was blocked by mixing at 25 °C for 16 h. The microspheres were magnetically separated to discard the supernatant; 0.8 mL of PBS-TBN buffer was added to resuspend to obtain granzyme B antibody-conjugated magnetically fluorescent encoded microspheres, which were stored at 4 °C;

[0167] The magnetically fluorescent encoded microspheres have a core embedded with Fe 3 O 4 , and the outer layer is encoded with different ratios of APC and APC-Cy7 dyes, and the surface has carboxyl-modified polystyrene microparticles.

[0168] Among them, MES buffer: 50 mM, pH 4.75;

[0169] PBS buffer: 10 mM, pH 7.4;

[0170] EDC solution: 50 mg / mL, EDC + MES buffer (pH 4.75);

[0171] Sulfo-NHS solution: 50 mg / mL, Sulfo-NHS + MES buffer (pH 4.75);

[0172] TBS-B buffer: Tris buffer (pH 8.2) + 1% BSA;

[0173] Tris buffer: 50 mM, pH 8.2;

[0174] PBS-TBN buffer: PBS buffer (pH 7.4) + 0.1% BSA + 0.02% Tween-20 + 0.1% Proclin300 + HBR-5 (50 μg / mL);

[0175] S2. Dissolve biotin in DMSO (dimethyl sulfoxide) solution to obtain a biotin solution with a concentration of 4 mg / mL. Dilute the granzyme B detection antibody with 10 mM PBS buffer at pH 7.4 to obtain a granzyme B detection antibody with a concentration of 1 mg / mL. According to the molar ratio of biotin to the granzyme B detection antibody of 50:1, add the biotin solution and the diluted granzyme B detection antibody into a reaction vessel and mix and react at room temperature for 2 h. Centrifuge the reaction solution at 2000 r / min for 5 min using a 50 KD ultrafiltration tube, and concentrate the liquid to about 50 μL. The concentrated solution obtained is the detection antibody.

[0176] S3. Mix the magnetic fluorescence-coded microspheres conjugated with granzyme B antibody, PBS buffer, stabilizer, preservative, surfactant, and blocker to obtain a working solution of magnetic microspheres, and then assemble it with the detection antibody, streptavidin-labeled phycoerythrin (SA-PE), freeze-dried calibrator, calibrator diluent, and washing solution to obtain a kit.

[0177] In the working solution of magnetic microspheres, the PBS buffer is 10 mM PBS buffer at pH 7.4, the stabilizer is 0.1% bovine serum albumin, the preservative is 0.1% proclin 300, the surfactant is 0.02% Tween-20, and the blocker is HBR-5 (50 μg / mL).

[0178] The freeze-dried calibrator is recombinant human granzyme B protein.

[0179] The calibrator diluent is serum.

[0180] The washing solution is PBS buffer (10 mM, pH 7.4).

[0181] Example 4

[0182] The method for detecting serum granzyme B using the kit prepared in Example 1 includes the following steps:

[0183] 1) Add 50 μL of magnetic microsphere working solution (including magnetic fluorescent encoded microspheres conjugated with granzyme B antibody, PBS buffer (10 mM, pH 7.4), 0.1% bovine serum albumin, 0.1% proclin 300, 0.02% Tween-20, and 50 μg / mL blocker HBR-5) into the reaction tubes. The magnetic microsphere working solution needs to be vortexed and mixed well for more than 30 s before adding it into the reaction tubes. Then add 50 μL of serum samples with different granzyme B concentrations (the concentrations of Granzyme B in the serum samples are respectively: 0 pg / mL, 4.096 pg / mL, 10.24 pg / mL, 25.6 pg / mL, 64 pg / mL, 160 pg / mL, 400 pg / mL, 1000 pg / mL, and 2500 pg / mL), mix well, and incubate at 25°C in the dark with gentle mixing for 40 minutes;

[0184] 2) Separate the magnetic beads and remove the supernatant, and wash once with 200 μL of magnetic bead washing solution; separate the magnetic beads and remove the supernatant, add 100 μL of biotinylated anti-human granzyme B monoclonal antibody, mix well, and incubate at 25°C in the dark with gentle mixing for 40 minutes;

[0185] 3) Separate the magnetic beads and remove the supernatant, wash once with 200 μL of magnetic bead washing solution, separate the magnetic beads and remove the supernatant again, add 100 μL of streptavidin-labeled phycoerythrin (SA-PE), and incubate at 25°C in the dark with gentle mixing for 20 minutes;

[0186] 4) Separate the magnetic beads and remove the supernatant, wash once with 200 μL of magnetic bead washing solution, separate the magnetic beads and remove the supernatant again, resuspend the microspheres with 200 μL of magnetic bead washing solution, and detect with a flow cytometer.

[0187] Example 5

[0188] The method for detecting serum granzyme B using the kit prepared in Example 2 includes the following steps:

[0189] 1) Add 50 μL of magnetic microsphere working solution (including magnetic fluorescent encoded microspheres conjugated with granzyme B antibody, PBS buffer (10 mM, pH 7.4), 0.1% bovine serum albumin, 0.1% proclin 300, 0.02% Tween-20, and 50 μg / mL blocker HBR-5) into the reaction tubes. The magnetic microsphere working solution needs to be vortexed and mixed well for more than 30 s before adding it into the reaction tubes. Then add 50 μL of serum samples with different granzyme B concentrations (the concentrations of Granzyme B in the serum samples are respectively: 0 pg / mL, 4.096 pg / mL, 10.24 pg / mL, 25.6 pg / mL, 64 pg / mL, 160 pg / mL, 400 pg / mL, 1000 pg / mL, and 2500 pg / mL), mix well, and incubate at 25°C in the dark with gentle mixing for 40 minutes;

[0190] 2) Magnetic separation to remove the supernatant, and wash once with 200 μL of magnetic bead washing solution; magnetic separation to remove the supernatant, add 100 μL of biotin-labeled anti-human granzyme B monoclonal antibody, mix well, and incubate at 25 °C in the dark for 40 minutes;

[0191] 3) Magnetic separation to remove the supernatant, wash once with 200 μL of magnetic bead washing solution, magnetic separation to remove the supernatant again, add 100 μL of streptavidin-labeled phycoerythrin (SA-PE), mix well, and incubate at 25 °C in the dark for 20 minutes;

[0192] 4) Magnetic separation to remove the supernatant, wash once with 200 μL of magnetic bead washing solution, magnetic separation to remove the supernatant again, resuspend the microspheres with 200 μL of magnetic bead washing solution, and detect by flow cytometry.

[0193] Example 6

[0194] The method for detecting serum granzyme B using the kit prepared in Example 3 includes the following steps:

[0195] 1) Add 50 μL of magnetic microsphere working solution (including magnetic fluorescent encoded microspheres conjugated with granzyme B antibody, PBS buffer (10 mM, pH 7.4), 0.1% bovine serum albumin, 0.1% proclin 300, 0.02% Tween-20, and 50 μg / mL blocker HBR-5) to the reaction tubes respectively. The magnetic microsphere working solution needs to be vortexed for more than 30 s before adding to the reaction tubes, and then add 50 μL of serum samples with different granzyme B concentrations (the concentrations of Granzyme B in the serum samples are: 0 pg / mL, 4.096 pg / mL, 10.24 pg / mL, 25.6 pg / mL, 64 pg / mL, 160 pg / mL, 400 pg / mL, 1000 pg / mL, and 2500 pg / mL), mix well, and incubate at 25 °C in the dark for 40 minutes;

[0196] 2) Magnetic separation to remove the supernatant, and wash once with 200 μL of magnetic bead washing solution; magnetic separation to remove the supernatant, add 100 μL of biotin-labeled anti-human granzyme B monoclonal antibody, mix well, and incubate at 25 °C in the dark for 40 minutes;

[0197] 3) Magnetic separation to remove the supernatant, wash once with 200 μL of magnetic bead washing solution, magnetic separation to remove the supernatant again, add 100 μL of streptavidin-labeled phycoerythrin (SA-PE), mix well, and incubate at 25 °C in the dark for 20 minutes;

[0198] 4) Magnetic separation to remove the supernatant, wash once with 200 μL of magnetic bead washing solution, magnetic separation to remove the supernatant again, resuspend the microspheres with 200 μL of magnetic bead washing solution, and detect by flow cytometry. The results are shown in Table 2.

[0199] Table 2 shows the fluorescence intensity detection results of Example 4, Example 5, and Example 6.

[0200]

[0201]

[0202] It can be analyzed from Table 2 that when the Granzyme B concentration is 4.096 pg / mL, the signal-to-noise ratios in Example 4, Example 5, and Example 6 are 2.15, 2.07, and 2.02 respectively. When the Granzyme B concentration is 2500 pg / mL, the signal-to-noise ratios in Example 4, Example 5, and Example 6 are 507.7, 373.7, and 431.0 respectively. Therefore, it is proved that the signal-to-noise ratio in Example 4 is higher, the background of the curve is lower, and the sensitivity of the kit prepared in Example 4 is higher.

[0203] Example 7

[0204] A method for detecting Granzyme B in serum using the kit prepared in Example 1, comprising the following steps:

[0205] 1) Add 50 μL of magnetic microsphere working solution (including magnetic fluorescent encoded microspheres conjugated with Granzyme B antibody, PBS buffer (10 mM, pH 7.4), 0.1% bovine serum albumin, 0.1% proclin 300, 0.02% Tween-20, and 50 μg / mL blocker HBR-5) to the reaction tube. The magnetic microsphere working solution needs to be vortexed and mixed for more than 30 s before adding it to the reaction tube. Then add 50 μL of serum samples with different Granzyme B concentrations (the Granzyme B concentrations in the serum samples are: 0 pg / mL, 5 pg / mL, 50 pg / mL, 500 pg / mL, and 5000 pg / mL) and three actual serum samples (labeled as Sample 1, Sample 2, and Sample 3) respectively. Mix the samples well and incubate them at 25°C in the dark for 40 minutes.

[0206] 2) Separate the magnetic beads and remove the supernatant, and wash once with 200 μL of magnetic bead washing solution; separate the magnetic beads and remove the supernatant, add 100 μL of biotin-labeled anti-human Granzyme B monoclonal antibody, mix well, and incubate at 25°C in the dark for 40 minutes.

[0207] 3) Separate the magnetic beads and remove the supernatant, wash once with 200 μL of magnetic bead washing solution, separate the magnetic beads again and remove the supernatant, add 100 μL of streptavidin-labeled phycoerythrin (SA-PE), and incubate at 25°C in the dark for 20 minutes.

[0208] 4) Separate the magnetic beads and remove the supernatant, wash once with 200 μL of magnetic bead washing solution, separate the magnetic beads again and remove the supernatant, resuspend the microspheres with 200 μL of magnetic bead washing solution, and detect them by flow cytometer. The results are shown in Table 3 and Table 4.

[0209] Example 8

[0210] A method for detecting granzyme B in serum using the kit prepared in Example 1, comprising the following steps:

[0211] 1) Add 50 μL of magnetic microsphere working solution (including magnetic fluorescent encoded microspheres conjugated with granzyme B antibody, PBS buffer (10 mM, pH 7.4), 0.1% bovine serum albumin, 0.1% proclin 300, 0.02% Tween-20 and 50 μg / mL blocker HBR-5) to the reaction tube. The magnetic microsphere working solution needs to be vortexed and mixed for more than 30 s before adding to the reaction tube. Then add 50 μL of serum samples with different granzyme B concentrations (the concentrations of Granzyme B in the serum samples are 0 pg / mL, 5 pg / mL, 50 pg / mL, 500 pg / mL and 5000 pg / mL) and three actual serum samples (labeled as Sample 1, Sample 2 and Sample 3) respectively. Mix the samples thoroughly and incubate in the dark at 37 °C for 40 minutes;

[0212] 2) Magnetically separate and remove the supernatant, and wash once with 200 μL of magnetic bead washing solution; magnetically separate and remove the supernatant, add 100 μL of biotin-labeled anti-human granzyme B monoclonal antibody, mix thoroughly, and incubate in the dark at 37 °C for 40 minutes;

[0213] 3) Magnetically separate and remove the supernatant, wash once with 200 μL of magnetic bead washing solution, magnetically separate and remove the supernatant again, add 100 μL of streptavidin-labeled phycoerythrin (SA-PE), and incubate in the dark at 37 °C for 20 minutes;

[0214] 4) Magnetically separate and remove the supernatant, wash once with 200 μL of magnetic bead washing solution, magnetically separate and remove the supernatant again, resuspend the microspheres with 200 μL of magnetic bead washing solution, and detect with a flow cytometer. The results are shown in Table 3.

[0215] Table 3 shows the fluorescence intensity detection results in Example 7 and Example 8

[0216]

[0217] Analysis from Table 3 shows that when the concentration of Granzyme B is 5 pg / mL, the signal-to-noise ratios in Example 7 and Example 8 are 1.72 and 1.30 respectively. When the concentration of Granzyme B is 5000 pg / mL, the signal-to-noise ratios in Example 7 and Example 8 are 494.13 and 572.38 respectively. That is to say, when the reaction temperature is 25 °C, the signal-to-noise ratio of the sample with low concentration of Granzyme B is higher, and for the actually selected Samples 1, 2 and 3, the fluorescence intensity is also higher, which proves that when detecting the concentration of Granzyme B in serum samples, setting the reaction temperature at 25 °C results in higher sensitivity.

[0218] Example 9

[0219] A method for detecting granzyme B in serum using the kit prepared in Example 1, comprising the following steps:

[0220] 1) Add 50 μL of magnetic microsphere working solution (including magnetic fluorescent encoded microspheres conjugated with granzyme B antibody, PBS buffer (10 mM, pH 7.4), 0.1% bovine serum albumin, 0.1% proclin 300, 0.02% Tween-20 and 50 μg / mL blocker HBR-5) to the reaction tube. The magnetic microsphere working solution needs to be vortexed and mixed for more than 30 s before adding to the reaction tube. Then add 50 μL of serum samples with different granzyme B concentrations (the concentrations of Granzyme B in the serum samples are: 0 pg / mL, 5 pg / mL, 50 pg / mL, 500 pg / mL and 5000 pg / mL) and three actual serum samples (labeled as Sample 1, Sample 2 and Sample 3 respectively). Mix the samples well and incubate in the dark at 25 °C for 60 minutes;

[0221] 2) Magnetically separate and remove the supernatant, and wash once with 200 μL of magnetic bead washing solution; magnetically separate and remove the supernatant, add 100 μL of biotin-labeled anti-human granzyme B monoclonal antibody, mix well, and incubate in the dark at 25 °C for 60 minutes;

[0222] 3) Magnetically separate and remove the supernatant, wash once with 200 μL of magnetic bead washing solution, magnetically separate and remove the supernatant again, add 100 μL of streptavidin-labeled phycoerythrin (SA-PE), and incubate in the dark at 25 °C for 30 minutes;

[0223] 4) Magnetically separate and remove the supernatant, wash once with 200 μL of magnetic bead washing solution, magnetically separate and remove the supernatant again, resuspend the microspheres with 200 μL of magnetic bead washing solution, and detect with a flow cytometer. The results are shown in Table 4.

[0224] Table 4 shows the detection results in Example 7 and Example 9

[0225]

[0226] As can be seen from the comparative analysis in Table 4, when the concentration of Granzyme B is 5 pg / mL, the signal-to-noise ratios in Example 7 and Example 9 are 1.72 and 2.04 respectively. When the concentration of Granzyme B is 5000 pg / mL, the signal-to-noise ratios in Example 7 and Example 9 are 494.13 and 784.25 respectively. That is to say, when the reaction time is 60 min + 60 min + 30 min, the signal-to-noise ratio is higher, and for the actually selected samples 1, 2, and 3, the fluorescence intensity is also higher, which proves that when detecting the concentration of Granzyme B in serum samples, the sensitivity is higher when the reaction time is set to 60 min + 60 min + 30 min.

[0227] Drawing of the spectrogram of Granzyme B fluorescently encoded microspheres

[0228] Taking APC-Cy7-H as the vertical coordinate and APC-H as the horizontal coordinate to establish a scatter plot, and using a rectangular gate to circle the aggregated particle clusters.

[0229] Drawing of the calibration curve of Granzyme B

[0230] The specific operation steps are as follows:

[0231] Preparation of calibration products

[0232] 1. Prepare 8 0.6 mL centrifuge tubes as calibration product gradient tubes, and label them S0 - S7; the S7 tube is left empty, and 150 μL of calibration product diluent is added to the S0 - S6 tubes.

[0233] 2. Add 0.5 mL of calibration product diluent to the calibration product bottle, fully dissolve it, let it stand at room temperature for 15 min, mix well and transfer 200 μL to the S7 tube to obtain the highest concentration calibration solution;

[0234] 3. Take 100 μL of the calibration product solution in the S7 tube and transfer it to the S6 tube to mix well, which is the 1:2.5 diluted calibration solution; sequentially perform gradient dilution to obtain the calibration solutions in the S6 - S1 tubes; the calibration product diluent in the S0 tube is used as the 0 concentration calibration solution.

[0235] Detection

[0236] 1) Add 50 μL of capture microsphere suspension (vortex and mix well for more than 30 s before adding) and 50 μL of calibration solution (S0 - S7) to the reaction tube, mix well, and incubate at 25°C in the dark for 60 minutes;

[0237] 2) Perform magnetic separation to remove the supernatant, wash once with 200 μL of magnetic bead washing solution; perform magnetic separation to remove the supernatant, add 100 μL of biotin-labeled antibody, mix well, and incubate at 25°C in the dark for 60 minutes;

[0238] 3) Magnetic separation to remove the supernatant, wash once with 200 μL of magnetic bead washing solution, magnetic separate again to remove the supernatant, add 100 μL of SA-PE, mix well and incubate in the dark at 25 °C for 30 minutes;

[0239] 4) Magnetic separation to remove the supernatant, wash once with 200 μL of magnetic bead washing solution, magnetic separate again to remove the supernatant, resuspend the microspheres with 200 μL of magnetic bead washing solution, and detect with a flow cytometer. The results are shown in Table 5.

[0240] Table 5 shows the detection results of the calibration samples

[0241] Sample Number Concentration (pg / mL) Fluorescence Signal (MFI) S0 0 103 S1 10.24 295 S2 25.6 473 S3 64 999 S4 160 2465 S5 400 5605 S6 1000 13128 S7 2500 27673

[0242] According to the data in Table 5, perform linear fitting to obtain the granzyme B calibration curve as Figure 1 shown. The equation of the granzyme B calibration curve is y = 11.11180x + 564.71165, where x represents the concentration of serum granzyme B and y represents the signal value. The R 2 of the granzyme B calibration curve is 0.99465, indicating that the calibration curve has a good fit.

[0243] Determination of the detection limit

[0244] Use the calibration diluent as a sample for detection, repeat the determination 20 times, obtain the MFI values (A values) of the 20 measurement results, as shown in Table 6, calculate their average value (M) and standard deviation (SD), obtain the A value corresponding to M + 2SD, and substitute the A value corresponding to M + 2SD into the above equation according to the calibration curve equation of the standard product used in the kit to obtain the corresponding concentration value, which is the detection limit (LOD) value.

[0245] Table 6 shows the determination results of the MFI values

[0246]

[0247]

[0248] According to the data in Table 6, the calculated LOD is 0.436 pg / mL. The detection linear range is 0.436 pg / mL to 2500 pg / mL. It is wider than the detection linear range of traditional kits for detecting serum granzyme B (i.e., 0.5 pg / mL to 15 pg / mL). The correlation coefficient R 2 is 0.99465.

[0249] In summary, the kit for detecting granzyme B of the present invention is applicable to flow cytometers configured with 2 lasers and 4 channels or more. The flow cytometer greatly saves experimental time and improves the convenience of operation through its high-speed sample collection, simplified sample processing steps, automated and intelligent design, multi-parameter analysis ability, and rapid data processing. The quantitative detection of serum granzyme B can be completed in 3 to 4 hours, making the detection faster, more reliable, and more stable.

[0250] The kit for detecting granzyme B of the present invention is based on the flow cytometry encoded microsphere chip technology, thus having the high sensitivity and high precision of chemiluminescence, and at the same time having a wider detection range and better repeatability. The flow cytometry encoded microsphere chip technology enables the analysis sensitivity of protein quantification to reach the pg / ml level, and the detection range is 0.436 - 2500 pg / ml. The target protein is quantified according to the fluorescence intensity of the antibody, excluding the false positives generated by enzyme-linked amplification in the ELISA reaction. This makes the flow cytometry encoded microsphere chip technology perform excellently in detecting trace biomarkers, especially suitable for early disease screening.

[0251] The kit for detecting granzyme B of the present invention is based on the flow cytometry encoded microsphere chip technology and has the advantage of fewer operation steps. It mainly involves sample addition, incubation, and result reading, simplifies the experimental process, and reduces the result deviation caused by manual operation. ELISA requires multiple washing steps and incubation processes, with relatively complex operations, and each step may affect the accuracy of the final result.

[0252] The magnetic fluorescent encoded microspheres used in the kit for detecting granzyme B of the present invention can be highly purified within a short time through a simple magnetic separation device, providing convenience in operation and avoiding the steps of adjusting complex parameters such as centrifugation speed, time, and temperature during the separation of traditional polystyrene microspheres. Thereby, it reduces the sample loss caused by centrifugation and maintains the integrity of the sample, and has the value of popularization and application in the field of immunoassay technology.

[0253] The above embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention.

Claims

1. A kit for detecting granzyme B, characterized in that: include: Magnetic microsphere working solution, detection antibody and streptavidin-labeled phycoerythrin; The components of the magnetic microsphere working solution include magnetic fluorescent coding microspheres coupled with granzyme B antibodies; The detection antibody is selected from the group consisting of granzyme B detection antibody and biotin.

2. The kit for detecting granzyme B according to claim 1, characterized in that: The granzyme B antibody-coupled magnetic fluorescent coded microspheres are selected from magnetic fluorescent coded microspheres coated with granzyme B capture antibodies on the surface; And / or, the detection antibody is selected from biotin-labeled anti-human granzyme B monoclonal antibody; And / or, the components of the magnetic microsphere working solution further include PBS buffer, stabilizer, preservative, surfactant and blocking agent; And / or, the granzyme B antibody is an IgG type antibody.

3. The kit for detecting granzyme B according to claim 1, characterized in that: The kit also includes a lyophilized calibrator, a calibrator diluent, and a cleaning solution.

4. The kit for detecting granzyme B according to claim 3, characterized in that: The lyophilized calibrator is selected from recombinant human granzyme B protein; And / or, the calibrator diluent is selected from serum; And / or, the cleaning solution is selected from PBS buffer.

5. A method for preparing a kit for detecting granzyme B according to any one of claims 1 to 4, characterized in that: The following steps are involved: The magnetic fluorescent coded microspheres are added to MES buffer, resuspended, and EDC solution and Sulfo-NHS solution are added, and the supernatant is discarded after magnetic separation; then, granzyme B antibody is added, and MES buffer is supplemented, and the reaction is carried out under the first temperature condition, and the supernatant is discarded after magnetic separation; TBS-B buffer is added, and the reaction is blocked under the first temperature condition, and the supernatant is discarded after magnetic separation; PBS-TBN buffer is added and resuspended to obtain granzyme B antibody-coupled magnetic fluorescent coded microspheres; Dissolving biotin in a DMSO solution to obtain a biotin solution, diluting the granzyme B detection antibody with a PBS buffer to obtain a diluted granzyme B detection antibody; mixing the biotin solution and the diluted granzyme B detection antibody to obtain a detection antibody; Granzyme B antibody-coupled magnetic fluorescent coded microspheres, PBS buffer, stabilizer, preservative, surfactant and blocking agent are mixed to obtain a magnetic microsphere working solution, which is then assembled with a detection antibody and streptavidin-labeled phycoerythrin to obtain a kit.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the magnetic fluorescent encoded microspheres to the granzyme B antibody is 1.0 mg: 40 μg; And / or, the molar ratio of the biotin to the granzyme B detection antibody is 50:

1.

7. The preparation method according to claim 5, characterized in that: The EDC solution is EDC + MES buffer, with a concentration of 50 mg / mL and a pH value of 4.75; and / or, the Sulfo-NHS solution is Sulfo-NHS + MES buffer with a concentration of 50 mg / mL and a pH of 4.75; And / or, the concentration of the MES buffer is 50 mM and the pH value is 4.75; And / or, the TBS-B buffer is Tris buffer + 1% BSA, and the pH value of the Tris buffer is 8.2; And / or, the PBS-TBN buffer is PBS buffer + 0.1% BSA + 0.02% Tween-20 + 0.1% Proclin300 + HBR-5, and the pH value of the PBS buffer is 7.4; And / or, the concentration of the PBS buffer is 10 mM and the pH value is 7.

4.

8. The preparation method according to claim 5, characterized in that: The first temperature is 25°C; And / or, the reaction time under the first temperature condition is 3h; And / or, the blocking reaction time under the first temperature condition is 12 to 18 hours; And / or, the temperature of the mixed reaction of the biotin solution and the diluted granzyme B detection antibody is room temperature, and the mixed reaction time is 2 hours.

9. Use of a kit prepared by the preparation method according to any one of claims 5 to 8, characterized in that: The kit is used for detecting serum granzyme B.

10. The use according to claim 9, characterized in that: The kit is combined with flow cytometry to detect serum granzyme B; And / or, the detection range of the kit is 0.436~2500pg / ml.

Images