Kit for joint detection of interleukin 6 and procalcitonin, preparation method and detection method

Abstract

The invention belongs to the technical field of in-vitro diagnosis and rapid detection, and relates to an interleukin 6 and procalcitonin joint detection kit, a preparation method and a detection method, the interleukin 6 and procalcitonin joint detection kit comprises a Ra1 reagent, a Ra2 reagent, an Rb1 reagent, an Rb2 reagent and a substrate solution; the Ra1 reagent is an interleukin 6 antibody magnetic bead labeling solution, and is prepared from a biotin labeled interleukin 6 first antibody solution and a streptavidin magnetic bead solution; the Ra2 reagent is a procalcitonin antibody magnetic bead labeling solution, and is prepared from a biotin labeled procalcitonin first antibody solution and a streptavidin magnetic bead solution; the Rb1 reagent is prepared from an alkaline phosphatase labeled interleukin 6 second antibody solution; the Rb2 reagent is prepared from a second procalcitonin antibody solution labeled by horse radish peroxidase; the substrate solution comprises a first substrate solution and a second substrate solution. The detection sensitivity can be improved, the detection time is shortened, the detection efficiency is improved, and the operation is simple.

Description

Technical Field

[0001] The present application relates to the technical field of rapid in vitro diagnostic testing, and in particular to a kit for the combined detection of interleukin-6 and procalcitonin, and a preparation method and a detection method thereof. Background Art

[0002] Procalcitonin (PCT) is the propeptide of calcitonin, which is secreted by thyroid C cells. Under physiological conditions, the content in the body is trace, while under pathological conditions, the amount of PCT produced in the body increases. PCT is a sensitive indicator for identifying systemic bacterial infection and monitoring local bacterial infection. It has high sensitivity and specificity for distinguishing bacterial infection from other inflammatory response states, and can be used to monitor the occurrence and development of infectious diseases, and reflect the inflammatory activity and the severity of the disease.

[0003] Interleukin-6 (IL-6) is an important mediator produced by the body in the immune response. It can act on a variety of target cells and is a polypeptide cytokine with a variety of complex physiological functions. IL-6 is mainly produced by macrophages, T lymphocytes, and B lymphocytes. It is the earliest marker to increase when inflammation occurs. It has a half-life of 60 minutes and can be used to evaluate the effectiveness of treatment. During acute inflammation, interleukin-6 is rapidly produced, and its elevated level is consistent with the severity of the infection and can be expressed at a high level for a long time in critically ill patients. Interleukin-6 participates in the body's inflammatory response and anti-infection effects, and is one of the most widely used inflammatory indicators.

[0004] The combined detection of IL-6 and PCT can complement each other's strengths and weaknesses, improve the accuracy of diagnosis, and have a higher value for combined diagnosis. The market is huge. With the continuous deepening of medical research, the value of the combined detection of IL-6 and PCT will also bring more and more medical value and benefits to clinicians and patients.

[0005] At present, among the commonly used methods for the joint detection of interleukin-6 and procalcitonin, the colloidal gold method is easy to operate and has a fast detection speed, but it can only perform qualitative or semi-quantitative detection; the chemiluminescence method is mostly a single-component detection, that is, each analysis process only determines the content of one component, and multiple measurements are performed to finally obtain the content of all required components. The analysis time is long, the reagent consumption is high, the analysis throughput is low, and the labor workload is high; compared with the chemiluminescence method, the fluorescence immunochromatography method has lower sensitivity, a narrower linear range, and a larger error.

[0006] Based on the above-mentioned deficiencies, there is an urgent need for a combined detection method for interleukin-6 and procalcitonin that has high detection sensitivity, high detection efficiency and high detection accuracy. Summary of the invention

[0007] The purpose of the embodiments of the present application is to propose a kit for the combined detection of interleukin 6 and procalcitonin, a preparation method and a detection method, so as to solve the technical problems of low detection sensitivity, low detection efficiency and poor detection accuracy in the combined detection method of interleukin 6 and procalcitonin in the prior art.

[0008] In order to solve the above technical problems, the present application embodiment provides a kit for the combined detection of interleukin-6 and procalcitonin, which adopts the following technical solution:

[0009] It includes Ra1 reagent, Ra2 reagent, Rb1 reagent, Rb2 reagent and substrate solution;

[0010] Wherein, the Ra1 reagent is an interleukin-6 antibody magnetic bead labeling solution, which is prepared from a biotin-labeled interleukin-6 first antibody solution and a streptavidin magnetic bead solution;

[0011] The Ra2 reagent is a procalcitonin antibody magnetic bead labeling solution, which is prepared from a biotin-labeled procalcitonin first antibody solution and a streptavidin magnetic bead solution;

[0012] The Rb1 reagent is prepared from an alkaline phosphatase-labeled interleukin-6 secondary antibody solution;

[0013] The Rb2 reagent is prepared from a procalcitonin second antibody solution labeled with horseradish peroxidase;

[0014] The substrate liquid includes a first substrate liquid and a second substrate liquid.

[0015] Furthermore, the components of the first substrate solution include 1,2-dioxetane derivatives; and the components of the second substrate solution include 3-amino-phthalic acid hydrazide.

[0016] Further, the pH value of the streptavidin magnetic bead solution is 7.3-7.5, and the components of the streptavidin magnetic bead solution include streptavidin-modified magnetic beads, Tween 80 and Proclin300;

[0017] The pH value of the biotin-labeled interleukin 6 first antibody solution is 7.3-7.5, and the components of the biotin-labeled interleukin 6 first antibody solution include biotin-labeled interleukin 6 first antibody, PBS buffer, bovine serum albumin, sucrose and Proclin300;

[0018] The pH value of the biotin-labeled procalcitonin first antibody solution is 7.3-7.5, and the components of the biotin-labeled procalcitonin first antibody solution include biotin-labeled procalcitonin first antibody, PBS buffer, bovine serum albumin, sucrose and Proclin300;

[0019] The pH value of the alkaline phosphatase-labeled interleukin 6 second antibody solution is 7.3-7.5, and the components of the alkaline phosphatase-labeled interleukin 6 second antibody solution include alkaline phosphatase-labeled interleukin 6 second antibody, PBS buffer, bovine serum albumin, sucrose and Proclin300;

[0020] The pH value of the horseradish peroxidase-labeled procalcitonin second antibody solution is 7.3-7.5, and the components of the horseradish peroxidase-labeled procalcitonin second antibody solution include horseradish peroxidase-labeled procalcitonin second antibody, PBS buffer, sodium chloride, bovine serum albumin, Proclin300 and ethylenediaminetetraacetic acid.

[0021] Further, the concentration of the streptavidin-modified magnetic beads is 0.28-0.32 mg / mL;

[0022] The concentration of the biotin-labeled interleukin-6 first antibody is 8-12 μg / mL;

[0023] The concentration of the biotin-labeled procalcitonin first antibody is 9-12 μg / mL;

[0024] The concentration of the alkaline phosphatase-labeled interleukin-6 second antibody is 4 to 6 μg / mL;

[0025] The concentration of the horseradish peroxidase-labeled procalcitonin second antibody is 0.8-1.3 μg / mL.

[0026] Furthermore, the kit also includes a cleaning solution, a magnetic bead diluent and a sample diluent;

[0027] The cleaning solution is a PBS buffer containing sodium chloride and a surfactant;

[0028] The magnetic bead diluent is a PBS buffer containing a surfactant;

[0029] The sample diluent is a physiological saline solution containing bovine serum albumin.

[0030] Furthermore, the kit also includes at least one reagent strip having a plurality of reagent wells, and the Ra1 reagent, the Ra2 reagent, the Rb1 reagent, the Rb2 reagent, the first substrate solution and the second substrate solution are respectively contained in different reagent wells.

[0031] In order to solve the above technical problems, the present application also provides a method for preparing a kit for the combined detection of interleukin-6 and procalcitonin, which adopts the following technical solution:

[0032] Prepare streptavidin magnetic bead solution, biotin-labeled interleukin 6 first antibody solution and biotin-labeled procalcitonin first antibody solution, alkaline phosphatase-labeled interleukin 6 second antibody solution and horseradish peroxidase-labeled procalcitonin second antibody solution respectively;

[0033] The streptavidin magnetic bead solution and the biotin-labeled interleukin-6 first antibody solution are mixed to prepare Ra1 reagent;

[0034] Mixing the streptavidin magnetic bead solution and the biotin-labeled procalcitonin first antibody solution to obtain Ra2 reagent;

[0035] The alkaline phosphatase-labeled interleukin-6 second antibody solution is used as Rb1 reagent, and the horseradish peroxidase-labeled procalcitonin second antibody solution is used as Rb1 reagent;

[0036] The Ra1 reagent, the Ra2 reagent, the Rb1 reagent, the Rb2 reagent, the first substrate solution and the second substrate solution are packaged according to the loading amount to obtain a reagent kit.

[0037] Furthermore, the streptavidin magnetic bead solution is prepared by the following preparation steps:

[0038] Dissolving streptavidin-modified magnetic beads, Tween 80 and Proclin 300 in purified water in sequence, dissolving and mixing, to prepare the streptavidin magnetic bead solution; and / or

[0039] The biotin-labeled interleukin-6 first antibody solution is prepared by the following preparation steps:

[0040] Take interleukin 6 primary antibody, add PBS buffer, and obtain interleukin 6 primary antibody solution;

[0041] Prepare activated biotin solution;

[0042] adding the activated biotin solution to the interleukin-6 first antibody solution for reaction, and dialyzing to obtain an interleukin-6 antibody labeled solution;

[0043] Measure a preset first volume of interleukin-6 antibody labeling solution to obtain a biotin-labeled interleukin-6 first antibody solution; and / or

[0044] The biotin-labeled procalcitonin first antibody solution is prepared by the following preparation steps:

[0045] Take the procalcitonin primary antibody, add PBS buffer to obtain the procalcitonin primary antibody solution;

[0046] Adding a pre-prepared activated biotin solution to the procalcitonin first antibody solution to react, and dialyzing to obtain a procalcitonin antibody labeling solution;

[0047] Measuring a preset first volume of the procalcitonin antibody labeling solution to obtain a biotin-labeled procalcitonin first antibody solution; and / or

[0048] The alkaline phosphatase-labeled interleukin-6 second antibody solution is prepared by the following preparation steps:

[0049] Dissolve bovine serum albumin, sucrose, Proclin300 and alkaline phosphatase-labeled interleukin-6 secondary antibody in PBS buffer in sequence and mix well to prepare an alkaline phosphatase-labeled interleukin-6 secondary antibody solution; and / or

[0050] The horseradish peroxidase-labeled procalcitonin second antibody solution is prepared by the following preparation steps:

[0051] Sodium chloride, bovine serum albumin, ethylenediaminetetraacetic acid, Proclin300 and horseradish peroxidase-labeled procalcitonin secondary antibody were sequentially dissolved in PBS buffer and mixed evenly to prepare horseradish peroxidase-labeled procalcitonin secondary antibody solution.

[0052] Furthermore, the alkaline phosphatase-labeled interleukin-6 second antibody is prepared by the following preparation steps:

[0053] Dissolving alkaline phosphatase in hydrochloric acid buffer, and adding a first oxidant to perform an oxidation reaction to obtain an oxidized alkaline phosphatase solution;

[0054] Adding an alkoxide solution and anhydrous ethanol to the oxidized alkaline phosphatase solution, mixing the mixture, and subjecting the mixture to a centrifugal reaction to obtain the oxidized alkaline phosphatase;

[0055] Adding carbonate buffer and glutaraldehyde to the oxidized alkaline phosphatase to mix well, and adding interleukin 6 secondary antibody to react to obtain an interleukin 6 enzyme-labeled antibody solution;

[0056] Adding a reducing agent and anhydrous ethanol to the interleukin-6 enzyme-labeled antibody solution in sequence, and obtaining an alkaline phosphatase-labeled interleukin-6 secondary antibody after centrifugation; and / or

[0057] The horseradish peroxidase-labeled procalcitonin second antibody is prepared by the following preparation steps:

[0058] preparing a horseradish peroxidase solution and a second oxidant solution, and mixing the horseradish peroxidase solution and the oxidant solution to obtain an oxidized horseradish peroxidase solution;

[0059] adding ethylene glycol to the oxidized horseradish peroxidase solution;

[0060] diluting the procalcitonin second antibody with a carbonate buffer, and adding the oxidized horseradish peroxidase solution to react to obtain a reaction solution;

[0061] preparing a reducing agent solution, and adding the reducing agent solution into the reaction solution to react, thereby obtaining a procalcitonin enzyme-labeled antibody solution;

[0062] The procalcitonin enzyme-labeled antibody solution was dialyzed to obtain the procalcitonin secondary antibody labeled with horseradish peroxidase.

[0063] In order to solve the above technical problems, the present application also provides a detection method of a kit for the combined detection of interleukin-6 and procalcitonin, which adopts the following technical solution:

[0064] Collect samples to be tested;

[0065] The sample to be tested reacts with the Ra1 reagent and the Ra2 reagent in the kit respectively to obtain a first antibody-antigen complex and a second antibody-antigen complex;

[0066] reacting the first antibody-antigen complex and the second antibody-antigen complex with the corresponding Rb1 reagent and Rb2 reagent to obtain the first antibody-antigen-alkaline phosphatase labeled antibody complex and the second antibody-antigen-horseradish peroxidase labeled antibody complex;

[0067] The first antibody-antigen-alkaline phosphatase labeled antibody complex and the second antibody-antigen-horseradish peroxidase labeled antibody complex are reacted with the corresponding first substrate solution and the second substrate solution, and the luminescence value is measured to obtain the luminescence value to be measured;

[0068] According to the luminescence value to be measured, the interleukin-6 content and the procalcitonin content of the sample to be measured are obtained according to the standard curve.

[0069] Compared with the prior art, this application has the following beneficial effects:

[0070] The kit for the combined detection of interleukin-6 and procalcitonin provided in the present application can improve the detection sensitivity by coating the biotin-labeled interleukin-6 first antibody and the biotin-labeled procalcitonin first antibody with streptavidin magnetic beads respectively. At the same time, under the action of an external magnetic field, unbound substances can be fully automatically cleaned to greatly improve the detection efficiency; secondly, IL-6 and PCT tests can be performed simultaneously on one test card, and only one sample needs to be added to detect two results at the same time, which has the advantages of simple operation, short detection time and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] In order to more clearly illustrate the scheme in the present application, a brief introduction is given below to the drawings required for use in the description of the embodiments of the present application. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0072] Figure 1 It is a schematic diagram of the structure of the reagent strip of the kit for combined detection of interleukin-6 and procalcitonin provided in the present application. DETAILED DESCRIPTION

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of the present application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application; the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of the present application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0074] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0075] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0076] The present application provides a kit for the combined detection of interleukin-6 and procalcitonin, the kit comprising Ra1 reagent, Ra2 reagent, Rb1 reagent, Rb2 reagent and substrate solution;

[0077] Among them, Ra1 reagent is interleukin 6 antibody magnetic bead labeling liquid, which is prepared by biotin-labeled interleukin 6 first antibody solution and streptavidin magnetic bead solution; Ra2 reagent is procalcitonin antibody magnetic bead labeling liquid, which is prepared by biotin-labeled procalcitonin first antibody solution and streptavidin magnetic bead solution; Rb1 reagent is prepared by alkaline phosphatase-labeled interleukin 6 second antibody solution; Rb2 reagent is prepared by horseradish peroxidase-labeled procalcitonin second antibody solution; the substrate solution includes a first substrate solution and a second substrate solution.

[0078] Among them, in the Ra1 reagent, the biotin-labeled interleukin 6 first antibody in the biotin-labeled interleukin 6 first antibody solution coats the streptavidin magnetic beads in the streptavidin magnetic bead solution to form interleukin 6 antibody-labeled magnetic beads; in the Ra2 reagent, the biotin-labeled procalcitonin first antibody in the biotin-labeled procalcitonin first antibody solution coats the streptavidin magnetic beads in the streptavidin magnetic bead solution to form procalcitonin antibody-labeled magnetic beads.

[0079] The magnetic beads are hydrophilic magnetic beads coupled with streptavidin, which can inhibit the nonspecific adsorption of proteins, nucleic acids, etc. on the surface of the magnetic beads. Therefore, biotin-labeled antibodies can be specifically recovered with high purity, enhancing the antigen-antibody reactivity and improving the detection sensitivity.

[0080] The alkaline phosphatase-labeled interleukin-6 secondary antibody reacts to form a magnetic bead-coated antibody-antigen to be tested-enzyme-labeled antibody complex, and the corresponding substrate solution is added. The alkaline phosphatase causes the luminescent agent in the substrate solution to remove the phosphate group and emit light; similarly, the horseradish peroxidase-labeled procalcitonin secondary antibody and the horseradish peroxidase (HRP)-catalyzed substrate solution emit light, and the luminescence intensity is measured by an instrument to achieve the detection of procalcitonin.

[0081] In this embodiment, the components of the first substrate solution include 1,2-dioxetane derivatives, namely AMPPD; the components of the second substrate solution include 3-amino-phthalhydrazide, namely luminol.

[0082] Specifically, alkaline phosphatase causes AMPPD to remove the phosphate group and emit light; under the catalysis of horseradish peroxidase, luminol reacts with hydrogen peroxide to generate a peroxide. The peroxide is unstable and decomposes immediately to form a luminescent electronic excited intermediate. When the latter returns from the excited state to the ground state, fluorescence is generated.

[0083] The present application forms a biotin-streptavidin detection system by coupling the linked avidin on the surface of magnetic beads and the biotin of the labeled antibody, so that the stability of the combined detection of interleukin 6 and procalcitonin is better.

[0084] In some embodiments, the pH value of the streptavidin magnetic bead solution is 7.3-7.5, and the components of the streptavidin magnetic bead solution include streptavidin-modified magnetic beads, Tween 80 and Proclin300.

[0085] The pH value of the biotin-labeled interleukin 6 first antibody solution is 7.3-7.5, and the components of the biotin-labeled interleukin 6 first antibody solution include biotin-labeled interleukin 6 first antibody, PBS buffer, bovine serum albumin, sucrose and Proclin300.

[0086] The pH value of the biotin-labeled procalcitonin first antibody solution is 7.3-7.5. The components of the biotin-labeled procalcitonin first antibody solution include biotin-labeled procalcitonin first antibody, PBS buffer, bovine serum albumin, sucrose and Proclin300.

[0087] The pH value of the alkaline phosphatase-labeled interleukin 6 second antibody solution is 7.3-7.5, and the components of the alkaline phosphatase-labeled interleukin 6 second antibody solution include alkaline phosphatase-labeled interleukin 6 second antibody, PBS buffer, bovine serum albumin, sucrose and Proclin300.

[0088] The pH value of the horseradish peroxidase-labeled procalcitonin second antibody solution is 7.3-7.5, and the components of the horseradish peroxidase-labeled procalcitonin second antibody solution include horseradish peroxidase-labeled procalcitonin second antibody, PBS buffer, sodium chloride, bovine serum albumin, Proclin 300 and ethylenediaminetetraacetic acid (EDTA).

[0089] The concentration of PBS buffer is 0.01 M, and its components include sodium chloride, potassium chloride, anhydrous disodium hydrogen phosphate and anhydrous potassium dihydrogen phosphate.

[0090] Bovine serum albumin (BSA), sucrose and sodium chloride are used as stabilizers, Tween 80 is used as a surfactant, and Proclin 300 is used as a preservative to improve the stability of the solution and eliminate various external interferences to the measurement results.

[0091] In some embodiments, the concentration of streptavidin-modified magnetic beads is 0.28-0.32 mg / mL;

[0092] The concentration of the biotinylated interleukin-6 primary antibody was 8 to 12 μg / mL;

[0093] The concentration of the biotin-labeled procalcitonin primary antibody was 9 to 12 μg / mL;

[0094] The concentration of the alkaline phosphatase-labeled interleukin-6 secondary antibody was 4 to 6 μg / mL;

[0095] The concentration of the horseradish peroxidase-labeled procalcitonin secondary antibody was 0.8-1.3 μg / mL.

[0096] In some embodiments, the kit for the combined detection of interleukin-6 and procalcitonin further comprises a washing solution, a magnetic bead diluent and a sample diluent.

[0097] The cleaning solution is a PBS buffer solution containing sodium chloride and a surfactant; the magnetic bead diluent is a PBS buffer solution containing a surfactant; and the sample diluent is a physiological saline solution containing bovine serum albumin.

[0098] Surfactants include, but are not limited to, emulsifier OP, SDS (Sodium dodecyl sulfate), Tween20, Tween80, Brij35 (dodecyl polyethylene glycol ether), Brij98 (oleyl alcohol polyoxyethylene ether), Tritonx-100 (polyethylene glycol octylphenyl ether) and Tritonx-405 (polyoxyethylene isooctyl cyclohexyl ether), etc.

[0099] In some embodiments, the kit further comprises at least one reagent strip, see Figure 1 As shown, a plurality of reagent wells are arranged on the reagent strip, and Ra1 reagent, Ra2 reagent, Rb1 reagent, Rb2 reagent, the first substrate solution and the second substrate solution are respectively loaded in different reagent wells.

[0100] Specifically, the reagent strip is provided with holes 1 to 12, wherein the Ra1 reagent is installed in hole 2; the Ra2 reagent is installed in hole 3; the Rb1 reagent is installed in hole 4; the Rb2 reagent is installed in hole 5; the first substrate liquid is installed in hole 6; the second substrate liquid is installed in hole 7; the magnetic bead diluent is installed in hole 8; the sample diluent is installed in hole 9; the cleaning liquid is installed in holes 10-11; hole 1 is a magnetic rod sleeve placement hole for installing the magnetic rod sleeve. After each reagent is installed in the corresponding reagent hole, it is packaged.

[0101] It should be understood that the reagent strip is a universal reagent strip, and the specific holes for packaging the reagents can be set according to actual conditions.

[0102] Based on the above-mentioned kit for combined detection of interleukin-6 and procalcitonin, the present application also provides a method for preparing a kit for combined detection of interleukin-6 and procalcitonin, comprising the following steps:

[0103] Prepare streptavidin magnetic bead solution, biotin-labeled interleukin 6 first antibody solution and biotin-labeled procalcitonin first antibody solution, alkaline phosphatase-labeled interleukin 6 second antibody solution and horseradish peroxidase-labeled procalcitonin second antibody solution respectively;

[0104] The streptavidin magnetic bead solution and the biotin-labeled interleukin-6 first antibody solution were mixed to prepare Ra1 reagent;

[0105] The streptavidin magnetic bead solution and the biotin-labeled procalcitonin first antibody solution were mixed to prepare Ra2 reagent;

[0106] The alkaline phosphatase-labeled interleukin-6 secondary antibody solution was used as the Rb1 reagent, and the horseradish peroxidase-labeled procalcitonin secondary antibody solution was used as the Rb1 reagent;

[0107] The Ra1 reagent, the Ra2 reagent, the Rb1 reagent, the Rb2 reagent, the first substrate solution and the second substrate solution are packaged according to the loading amounts to obtain a test kit.

[0108] Wherein, the streptavidin magnetic bead solution is prepared by the following preparation steps:

[0109] The streptavidin-modified magnetic beads, Tween 80 and Proclin 300 were dissolved in purified water in sequence, and the mixture was dissolved and mixed to prepare a streptavidin magnetic bead solution.

[0110] In some embodiments, the biotin-labeled interleukin-6 first antibody solution is prepared by the following preparation steps:

[0111] Take interleukin 6 primary antibody, add PBS buffer, and obtain interleukin 6 primary antibody solution;

[0112] Prepare activated biotin solution;

[0113] Adding an activated biotin solution to the interleukin-6 first antibody solution for reaction, and dialyzing to obtain an interleukin-6 antibody labeled solution;

[0114] A preset first volume of interleukin 6 antibody labeling solution is measured to obtain a biotin-labeled interleukin 6 first antibody solution.

[0115] For example, take IL-6 antibody (interleukin 6 first antibody) in a centrifuge tube, add 0.01M PBS buffer, mix well, and obtain interleukin 6 first antibody solution; take out 1 tube of activated biotin, add a certain amount of purified water in the tube according to its mass, and obtain activated biotin solution; add activated biotin solution to interleukin 6 first antibody solution, mix well, and rotate at room temperature and avoid light to react; after the reaction is completed, transfer the solution into a cellulose dialysis bag and dialyze with 0.01M PBS buffer at 2°C to 8°C; after the dialysis is completed, obtain interleukin 6 antibody labeled solution, transfer the obtained interleukin 6 antibody labeled solution into a clean centrifuge tube, measure the first preset volume, add glycerol, and obtain biotin labeled IL-6 antibody concentrate, that is, biotin labeled interleukin 6 first antibody solution. Among them, glycerol is used to preserve the solution.

[0116] In some embodiments, the biotin-labeled procalcitonin first antibody solution is prepared by the following preparation steps:

[0117] Take the procalcitonin primary antibody, add PBS buffer to obtain the procalcitonin primary antibody solution;

[0118] Adding a pre-prepared activated biotin solution to the procalcitonin first antibody solution for reaction, dialyzing to obtain a procalcitonin antibody labeling solution;

[0119] A preset first volume of the procalcitonin antibody labeling solution is measured to obtain a biotin-labeled procalcitonin first antibody solution.

[0120] For example, PCT antibody (primary procalcitonin antibody) is placed in a centrifuge tube, 0.01M PBS buffer is added, and the mixture is mixed to obtain a primary procalcitonin antibody solution; 1 tube of packaged activated biotin is taken out, and a certain amount of purified water is added to the tube according to its mass to obtain an activated biotin solution; the activated biotin solution is added to the primary procalcitonin antibody solution, mixed, and reacted in a rotation manner at room temperature and away from light; after the reaction is completed, the solution is transferred into a cellulose dialysis bag, and dialyzed with 0.01M PBS buffer at 2°C to 8°C; after the dialysis is completed, a procalcitonin antibody labeled solution is obtained, and the obtained procalcitonin antibody labeled solution is transferred into a clean centrifuge tube, a first preset volume is measured, and glycerol is added to obtain a biotin-labeled procalcitonin antibody concentrate, i.e., a biotin-labeled procalcitonin first antibody solution.

[0121] In some embodiments, the alkaline phosphatase-labeled interleukin-6 secondary antibody solution is prepared by the following preparation steps:

[0122] Bovine serum albumin, sucrose, Proclin300 and alkaline phosphatase-labeled interleukin-6 secondary antibody were dissolved in PBS buffer in sequence and mixed evenly to prepare an alkaline phosphatase-labeled interleukin-6 secondary antibody solution.

[0123] The concentration of PBS buffer is 0.01M.

[0124] In some embodiments, the horseradish peroxidase-labeled procalcitonin secondary antibody solution is prepared by the following preparation steps:

[0125] Sodium chloride, bovine serum albumin, ethylenediaminetetraacetic acid, Proclin300 and horseradish peroxidase-labeled procalcitonin secondary antibody were sequentially dissolved in PBS buffer and mixed evenly to prepare horseradish peroxidase-labeled procalcitonin secondary antibody solution.

[0126] The concentration of PBS buffer is 0.01M.

[0127] In some embodiments, the alkaline phosphatase-labeled interleukin-6 secondary antibody is prepared by the following preparation steps:

[0128] Dissolving alkaline phosphatase in hydrochloric acid buffer, and adding a first oxidant to perform an oxidation reaction to obtain an oxidized alkaline phosphatase solution;

[0129] Adding an alkoxide solution and anhydrous ethanol to the oxidized alkaline phosphatase solution, mixing the mixture, and subjecting the mixture to a centrifugal reaction to obtain the oxidized alkaline phosphatase;

[0130] Adding carbonate buffer and glutaraldehyde to the oxidized alkaline phosphatase to mix well, and adding interleukin 6 secondary antibody to react to obtain an interleukin 6 enzyme-labeled antibody solution;

[0131] The reducing agent and anhydrous ethanol were sequentially added to the interleukin-6 enzyme-labeled antibody solution, and the alkaline phosphatase-labeled interleukin-6 secondary antibody was obtained after centrifugation.

[0132] For example, 5 mg of alkaline phosphatase is dissolved in 0.5 mL of 0.05 M NaAc solution (hydrochloric acid buffer), an equal volume of sodium periodate (first oxidant) solution is added, and an oxidation reaction is carried out at 4°C for 20 minutes to obtain an oxidized alkaline phosphatase solution. 0.5 mL of ethylene glycol-NaCl solution (alcohol salt solution) is added to the oxidized alkaline phosphatase solution, and then 1.5 mL of ice-cold anhydrous ethanol is added, mixed, and allowed to stand at -20°C for 1 hour, and centrifuged at 4000g for 10 minutes to obtain oxidized alkaline phosphatase. Add 0.5 mL of carbonate buffer with a concentration of 0.05 M and a pH value of 8-10 to dissolve the oxidized alkaline phosphatase obtained by precipitation, and add 0.1 mL of glutaraldehyde, mix well, add 5 mg of the interleukin 6 second antibody to be labeled, react for 2 hours; add 0.4 mg of NaBH4 solution (reducing agent), and place at room temperature for 2 hours; add 0.6 mL of ice-cold anhydrous ethanol, and stand at -20°C for 1 hour, centrifuge at 4000g for 10 minutes, and obtain the alkaline phosphatase-labeled interleukin 6 second antibody. The required alkaline phosphatase-labeled interleukin 6 second antibody is dissolved in PBS buffer or freeze-dried for storage.

[0133] In some embodiments, the horseradish peroxidase-labeled procalcitonin secondary antibody is prepared by the following preparation steps:

[0134] preparing a horseradish peroxidase solution and a second oxidant solution, and mixing the horseradish peroxidase solution and the oxidant solution to obtain an oxidized horseradish peroxidase solution;

[0135] Ethylene glycol was added to the oxidized horseradish peroxidase solution;

[0136] Diluting the procalcitonin secondary antibody with carbonate buffer, and adding the oxidized horseradish peroxidase solution to react to obtain a reaction solution;

[0137] preparing a reducing agent solution, and adding the reducing agent solution into the reaction solution to react, thereby obtaining a procalcitonin enzyme-labeled antibody solution;

[0138] The procalcitonin enzyme-labeled antibody solution was dialyzed to obtain the procalcitonin secondary antibody labeled with horseradish peroxidase.

[0139] For example, 2 mg HRP dry powder was weighed and dissolved in 100 μL ddH2O (double distilled water) to prepare a horseradish peroxidase solution, and then 21 mg NaIO4 (second oxidant) was weighed and dissolved in 1 mL ddH2O to prepare a second oxidant solution. 100 μL NaIO4 solution was slowly mixed with 100 μL HRP solution, and allowed to stand at 4°C for 30 min to obtain an oxidized horseradish peroxidase solution. 2 μL ethylene glycol was slowly added to the oxidized horseradish peroxidase solution, and allowed to stand at room temperature in the dark for 30 min. The high concentration of procalcitonin second antibody was diluted to 2 mg / mL with 0.5 M carbonate buffer (pH 9.6), and the oxidized horseradish peroxidase solution was directly added to the treated antibody solution, and the reaction was carried out at room temperature for 2 h to obtain a reaction solution. Weigh 0.4 mg of NaBH4 (reducing agent) and dissolve it in 20 μL of ddH2O to prepare a reducing agent solution. Add all of the reducing agent solution to the reaction solution in the previous step, let it stand at 4°C for 2 hours, and shake it every 30 minutes. Dialyze it overnight against PBS buffer (pH 7.2) to obtain a horseradish peroxidase-labeled procalcitonin secondary antibody, add 30% to 50% glycerol by volume, mix well, and store at -20°C.

[0140] The present application also provides a detection method of a kit for the combined detection of interleukin-6 and procalcitonin, comprising the following steps:

[0141] Collect samples to be tested;

[0142] The sample to be tested is reacted with the Ra1 reagent and the Ra2 reagent in the kit respectively to obtain a first antibody-antigen complex and a second antibody-antigen complex;

[0143] reacting the first antibody-antigen complex and the second antibody-antigen complex with the corresponding Rb1 reagent and Rb2 reagent to obtain the first antibody-antigen-alkaline phosphatase labeled antibody complex and the second antibody-antigen-horseradish peroxidase labeled antibody complex;

[0144] The first antibody-antigen-alkaline phosphatase labeled antibody complex and the second antibody-antigen-horseradish peroxidase labeled antibody complex are reacted with the corresponding first substrate solution and the second substrate solution, and the luminescence value is measured to obtain the luminescence value to be measured;

[0145] According to the luminescence value to be tested, the interleukin-6 content and procalcitonin content of the sample to be tested are obtained according to the standard curve.

[0146] The sample to be tested is at least one of a human serum sample, a plasma sample, and a whole blood sample. The detection method of this embodiment adopts magnetic particle chemiluminescence method, which has a simple operation process, short detection time and high sensitivity.

[0147] The following is a more specific description of the content of the present application in conjunction with specific embodiments, and further elaboration of the present application, but these embodiments are by no means limiting the present application.

[0148] Example 1

[0149] This embodiment is a method for preparing a kit for the combined detection of interleukin-6 and procalcitonin. The components in the kit are prepared according to the following table.

[0150] Ra1 reagent is prepared from a biotin-labeled interleukin 6 first antibody solution and a streptavidin magnetic bead solution, wherein the components of the streptavidin magnetic bead solution are shown in Table 1, and the interleukin 6 first antibody solution is shown in Table 2.

[0151] Table 1

[0152]

[0153] Table 2

[0154]

[0155] The Ra2 reagent is prepared from a streptavidin magnetic bead solution and a biotin-labeled procalcitonin first antibody solution, wherein the components of the streptavidin magnetic bead solution are shown in Table 1, and the biotin-labeled procalcitonin first antibody solution is shown in Table 3.

[0156] Table 3

[0157]

[0158] The components of the Rb1 reagent are shown in Table 4.

[0159] Table 4

[0160]

[0161] The composition of Rb2 reagent is shown in Table 5.

[0162] Table 5

[0163]

[0164] The composition of the cleaning solution is shown in Table 6.

[0165] Table 6

[0166]

[0167]

[0168] The components of 0.01M PBS buffer are shown in Table 7.

[0169] Table 7

[0170]

[0171] The magnetic bead diluent and sample diluent are conventional magnetic bead diluent and sample diluent.

[0172] Example 2

[0173] This example describes in detail the detection method of the kit for the combined detection of interleukin-6 and procalcitonin provided by the present application.

[0174] Step S1, sample collection

[0175] (1) Whole blood collection: Collect venous blood from the subject using an anticoagulant tube containing lithium heparin, EDTA-K2 or sodium citrate, and shake the collected blood sample for later use;

[0176] (2) Plasma collection: Collect venous blood from the subject using an anticoagulant tube containing lithium heparin, EDTA-K2 or sodium citrate. Plasma should be separated as soon as possible after blood collection to avoid hemolysis.

[0177] (3) Serum collection: Collect venous blood from the subject using a serum tube. The serum should be separated as soon as possible after blood collection to avoid hemolysis.

[0178] Step S2, sample detection

[0179] (1) Select Add a new batch of reagents and scan the master curve card in the kit with a scanner;

[0180] (2) Place the collected sample (sample volume not less than 300 μL) on the instrument sample rack and add the sample number on the instrument;

[0181] (3) After placing the magnetic rod cover in the corresponding hole of the test strip, insert the test strip into the instrument and wait for more than 2 minutes;

[0182] (4) Click the Start Test button on the instrument screen and the instrument will automatically start testing.

[0183] Step S3, calibration curve and calibration

[0184] Calibration tests were performed using the matching kit and calibrators.

[0185] The master calibration curve is obtained by scanning the master curve card in the kit and directly generating it, and the calibration material assignment information is scanned and imported into the system. The fully automatic chemiluminescence immunoassay analyzer obtains the current working calibration curve by two-point calibration through the test results of the calibration material.

[0186] The test kit in this application is suitable for the New Health ECL1600, Daan Gene Hilight-16, and Yingkai Shinei1900 fully automatic chemiluminescence immunoassay analyzers.

[0187] Step S4, result output

[0188] The fully automatic chemiluminescence immunoassay analyzer automatically calculates the concentration of each sample with the aid of a working calibration curve. The results of IL-6 (interleukin 6) are expressed in pg / mL, and the results of PCT (procalcitonin) are expressed in ng / mL.

[0189] Example 3

[0190] This example evaluates the analytical performance of a kit for the combined detection of interleukin-6 and procalcitonin.

[0191] Analytical Performance 1: Detection Limit

[0192] Performance requirements: IL-6 should not be higher than 3.00pg / mL; PCT should not be higher than 0.05ng / mL.

[0193] Test method: Test 5 low-value samples with concentrations close to the detection limit (LOD). Repeat the test for 5 times for each sample and sort the test results by size. When the number of test results that are less than the blank limit (LOB) among the 25 test values ​​is less than or equal to 3, it can be considered that the settings of the blank limit and the detection limit are basically reasonable.

[0194] Test results: As shown in Table 8, the test results of the detection limit of the kit meet the requirements.

[0195] Table 8 Kit detection limit test results

[0196]

[0197] Analytical Performance II: Linearity

[0198] (1) Performance requirements:

[0199] When IL-6 is in the range of [3.00, 5000.00] pg / mL, the linear correlation coefficient (r) should not be less than 0.9900; when PCT is in the range of [0.10, 100.00] ng / mL, the linear correlation coefficient (r) should not be less than 0.9900.

[0200] (2) Test method: Operate according to the reagent instructions on the fully automatic chemiluminescence immunoassay analyzer, repeat the test for 3 times for each concentration of the company's linear reference product, and calculate the linear correlation coefficient r according to the following formula. The result should meet the linear performance requirements.

[0201] Among them, the concentrations of IL-6 linear reference substances are approximately 5000.00pg / mL, 1000.00pg / mL, 100.00pg / mL, 10.00pg / mL, and 3.00pg / mL; the concentrations of PCT linear reference substances are approximately 100.00ng / mL, 50.00ng / mL, 10.00ng / mL, 0.50ng / mL, and 0.10ng / mL. The specific concentrations shall be subject to the results indicated in the company's standardized records.

[0202] B=(MT) / T×100%

[0203] Where:

[0204] B——relative deviation;

[0205] M——the average value of the test results;

[0206] T——theoretical value.

[0207]

[0208] In the formula, r is the linear correlation coefficient;

[0209] X i ——theoretical concentration;

[0210] Y i ——The mean of repeated test results corresponding to the theoretical concentration;

[0211] ——X i The average value of

[0212] ——Y i The average value of

[0213] i——1, 2, 3,…n.

[0214] (3) Test results: As shown in Table 9, the linearity test results of the kit met the requirements.

[0215] Table 9 Kit linearity test results

[0216]

[0217] Analytical performance three: repeatability

[0218] (1) Performance requirements:

[0219] The intra-batch coefficient of variation (CV) of IL-6 should be ≤8%; the intra-batch coefficient of variation (CV) of PCT should be ≤10%.

[0220] (2) Test method: Take three batches of test kits and test the company's reference products with low and high concentrations respectively, repeat 10 times each time, calculate the mean value M and standard deviation SD of the 10 measurement results respectively, and calculate the coefficient of variation (CV) according to the following formula. The results should meet the requirements of repeatability performance.

[0221] Among them, the IL-6 corporate reference concentrations are approximately 10.00pg / mL and 1000.00pg / mL, and the PCT concentrations are approximately 0.50ng / mL and 10.00ng / mL. The specific concentrations shall be based on the results indicated in the company's standardized records.

[0222] CV=SD / M×100%

[0223] Where:

[0224] CV—coefficient of variation;

[0225] SD—standard deviation of the measurement results;

[0226] M—The average value of the measurement results.

[0227] (3) Test results: As shown in Tables 10-11, the repeatability test results of the kit met the requirements.

[0228] Table 10 IL-6 repeatability test

[0229]

[0230] Table 11 PCT repeatability test

[0231]

[0232]

[0233] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A kit for the combined detection of interleukin-6 and procalcitonin, characterized in that: include: Ra1 reagent, Ra2 reagent, Rb1 reagent, Rb2 reagent and substrate solution; Wherein, the Ra1 reagent is an interleukin-6 antibody magnetic bead labeling solution, which is prepared from a biotin-labeled interleukin-6 first antibody solution and a streptavidin magnetic bead solution; The Ra2 reagent is a procalcitonin antibody magnetic bead labeling solution, which is prepared from a biotin-labeled procalcitonin first antibody solution and a streptavidin magnetic bead solution; The Rb1 reagent is prepared from an alkaline phosphatase-labeled interleukin-6 secondary antibody solution; The Rb2 reagent is prepared from a procalcitonin second antibody solution labeled with horseradish peroxidase; The substrate liquid includes a first substrate liquid and a second substrate liquid.

2. The kit for combined detection of interleukin-6 and procalcitonin according to claim 1, characterized in that: The components of the first substrate solution include 1,2-dioxetane derivatives; the components of the second substrate solution include 3-amino-phthalic acid hydrazide.

3. The kit for combined detection of interleukin-6 and procalcitonin according to claim 1, characterized in that: The pH value of the streptavidin magnetic bead solution is 7.3-7.5, and the components of the streptavidin magnetic bead solution include streptavidin-modified magnetic beads, Tween 80 and Proclin 300; The pH value of the biotin-labeled interleukin 6 first antibody solution is 7.3-7.5, and the components of the biotin-labeled interleukin 6 first antibody solution include biotin-labeled interleukin 6 first antibody, PBS buffer, bovine serum albumin, sucrose and Proclin300; The pH value of the biotin-labeled procalcitonin first antibody solution is 7.3-7.5, and the components of the biotin-labeled procalcitonin first antibody solution include biotin-labeled procalcitonin first antibody, PBS buffer, bovine serum albumin, sucrose and Proclin300; The pH value of the alkaline phosphatase-labeled interleukin 6 second antibody solution is 7.3-7.5, and the components of the alkaline phosphatase-labeled interleukin 6 second antibody solution include alkaline phosphatase-labeled interleukin 6 second antibody, PBS buffer, bovine serum albumin, sucrose and Proclin300; The pH value of the horseradish peroxidase-labeled procalcitonin second antibody solution is 7.3-7.5, and the components of the horseradish peroxidase-labeled procalcitonin second antibody solution include horseradish peroxidase-labeled procalcitonin second antibody, PBS buffer, sodium chloride, bovine serum albumin, Proclin300 and ethylenediaminetetraacetic acid.

4. The kit for combined detection of interleukin-6 and procalcitonin according to claim 3, characterized in that: The concentration of the streptavidin-modified magnetic beads is 0.28-0.32 mg / mL; The concentration of the biotin-labeled interleukin-6 first antibody is 8-12 μg / mL; The concentration of the biotin-labeled procalcitonin first antibody is 9-12 μg / mL; The concentration of the alkaline phosphatase-labeled interleukin-6 second antibody is 4 to 6 μg / mL; The concentration of the horseradish peroxidase-labeled procalcitonin second antibody is 0.8-1.3 μg / mL.

5. The kit for combined detection of interleukin-6 and procalcitonin according to claim 1, characterized in that: The kit also includes a cleaning solution, a magnetic bead diluent and a sample diluent; The cleaning solution is a PBS buffer containing sodium chloride and a surfactant; The magnetic bead diluent is a PBS buffer containing a surfactant; The sample diluent is a physiological saline solution containing bovine serum albumin.

6. The kit for combined detection of interleukin-6 and procalcitonin according to any one of claims 1 to 5, characterized in that: The reagent kit further includes at least one reagent strip having a plurality of reagent wells disposed thereon, and the Ra1 reagent, the Ra2 reagent, the Rb1 reagent, the Rb2 reagent, the first substrate solution and the second substrate solution are correspondingly loaded in different reagent wells.

7. A method for preparing a kit for the combined detection of interleukin-6 and procalcitonin, characterized in that: The following steps are involved: Prepare streptavidin magnetic bead solution, biotin-labeled interleukin 6 first antibody solution and biotin-labeled procalcitonin first antibody solution, alkaline phosphatase-labeled interleukin 6 second antibody solution and horseradish peroxidase-labeled procalcitonin second antibody solution respectively; The streptavidin magnetic bead solution and the biotin-labeled interleukin-6 first antibody solution are mixed to prepare Ra1 reagent; The streptavidin magnetic bead solution and the biotin-labeled procalcitonin first antibody solution are mixed to prepare Ra2 reagent; The alkaline phosphatase-labeled interleukin-6 second antibody solution is used as Rb1 reagent, and the horseradish peroxidase-labeled procalcitonin second antibody solution is used as Rb1 reagent; The Ra1 reagent, the Ra2 reagent, the Rb1 reagent, the Rb2 reagent, the first substrate solution and the second substrate solution are packaged according to the loading amount to obtain a reagent kit.

8. The method for preparing a kit for the combined detection of interleukin-6 and procalcitonin according to claim 7, characterized in that: The streptavidin magnetic bead solution is prepared by the following preparation steps: Dissolving streptavidin-modified magnetic beads, Tween 80 and Proclin 300 in purified water in sequence, dissolving and mixing, to prepare the streptavidin magnetic bead solution; and / or The biotin-labeled interleukin-6 first antibody solution is prepared by the following preparation steps: Take interleukin 6 primary antibody, add PBS buffer, and obtain interleukin 6 primary antibody solution; Prepare activated biotin solution; adding the activated biotin solution to the interleukin-6 first antibody solution for reaction, and dialyzing to obtain an interleukin-6 antibody labeled solution; Measure a preset first volume of interleukin-6 antibody labeling solution to obtain a biotin-labeled interleukin-6 first antibody solution; and / or The biotin-labeled procalcitonin first antibody solution is prepared by the following preparation steps: Take the procalcitonin primary antibody, add PBS buffer to obtain the procalcitonin primary antibody solution; Adding a pre-prepared activated biotin solution to the procalcitonin first antibody solution to react, and dialyzing to obtain a procalcitonin antibody labeling solution; Measuring a preset first volume of the procalcitonin antibody labeling solution to obtain a biotin-labeled procalcitonin first antibody solution; and / or The alkaline phosphatase-labeled interleukin-6 second antibody solution is prepared by the following preparation steps: Dissolve bovine serum albumin, sucrose, Proclin300 and alkaline phosphatase-labeled interleukin-6 secondary antibody in PBS buffer in sequence and mix well to prepare an alkaline phosphatase-labeled interleukin-6 secondary antibody solution; and / or The horseradish peroxidase-labeled procalcitonin second antibody solution is prepared by the following preparation steps: Sodium chloride, bovine serum albumin, ethylenediaminetetraacetic acid, Proclin300 and horseradish peroxidase-labeled procalcitonin secondary antibody were sequentially dissolved in PBS buffer and mixed evenly to prepare horseradish peroxidase-labeled procalcitonin secondary antibody solution.

9. The method for preparing a kit for the combined detection of interleukin-6 and procalcitonin according to claim 8, characterized in that: The alkaline phosphatase-labeled interleukin-6 secondary antibody is prepared by the following preparation steps: Dissolving alkaline phosphatase in hydrochloric acid buffer, and adding a first oxidant to perform an oxidation reaction to obtain an oxidized alkaline phosphatase solution; Adding an alkoxide solution and anhydrous ethanol to the oxidized alkaline phosphatase solution, mixing the mixture, and subjecting the mixture to a centrifugal reaction to obtain the oxidized alkaline phosphatase; Adding carbonate buffer and glutaraldehyde to the oxidized alkaline phosphatase to mix well, and adding interleukin 6 secondary antibody to react to obtain an interleukin 6 enzyme-labeled antibody solution; Adding a reducing agent and anhydrous ethanol to the interleukin-6 enzyme-labeled antibody solution in sequence, and obtaining an alkaline phosphatase-labeled interleukin-6 secondary antibody after centrifugation; and / or The horseradish peroxidase-labeled procalcitonin second antibody is prepared by the following preparation steps: preparing a horseradish peroxidase solution and a second oxidant solution, and mixing the horseradish peroxidase solution and the oxidant solution to obtain an oxidized horseradish peroxidase solution; adding ethylene glycol to the oxidized horseradish peroxidase solution; diluting the procalcitonin second antibody with a carbonate buffer, and adding the oxidized horseradish peroxidase solution to react to obtain a reaction solution; preparing a reducing agent solution, and adding the reducing agent solution into the reaction solution to react, thereby obtaining a procalcitonin enzyme-labeled antibody solution; The procalcitonin enzyme-labeled antibody solution was dialyzed to obtain the procalcitonin secondary antibody labeled with horseradish peroxidase.

10. A detection method of a kit for the combined detection of interleukin-6 and procalcitonin, characterized in that: The following steps are involved: Collect samples to be tested; The sample to be tested reacts with the Ra1 reagent and the Ra2 reagent in the kit respectively to obtain a first antibody-antigen complex and a second antibody-antigen complex; reacting the first antibody-antigen complex and the second antibody-antigen complex with the corresponding Rb1 reagent and Rb2 reagent to obtain the first antibody-antigen-alkaline phosphatase labeled antibody complex and the second antibody-antigen-horseradish peroxidase labeled antibody complex; The first antibody-antigen-alkaline phosphatase labeled antibody complex and the second antibody-antigen-horseradish peroxidase labeled antibody complex are reacted with the corresponding first substrate solution and the second substrate solution, and the luminescence value is measured to obtain the luminescence value to be measured; According to the luminescence value to be measured, the interleukin-6 content and the procalcitonin content of the sample to be measured are obtained according to the standard curve.

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