Kit for detecting marker S100 protein as well as preparation method and application of kit

Abstract

The invention relates to a kit for detecting marker S100 protein as well as a preparation method and application of the kit, and relates to the technical field of immunodetection. The kit comprises a reagent A and a reagent B, the reagent A comprises a magnetic bead working solution and a magnetic bead of an S100 monoclonal magnetic diluted microsphere coated antibody; and the reagent B comprises a labeled antibody diluent and an acridinium ester labeled anti-S100 antibody. The kit is more stable during transportation and storage, can be kept for a long time at the temperature of 2-8 DEG C, can be stabilized for 2 years, can be stably stored for 13 days in an aging test at the temperature of 37 DEG C, and can be used after being opened in a liquid form without freeze-drying, and can be kept for a long time at the temperature of 2-8 DEG C, and can be stabilized for 2 years.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassays, and in particular to a kit for detecting marker S100 protein, and a preparation method and application thereof. Background Art

[0002] Craniocerebral injury is a common and frequently occurring condition in neurosurgery, with an incidence second only to limb fractures among all injuries, ranking second in overall body injury. Craniocerebral injury often occurs from traffic accidents, work-related injuries, falls, and direct blows with blunt force. Its incidence is increasing in my country, becoming a public health and socioeconomic issue and a major complication of primary neurosurgery. Craniocerebral injury, especially severe craniocerebral injury, carries a high rate of disability and mortality. Methods for diagnosing and evaluating brain injury primarily include clinical assessment of brain function (e.g., neurologic examination, pupillary response, Glasgow Coma Scale, Glasgow Outcome Scale) and neuroimaging techniques (e.g., CT, MRI, TCD, and PET). Other methods include electrophysiological testing (e.g., brainstem auditory evoked potentials, somatic evoked potentials, motor evoked potentials, and electroencephalography), hemodynamic monitoring (e.g., arterial blood pressure and jugular venous oxygen saturation), standard invasive neurological testing (e.g., intracranial pressure and cerebral perfusion pressure), and advanced invasive neurological testing (e.g., brain tissue oxygen tension and microdialysis). However, these current methods for assisting in the diagnosis of brain injury have varying degrees of lag, making them prone to delays in providing timely treatment and increased risk of trauma. For example, in the early postoperative period after cardiac surgery, patients are often under anesthesia, require ventilator support, or have unstable circulation, making them unsuitable for or unable to cooperate with clinical examinations such as neuropsychiatric examinations, electroencephalograms, CT scans, and MRIs. Therefore, the study of biochemical markers associated with brain injury has become a hot topic in recent years.

[0003] The S100 protein was discovered in bovine brain by Moore BW in 1965. It is primarily distributed in glial cells and Schwann cells of the central and peripheral nervous systems. It is named for its ability to dissolve in 100% ammonium sulfate. Since approximately 96% of S100 protein is primarily found in brain tissue, it is considered a brain-specific protein. Based on amino acid sequence homology and structural similarities, the S100 protein family currently comprises 19 members, including S100A1-A13, CP-10 protein, S100B, S100P, profilaggrin, trychohylin, and repetin. S100αα, S100αβ, and S100ββ dimers are formed by the binding of two α and β subunits. S100 protein is a recently studied marker of brain injury. While typically undetectable in the plasma of healthy adults, it can leak from the cytosol into the cerebrospinal fluid (CSF) and then into the bloodstream through a damaged blood-brain barrier in conditions such as shock, subarachnoid hemorrhage, brain trauma, or cardiopulmonary bypass. Serum S100 protein levels vary with the severity and extent of brain injury. S100 protein levels can be elevated in pathological conditions such as craniocerebral injury, cerebrovascular disease, multiple sclerosis, encephalitis, and neurodegenerative diseases, making it an important neurochemical marker for detecting brain injury.

[0004] As a biochemical marker of neurological injury, S100 protein possesses high specificity, sensitivity, and non-invasive properties. It is heat-resistant and unaffected by heparin and protamine. Similarly, erythrocyte lysis does not affect sample analysis, and its detection is simple. When glial cells are damaged and the blood-brain barrier is disrupted, S100 protein levels are significantly elevated in cerebrospinal fluid and blood. Studies have shown that S100 protein and NES (neuron-specific enolase) levels are significantly higher in patients with traumatic brain injury than in healthy controls, with levels increasing in order of severity for mild, moderate, and severe traumatic brain injury. Therefore, S100 protein measurement is increasingly being used in the clinical diagnosis and prognosis of brain injury, playing a crucial role in grading the severity of post-stroke brain injury, assessing treatment efficacy, and clinical prognosis. Studies have shown that S100 protein and NES levels in the cerebrospinal fluid of children with toxic encephalitis vary with disease progression and duration, confirming that S100 protein and NES levels in the cerebrospinal fluid can be used to diagnose and assess the severity of encephalitis.

[0005] Currently available commercially available detection products, such as colloidal gold immunochromatography, enzyme-linked immunosorbent assay (ELISA), and chemiluminescence, all present challenges. Colloidal gold immunochromatography suffers from significant batch-to-batch variability and low sensitivity. Its physical adsorption method facilitates antigen / antibody detachment from the gold particle surface, and the label is unstable, resulting in only qualitative or semi-quantitative results. ELISA also suffers from complex and time-consuming procedures, requiring specialized personnel. Furthermore, numerous factors influence the operation process, making it prone to false-positive and false-negative results. Chemiluminescence offers high sensitivity, good specificity, and stability, and is non-toxic to the environment and the human body. However, currently marketed products utilize a single sample type and can only detect marker concentrations in plasma samples, a single indicator, resulting in long detection times and poor reproducibility. Therefore, achieving rapid and accurate results for the diagnosis and treatment of Alzheimer's disease and guidance of later-stage treatment remains an urgent challenge in the field of clinical diagnostic product research. Accordingly, the present invention provides a kit for detecting the marker S100 protein, its preparation method, and its application. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a kit for detecting the marker S100 protein, and its preparation method and application. The technical solution of the present invention to solve the above technical problem is as follows:

[0007] In a first aspect, a kit for detecting the marker S100 protein is provided, the kit comprising reagent A and reagent B; the reagent A comprises magnetic bead working solution and magnetic beads coated with S100 monoclonal magnetic rare earth microspheres and antibodies; the reagent B comprises labeled antibody diluent and acridinium ester-labeled anti-S100 antibody.

[0008] On the basis of the above technical solution, the present invention can also be improved as follows.

[0009] Furthermore, each 1L of the magnetic bead working solution includes the following components: HEPES 11.91g, sodium chloride 9.00g, BSA 25.00g, trehalose 100.00g, glucose 80.00g, Tween-20 1mL, Proclin 300 1mL, polyethylene glycol-20000 80.00g, alkyl polyglycoside glycerol ether 7.2g, pH = 7.4;

[0010] Each 1 L of the labeled antibody dilution solution includes the following components: 2.42 g of tris(hydroxymethyl)aminomethane, 1.41 mL of hydrochloric acid, 9.00 g of sodium chloride, 10.00 g of BSA, 0.5 mL of Proclin 300, 4.5 g of dextran, 7.4 g of isodecyl citrate monoester, and 0.01 g of amaranth.

[0011] Furthermore, the mass ratio of the S100 monoclonal magnetic rare earth microspheres coated antibodies to the magnetic beads in the S100 monoclonal magnetic rare earth microspheres coated antibodies is 100:3-8.

[0012] The ratio of acridinium ester to anti-S100 labeled antibody in the acridinium ester labeled anti-S100 antibody can be any ratio as long as it meets the requirements.

[0013] Furthermore, the kit also includes a calibrator, a quality control product and a chemiluminescent substrate.

[0014] In a second aspect, a method for preparing a kit for detecting the marker S100 protein comprises the following steps:

[0015] Magnetic bead coating: activating magnetic beads in a magnetic bead coating buffer using an activator to obtain activated magnetic beads; adding S100 monoclonal magnetic rare earth microspheres coated antibodies to the activated magnetic beads for coupling reaction to obtain S100 monoclonal magnetic rare earth microspheres coated antibodies magnetic beads; storing the S100 monoclonal magnetic rare earth microspheres coated antibodies magnetic beads in a magnetic bead working solution to obtain reagent A;

[0016] Acridinium ester labeling: An anti-S100 labeled antibody is subjected to a labeling reaction with acridinium in a luminophore labeling buffer to obtain an antibody labeling mixture; the antibody labeling mixture is ultrafiltered to obtain an acridinium ester-labeled anti-S100 antibody, and the acridinium ester-labeled anti-S100 antibody is stored in a labeled antibody diluent to obtain reagent B.

[0017] Furthermore, each 1 L of the magnetic bead coating buffer comprises the following components: MES 9.762 g, Triton-100 0.1 mL, alkyl polyglycoside sulfosuccinate disodium salt 5.00 g;

[0018] Each 1 L of the luminophore labeling buffer comprises the following components: 13.435 g of disodium hydrogen phosphate dodecahydrate, 0.8265 g of sodium dihydrogen phosphate dihydrate, 2.3 g of α-sulfo fatty acid methyl ester (mono) sodium salt, and 0.6 g of fatty alcohol sulfate (ester) salt.

[0019] Furthermore, the activator is EDC; the mass ratio of the activator to the magnetic beads is 10:0.5-2.

[0020] The dosage ratio of the S100 monoclonal magnetic rare earth microspheres coated with antibodies to the magnetic bead working solution only needs to meet the requirements.

[0021] Furthermore, the activation parameters are: room temperature, 20 to 40 minutes;

[0022] The coupling reaction parameters are: room temperature, 3 to 8 hours.

[0023] Furthermore, the parameters of the labeling reaction are: 37°C, 20-40 min;

[0024] The ultrafiltration parameters are: 14000xg, 5-15 min.

[0025] The dosage ratio of the acridinium ester-labeled anti-S100 antibody to the labeled antibody diluent only needs to meet the requirements.

[0026] In a third aspect, a kit for detecting the marker S100 protein is used to prepare a product for detecting the brain injury marker S100 protein.

[0027] Description of each component in magnetic bead working solution, labeled antibody diluent, magnetic bead coating buffer, and luminescent labeling buffer:

[0028] MES stands for sodium methyl ester sulfonate, a green and environmentally friendly surfactant synthesized from oil extracted from palm pulp.

[0029] Triton X-100 is a nonionic surfactant that is often used in biochemical applications to solubilize proteins;

[0030] Disodium salt of alkyl polyglycoside sulfosuccinate is an anionic surfactant; Disodium salt of alkyl polyglycoside sulfosuccinate is a derivative of alkyl polyglycoside, which is a new type of anionic surfactant;

[0031] HEPES (4-hydroxyethylpiperazineethanesulfonic acid) is used as a major buffer in biochemical research;

[0032] BSA is the abbreviation of bovine serum albumin;

[0033] Trehalose can form a protective film on the cell surface, effectively protecting the stability of the biological molecular structure and preventing it from being destroyed by other substances. Trehalose has a certain inhibitory effect on surface bacteria.

[0034] Tween-20 nonionic surfactant;

[0035] Proclin300; a highly effective water-soluble antibacterial agent / biopreservative;

[0036] Polyethylene glycol 20000 nonionic surfactant;

[0037] Alkyl polyglycoside glycerol ether is a new type of nonionic surfactant;

[0038] α-sulfo fatty acid methyl ester (mono) sodium salt is a surfactant, abbreviated as MES. It is a high-efficiency surfactant made from natural animal and plant oils and fats. It has good wetting, emulsifying, softening and hard water resistance properties, good solubility, high biodegradability, low skin irritation, and strong foaming power.

[0039] Fatty alcohol sulfate (ester) salt is one of the main ingredients in shampoo formula, including sodium salt, potassium salt, ammonium salt, monoethanolamine salt, diethanolamine salt and triethanolamine salt;

[0040] Tris(hydroxymethylaminomethane) is an organic compound with the chemical formula C4H 11 NO3 is a white crystalline powder at room temperature, easily soluble in water. It is a commonly used zwitterionic buffer. It is chemically stable at a pH of 7.4, can maintain the stability of biological molecules, and can effectively prevent deterioration and inactivation.

[0041] Isodecyl citrate monoester is a nonionic surfactant with excellent detergency, emulsification, dispersing, solubilizing, wetting and penetrating properties.

[0042] Amaranth, also known as Acid Red 27, Cockscomb Red, Blue Light Acid Red, p-Sulfonaphthalene Azo-R-Salt, and Edible Red No. 2, is a water-soluble azo dye. It is a reddish-brown or dark reddish-brown powder particle at room temperature. It is odorless, light-resistant, heat-resistant, and acid-resistant, and changes color when exposed to alkali.

[0043] The present invention utilizes a chemiluminescent immunosandwich assay to detect the concentration of S100 protein. Highly specific anti-S100 polyclonal antibodies are coupled to magnetic microspheres, and the highly specific anti-S100 antibodies are labeled with acridinium esters. A sample, magnetic beads coated with S100 monoclonal magnetic microspheres, and the acridinium ester-labeled anti-S100 antibody are added to reaction tubes. After incubation, the S100 protein in the sample binds to the magnetic beads coated with S100 monoclonal magnetic microspheres and simultaneously binds to the acridinium ester-labeled anti-S100 antibody to form an antibody-antigen-antibody sandwich complex. After the reaction is complete, the magnetic beads are attracted by a magnetic field, and unbound substances are washed away. Chemiluminescent substrates (pre-excitation solution / chemiluminescent substrate solution A, excitation solution / chemiluminescent substrate solution B) are added to reaction tubes to generate chemiluminescence. The number of photons produced by the reaction is measured by a photomultiplier tube, and the number of photons produced is proportional to the analyte concentration in the sample. The amount of analyte in the sample is determined by the calibration curve, and the instrument automatically calculates the S100 value based on the concentration in the detected sample.

[0044] The present invention has the following beneficial effects: the present invention adopts a highly specific anti-S100 antibody, can simultaneously detect the S100 value in human serum, plasma or urine samples with a single sampling; and adopts independently developed acridinium ester labeling buffer and magnetic bead coating buffer. At the same time, surfactants and antibody stabilizers are added to the acridinium ester and magnetic bead working solutions. The results are available in 8 minutes, the repeatability is within 5%, and the sensitivity reaches 1.0 pg / mL. At the same time, the kit of the present invention is more stable during transportation and storage. It can be kept stable for 2 years at 2-8°C for a long time and can be kept stable for 13 days after aging test at 37°C. The calibrators and quality control products matched with the reagents do not need to be lyophilized and are ready for use in liquid form. They can be kept stable for 2 years at 2-8°C for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a curve diagram of the S100 protein calibrator of the present invention;

[0046] Figure 2 This is the S100 linear correlation diagram of the present invention. DETAILED DESCRIPTION

[0047] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through regular channels.

[0048] Example

[0049] 1. Materials and methods

[0050] 1.1 Reagents and Materials

[0051] (1) Antigen and Antibody:

[0052] S100 monoclonal magnetic microsphere-coated antibody and anti-S100 labeled antibody were purchased from Beijing Baixinyi Biotechnology Co., Ltd. at a concentration of 10 mg / mL; S100 antigen was purchased from Beijing Aibosheng Biotechnology Co., Ltd. at a concentration of 5 mg / mL.

[0053] (2) Other reagents:

[0054] Acridinium ester (AE) was purchased from Helison (Xiamen) Biotechnology Co., Ltd.; carboxyl magnetic beads were purchased from Nanjing Ruibeixi Biotechnology Co., Ltd. at a concentration of 10 mg / mL; pre-stimulation solution / stimulation solution were purchased from Guangzhou Hongrunkang Technology Development Co., Ltd.; disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, HEPES (4-hydroxyethylpiperazineethanesulfonic acid), glucose, sodium chloride, sucrose, 3-morpholinepropanesulfonic acid (MOPS), N-tris(hydroxymethyl)methylglycine (Tricine), dextran, casein sodium, hydrazine yellow, fruit green, lactose, Tween-20, Triton-100, glycerol, tris(hydroxymethyl)aminomethane hydrochloride (Tris), 2-(N-morpholine)ethanesulfonic acid (MES), ethylene glycol, hydrochloric acid Conventional chemical reagents such as sodium periodate and sodium borohydride were purchased from Sinopharm Chemical Reagent Co., Ltd.; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), bovine serum albumin (BSA), Proclin-300, and polyethylene glycol 6000 were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; amaranth (standard solution) was purchased from Tianjin Guangfu Fine Chemical Research Institute; sorbitan polyoxyethylene ether tetraoleate, methyl glucoside sesquistearate (CAS: 68936-95-8), diethylene glycol monolaurate (CAS: 141-20-8), and polyoxypropylene stearate (CAS: 25190-52-7) were purchased from Shanghai Kao Co., Ltd. Polypropylene fumarate used the compound from the article “Wu Yongchao, Zheng Qixin, Guo Xiaodong, et al. Synthesis, cross-linking, biomechanics and in vitro degradation detection of polypropylene fumarate [J]. Bio-Orthopedic Materials and Clinical Research, 2003.”; isodecyl citrate monoester and lauryl citrate monoester used the compound from the article “Huang Di et al. Study on solution properties of new surfactant citric acid monoester and its application in gel oil. Modern Food Science and Technology 34.5(2018):10.”; surfactant Coltide HSi used the compound from the article “Li Linping. Application of a new surfactant in laundry detergent. China Detergent Industry 8(2014):3.”

[0055] (3) Instruments:

[0056] SMART 500S fully automatic chemiluminescence immunoassay analyzer produced by Chongqing Cosmay Biotechnology Co., Ltd. (Yu Medical Device Registration No. 20192220077).

[0057] 1.2 Experimental method

[0058] 1.2.1 Reagent preparation:

[0059] (1) Magnetic bead coating buffer:

[0060] Add 9.762 g of MES, 0.1 mL of Triton-100, and 5.00 g of disodium alkyl polyglycoside sulfosuccinate, and dilute to 1 L with purified water.

[0061] (2) Magnetic bead working solution:

[0062] HEPES 11.91 g, sodium chloride 9.00 g, BSA 25.00 g, trehalose 100.00 g, glucose 80.00 g, Tween-20 1 mL, Proclin 300 1 mL, polyethylene glycol 20000 80.00 g; alkyl polyglycoside glycerol ether 7.2 g, adjust the pH to 7.4, and dilute to 1 L with purified water.

[0063] (3) Luminescent labeling buffer:

[0064] 13.435 g of disodium hydrogen phosphate dodecahydrate, 0.8265 g of sodium dihydrogen phosphate dihydrate, 2.3 g of α-sulfo fatty acid methyl ester (mono) sodium salt, 0.6 g of fatty alcohol sulfate (ester), and dilute to 1 L with purified water.

[0065] (4) Luminescent blocking buffer:

[0066] 12.1 g of tris(hydroxymethyl)aminomethane, 9.0 g of sodium chloride, 10.0 g of lysine, 100 g of BSA, 1.5 g of lauryl citrate, 5.0 g of sorbitan polyoxyethylene ether tetraoleate, and dilute to 1 L with purified water.

[0067] (5) Acridinium ester labeled antibody dilution solution:

[0068] 2.42 g of tris(hydroxymethyl)aminomethane, 1.41 mL of hydrochloric acid, 9.00 g of sodium chloride, 10.00 g of BSA, 0.5 mL of Proclin 300, 4.5 g of dextran, 7.4 g of isodecyl citrate, 0.01 g of amaranth, and dilute to 1 L with purified water.

[0069] (6) Calibrator diluent:

[0070] 8.19 g of 3-morpholinepropanesulfonic acid (MOPS), 9.00 g of sodium chloride, 26.00 g of sorbitol, 5.00 g of sodium caseinate, 5.0 g of glycine, 40 mL of β-mercaptoethanol, 1.45 g of magnesium chloride, 0.58 g of zinc chloride, 200 mL of glycerol, 5.00 g of hydrazine yellow, 5.00 g of fruit green, 1 mL of Proclin 300, and dilute to 1 L with purified water.

[0071] (7) Quality control diluent:

[0072] 8.19 g of 3-morpholinepropanesulfonic acid (MOPS), 9.00 g of sodium chloride, 26.00 g of sorbitol, 5.00 g of sodium caseinate, 5.0 g of glycine, 40 mL of β-mercaptoethanol, 1.45 g of magnesium chloride, 0.58 g of zinc chloride, 200 mL of glycerol, 5.00 g of hydrazine yellow, 5.00 g of fruit green, 1 mL of Proclin 300, and dilute to 1 L with purified water.

[0073] (8) Working cleaning fluid:

[0074] 9.76 g of N-tris(hydroxymethyl)methylglycine (Tricine), 0.70 mL of hydrochloric acid, 9.00 g of sodium chloride, 5.0 g of monolauryl phosphate MAP, 2.5 mL of Triton 100, 40 g of surfactant Coltide HSi and 1 mL of Proclin 300 were added, and the volume was adjusted to 1 L with purified water.

[0075] 1.2.2 Magnetic bead coating:

[0076] The preparation of S100 monoclonal magnetic microspheres coated with antibodies includes the following steps:

[0077] (1) Magnetic bead washing: Place 10 mL of magnetic beads on a magnetic rack. After all the magnetic beads are adsorbed, discard the supernatant. Add 20 mL of magnetic bead coating buffer and mix at 30 rpm / min at room temperature for 10 min. Place the magnetic beads on a magnetic rack. After all the magnetic beads are adsorbed, discard the supernatant. Repeat this washing process twice and discard the supernatant.

[0078] (2) Activation of magnetic beads: Weigh 10.00 mg of EDC and dissolve it in 1 mL of magnetic bead coating buffer. Once fully dissolved, add it to the magnetic beads. Simultaneously add 49.00 mL of magnetic bead coating buffer and mix at 30 rpm / min at room temperature for 30 min. Then, repeat step 1.2.2.1 to wash the magnetic beads and discard the supernatant.

[0079] (3) Magnetic Bead Coating: Add 5 mg of S100 magnetic microsphere coating antibody to the magnetic beads, then add 50 mL of magnetic bead coating buffer and mix at 30 rpm / min at room temperature for 5 h. Then, repeat step 1.2.2.1 to wash the magnetic beads and discard the supernatant.

[0080] (4) Magnetic bead storage: Add 500 mL of magnetic bead working solution to the magnetic beads at 30 rpm / min, mix at room temperature for 30 min, and store at 2-8°C.

[0081] 1.2.3 Preparation of Luminescent Markers: (Taking S100 Luminescent Marker Working Solution as an example)

[0082] (1) Antibody labeling:

[0083] (1-1) Take 1 μL of acridinium ester mother solution (5 mM) and add 9 μL of DMF (N,N-dimethylformamide) to prepare the acridinium ester working solution (acridinium ester mother solution: anhydrous dimethylformamide = 1:9).

[0084] (1-2) Add 50 μg of anti-S100 labeled antibody to a centrifuge tube, add luminescent labeling buffer to 300 μL (the volume here increases with the amount of antibody), add 30 μL of acridine working solution, and shake in a 37°C incubator for 30 minutes;

[0085] (1-3) Add 100 μL of luminescent blocking solution and shake in a 37°C incubator for 30 minutes;

[0086] (2) Ultrafiltration of antibody after labeling:

[0087] (2-1) Rinse: Pipette 500 μL of luminescent labeling buffer into the sample reservoir, close the lid, and centrifuge at 10,000 x g for 10 minutes. Aspirate the liquid from the filtrate receiver. Repeat the rinse cycle.

[0088] (2-2) Ultrafiltration: Add labeled antibodies to the sample pool, close the lid, and centrifuge at 14,000 x g for 10 minutes to concentrate the labeled antibodies in the sample pool;

[0089] (2-3) Washing: Add 200 μL of acridinium ester labeled antibody dilution solution to the sample pool, centrifuge at 14,000 × g for 20 minutes, and concentrate the labeled antibody in the sample pool after washing;

[0090] (2-4) Collection: Add 100 μL of acridinium ester-labeled antibody diluent to the sample pool, pipette and reconstitute (pipe and reconstitute more than 30 times to avoid bubbles), transfer to a 1.5 ml centrifuge tube, add acridinium ester-labeled antibody diluent to 1 ml, and store at -20°C; the concentration of the luminescent marker storage solution is 0.05 mg / ml.

[0091] 1.2.4 Preparation of luminescent marker working solution:

[0092] Take 5 mL of the luminescent marker stock solution stored at -20°C in the dark, add 495 mL of acridinium ester labeled antibody diluent, mix at 30 rpm / min, 2-8°C in the dark for 30 minutes, and then store at 2-8°C in the dark.

[0093] 1.2.5 Preparation of calibrators:

[0094] Add 0.5 μL of S100 antigen to 999 μL of Calibrator Diluent and mix thoroughly at room temperature to prepare a 2.5 μg / mL stock solution. Then, use Calibrator Diluent to serially dilute the stock solution to six concentration points: 1500 ng / mL, 750 ng / mL, 250 ng / mL, 50 ng / mL, 25 ng / mL, and 0 ng / mL. Store at 2-8°C.

[0095] 1.2.6 Preparation of quality control products:

[0096] Add 0.5 μL of S100 antigen to 999 μL of Calibrator Diluent and mix at room temperature to prepare a 2.5 μg / mL stock solution. Then, dilute the stock solution to 200 ng / mL for Control Level 1 and 600 ng / mL for Control Level 2 using Control Diluent. Store at 2-8°C.

[0097] 1.2.7 Detection method:

[0098] (1) Testing procedures:

[0099] When loading the reagent bottle onto the machine for the first time, it should be gently turned over 30 times before opening, and the reagent bottle should be observed to ensure that the magnetic beads are completely suspended. If the magnetic beads are still attached to the bottom of the reagent bottle, continue to turn the reagent bottle until they are completely suspended. If they still cannot be suspended, the bottle of reagent cannot be used.

[0100] The system requires a sample volume of 10 μL per measurement. Before applying for a test, you should prepare all the materials required for the test and carefully read the chemiluminescence immunoassay user manual to obtain information on system operating procedures, sample management, usage precautions, maintenance, and other information. The main steps for applying for a test are as follows:

[0101] ① Enter the sample application interface and enter the sample number, sample rack number, location number, sample type, remarks and other information according to the test requirements;

[0102] ② Select the test item, select the sample type in the item options, enter the number of repetitions and other information;

[0103] ③ After preparing the test sample and placing it correctly, click the start button to start the test. The chemiluminescence immunoassay analyzer will perform the following operations in sequence:

[0104] A. The sample handling system transfers the sample rack to the sample aspiration position;

[0105] B. The cuvette loading and scheduling system loads the cuvette from the loading area to the sample loading position;

[0106] C. The sample injection system completes the sample injection and cleaning of the sample needle;

[0107] D. The reagent processing system provides the reagents required for the test, mixes each reagent, and sends it to the reagent aspiration position for aspiration;

[0108] The sample injection system completes the reagent injection and cleaning of the reagent needle;

[0109] E. Mix the reaction mixture with the counter-liquid mixing system and incubate in the reaction plate;

[0110] F. After incubation is complete, the magnetic separation system uses a cleaning solution to clean and separate the reaction mixture;

[0111] G. Substrate system: Inject preheated substrate solution into the reaction cup after magnetic separation, mix, incubate, and wait for light detection;

[0112] H. The light measurement reaction system sends each reaction cup to the signal collection position to collect signals for calculating the luminescence value;

[0113] I. Calculate the amount of analyte in the sample.

[0114] (2) Calibration:

[0115] Perform a calibration test using the included kit and calibrator. Before starting the kit calibration, scan the master curve in the kit's 2D barcode and import it into the system.

[0116] The chemiluminescence immunoassay analyzer uses the results of calibrator tests to adjust the master curve and generate the calibration curve for the current system. The instrument operating software includes a calibration check function that automatically verifies the validity of the calibration curve. If calibration fails, consider the instrument settings, status, location of the calibrators (place low-value calibrators first, then high-value calibrators), and expiration dates.

[0117] A valid calibration curve is required before all tests. Recalibration is required in the following situations:

[0118] ① Use a new batch of test kit;

[0119] ② The same batch of test kits has been used on the analyzer for more than 30 days;

[0120] ③After instrument maintenance;

[0121] ④The quality control value exceeds the specified range;

[0122] (3) Quality Control:

[0123] To ensure the reliability of test results, it is recommended that both high and low level quality control products be tested once every 24 hours. In addition, quality control testing is also recommended after each calibration test, reagent batch change, maintenance and troubleshooting.

[0124] The test values of quality control samples should be within the specified range. If they are outside the specified range, the user should check the detection system, such as the location, expiration date, storage method, calibration process, instrument performance and status, and recalibrate if necessary. If the results are still outside the range after retesting, please contact customer service.

[0125] (4) Calculation:

[0126] Using stored calibration data, the system software automatically determines the sample test results using a weighted four-parameter logarithmic curve (4PLC, Y-weighted) mathematical method. Results are given in ng / mL.

[0127] 2. Experimental results.

[0128] 2.1 Establishment of calibration curve:

[0129] The calibration curves are shown in Table 1 and Figure 1 .

[0130] Table 1 Calibration results of reagent S100 of the present invention

[0131]

[0132] 2.2 Minimum detection limit:

[0133] Using PBS buffer as a blank sample, the detection and comparison reagents prepared in the present invention were simultaneously measured 20 times. The test values of the test strips prepared in the present invention plus twice the standard deviation were substituted into the fitted curve for regression. The results showed that the minimum detection limit of the S100 test kit prepared in the present invention was 0.019 ng / mL, demonstrating the high sensitivity of the test strip preparation method prepared in the present invention. The results are shown in Table 2.

[0134] Table 2 Results of minimum detection limits of reagents

[0135]

[0136] 2.3 Repeatability:

[0137] The test kit of the present invention was tested repeatedly for two levels of quality control 10 times, and the CV (coefficient of dispersion) was calculated. The results showed that the CV of the first batch of the quality control level of the S100 reagent of the present invention was 3.74%, and the CV of the second batch of the quality control level was 2.76%. It can be seen that the preparation method of the test strips prepared by the present invention has good repeatability. The results are shown in Table 3.

[0138] Table 3 S100 repeatability results

[0139]

[0140]

[0141] 2.4 Linearity:

[0142] A high-value sample containing 30 ng / mL of S100 was diluted with PBS buffer, and 6 samples (xi) with different gradients were diluted 2-fold to test the test strips prepared by the present invention. The sample of each dilution concentration was tested 3 times, and the mean value (yi) of the measurement results was calculated respectively. The linear regression equation and linear regression coefficient were calculated with the dilution concentration (xi) as the independent variable and the mean value (yi) of the measurement result as the dependent variable; the results showed that the reagent of the present invention, S100 was in the range of [0.019 ~ 30] ng / mL, and the linear correlation coefficient (r) was not less than 0.9990. It can be seen that the preparation method of the test strips prepared by the present invention has good linearity. The results are shown in Tables 4 and Figure 2 .

[0143] Table 4 Linearity test results of the invention reagent S100

[0144]

[0145] 2.5 Accuracy:

[0146] A high-concentration reference substance (Solution A) was added to a low-concentration reference substance (Solution B), with the volume ratio between the high-concentration reference substance (Solution A) and the low-concentration reference substance (Solution B) being no greater than 1:9. Each sample was measured three times and the average value was obtained. The results calculated according to formula (1) indicate that the S100 accuracy of the detection method prepared in the present invention is good. The results are shown in Table 5.

[0147]

[0148] Where: R is the recovery rate; V is the volume of solution A added; V0 is the volume of serum sample B; c is the detection concentration of serum sample after adding solution A; c0 is the detection concentration of serum sample B; c s is the concentration of solution A.

[0149] Table 5 Accuracy test results of the invention reagent S100

[0150]

[0151] 2.6 Stability:

[0152] 2.6.1 Long-term stability at 2-8°C:

[0153] (1) Three batches of the high-sensitivity chemiluminescent immunoassay kit for the brain injury marker S100 of the present invention were stored in a finished product warehouse at 2-8°C in a dry environment. The temperature of the finished product warehouse was monitored using a thermometer and required to be between 2-8°C to ensure compliance with the product storage conditions during the stability study.

[0154] (2) Three batches of the reagent of the present invention stored in a finished product warehouse at 2-8°C were randomly sampled for testing at month 0, month 6, month 12, month 18, month 24, and month 25. The test items were: appearance, detection limit, accuracy, linearity, repeatability, inter-batch variation, accuracy of calibrator value assignment, uniformity of calibrator, validity of target value of quality control product, and uniformity of quality control product.

[0155] (3) Judgment of test content and results

[0156] ①Appearance

[0157] A. All components of the test kit should be complete and intact, with no leakage of liquid; the Chinese packaging label should be clear and not worn;

[0158] B. The magnetic bead reagent component is a liquid containing brown solid particles. The magnetic beads are not agglomerated, and the liquid is free of flocs and foreign matter.

[0159] C. Other reagent components are clear and uniform liquids without precipitation or flocs.

[0160] ② Accuracy: The recovery rate should be within the range of 85% to 115%;

[0161] ③ Detection limit: no more than 0.019 ng / mL;

[0162] ④ Linearity: The detection range of the kit is S100 [0.019-30] ng / mL, and the correlation coefficient R of the linear interval should be no less than 0.9900;

[0163] ⑤ Repeatability: coefficient of variation CV is not greater than 5%;

[0164] ⑥ Inter-batch difference: inter-batch coefficient of variation CV is not greater than 8%;

[0165] ⑦ Accuracy of calibrator value assignment: The relative deviation of the results of the calibration sample detected by the chemiluminescence immunoassay analyzer calibrated with the calibrator is ±8%;

[0166] ⑧ Homogeneity of calibration material: homogeneity ≤ 8%

[0167] ⑨ Target value validity of quality control products: When quality control products are tested using a chemiluminescence immunoassay analyzer calibrated with the calibrator, the results should be within the target value range;

[0168] ⑩ Homogeneity of quality control products: Homogeneity ≤ 8%

[0169] (4) Three batches of the kits of the present invention were stored at 2-8°C for 25 months, and all performance characteristics met the requirements. This indicates that the kits are stable when stored at 2-8°C for 25 months. Therefore, the kits of the present invention are stored at 2-8°C and have a shelf life of 24 months. The results are detailed in Tables 6-8.

[0170] Table 6 Real-time stability test results (first batch)

[0171]

[0172]

[0173] Table 7 Real-time stability test results (second batch)

[0174]

[0175] Table 8 Real-time stability test results (third batch)

[0176]

[0177]

[0178] 2.6.2 37℃ aging stability:

[0179] (1) The main raw material of the detection kit of the present invention is the coated antibody. Since temperature has a great influence on the activity of the antibody, the higher the temperature, the more serious the attenuation of the antibody activity. The accelerated stability test design adopts the destruction test at 37°C. The stability study is carried out by examining the appearance, detection limit, accuracy, linearity, repeatability, batch difference, calibration value assignment accuracy, calibration uniformity, target value validity of the quality control product, and quality control product uniformity performance indicators, which are required to be no less than the product design requirements.

[0180] (2) Three batches of test kits, 4 boxes each, totaling 12 boxes, were placed in a 37°C incubator according to the commercial packaging for accelerated testing. The full performance (appearance, detection limit, accuracy, linearity, repeatability, inter-batch variation, accuracy of calibrator value assignment, uniformity of calibrator, validity of target values of quality control products, and uniformity of quality control products) of the test kits were tested on day 0, day 5, day 10, and day 13, respectively.

[0181] (3) Judgment of test content and results: consistent with long-term stability at 2-8°C;

[0182] (4) After accelerating the test kit at 37°C for 13 days, all performances of the three batches of the test kit of the present invention met the requirements, indicating that the performance of the test kit is stable within 13 days of accelerating the test kit at 37°C. The results are shown in Tables 9 to 11.

[0183] Table 9 Accelerated stability test results (first batch)

[0184]

[0185]

[0186] Table 10 Accelerated stability test results (second batch)

[0187]

[0188] Table 11 Accelerated stability test results (third batch)

[0189]

[0190]

[0191] In summary, the kit of the present invention adopts the magnetic microparticle chemiluminescence-double antibody sandwich method. Compared with other methodologies such as enzyme-linked immunosorbent assay and fluorescent immunochromatography, it is simple to operate and can be tested on the machine directly after adding the sample. In addition, through the independently developed acridinium ester working solution, magnetic bead working solution and working cleaning solution, after adding the sample, the incubation time of the magnetic beads and acridinium ester with the sample is greatly shortened, and only 3 minutes is needed to reach the optimal reaction time. The subsequent cleaning process only requires 3 cleanings, each lasting 30 seconds, to remove unreacted impurities. The entire test only takes 360 seconds. Most similar chemiluminescence product detection reagents on the market require about 900 seconds. Therefore, the detection time is greatly shortened, achieving the purpose of real-time detection, fully meeting the diagnosis and treatment needs of time-sensitive diseases, and providing patients with accurate test results.

[0192] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A kit for detecting marker S100 protein, characterized in that: The kit comprises reagent A and reagent B; the reagent A comprises magnetic bead working solution and magnetic beads coated with S100 monoclonal magnetic rare earth microspheres and antibodies; the reagent B comprises labeled antibody diluent and acridinium ester labeled anti-S100 antibody.

2. A kit for detecting the marker S100 protein according to claim 1, characterized in that: Each 1L of the magnetic bead working solution includes the following components: HEPES 11.91g, sodium chloride 9.00g, BSA 25.00g, trehalose 100.00g, glucose 80.00g, Tween-20 1mL, Proclin 300 1mL, polyethylene glycol-20000 80.00g, alkyl polyglycoside glycerol ether 7.2g, pH = 7.4; Each 1 L of the labeled antibody dilution solution includes the following components: 2.42 g of tris(hydroxymethyl)aminomethane, 1.41 mL of hydrochloric acid, 9.00 g of sodium chloride, 10.00 g of BSA, 0.5 mL of Proclin 300, 4.5 g of dextran, 7.4 g of isodecyl citrate monoester, and 0.01 g of amaranth.

3. A kit for detecting the marker S100 protein according to claim 1, characterized in that: The mass ratio of the S100 monoclonal magnetic rare earth microspheres coated antibodies to the magnetic beads in the S100 monoclonal magnetic rare earth microspheres coated antibodies is 100:3-8.

4. A kit for detecting the marker S100 protein according to claim 1, characterized in that: The kit also includes a calibrator, a quality control product and a chemiluminescent substrate.

5. A method for preparing a kit for detecting the marker S100 protein according to any one of claims 1 to 4, characterized in that: The steps include: Magnetic bead coating: activating magnetic beads in a magnetic bead coating buffer using an activator to obtain activated magnetic beads; adding S100 monoclonal magnetic rare earth microspheres coated antibodies to the activated magnetic beads for coupling reaction to obtain S100 monoclonal magnetic rare earth microspheres coated antibodies magnetic beads; storing the S100 monoclonal magnetic rare earth microspheres coated antibodies magnetic beads in a magnetic bead working solution to obtain reagent A; Acridinium ester labeling: An anti-S100 labeled antibody is subjected to a labeling reaction with acridinium in a luminophore labeling buffer to obtain an antibody labeling mixture; the antibody labeling mixture is ultrafiltered to obtain an acridinium ester-labeled anti-S100 antibody, and the acridinium ester-labeled anti-S100 antibody is stored in a labeled antibody diluent to obtain reagent B.

6. The method for preparing a kit for detecting the marker S100 protein according to claim 5, characterized in that: Each 1 L of the magnetic bead coating buffer includes the following components: MES 9.762 g, Triton-100 0.1 mL, alkyl polyglycoside sulfosuccinate disodium salt 5.00 g; Each 1 L of the luminophore labeling buffer comprises the following components: 13.435 g of disodium hydrogen phosphate dodecahydrate, 0.8265 g of sodium dihydrogen phosphate dihydrate, 2.3 g of α-sulfo fatty acid methyl ester (mono) sodium salt, and 0.6 g of fatty alcohol sulfate (ester) salt.

7. The method for preparing a kit for detecting the marker S100 protein according to claim 5, characterized in that: The activator is EDC; the mass ratio of the activator to the magnetic beads is 10:0.5-2.

8. The method for preparing a kit for detecting the marker S100 protein according to claim 5, characterized in that: The activation parameters are: room temperature, 20 to 40 minutes; The coupling reaction parameters are: room temperature, 3 to 8 hours.

9. The method for preparing a kit for detecting the marker S100 protein according to claim 5, characterized in that: The labeling reaction parameters are: 37° C., 20 to 40 min; The ultrafiltration parameters are: 14000xg, 5-15 min.

10. Use of a kit for detecting marker S100 protein, characterized in that: The kit for detecting the marker S100 protein according to any one of claims 1 to 4 is used to prepare a product for detecting the brain injury marker S100 protein.

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