GFAP chemiluminescence detection kit and application thereof, and GFAP concentration detection method

Abstract

The invention belongs to the technical field of biology, and discloses a GFAP chemiluminiscence detection kit, application thereof and a GFAP concentration detection method. The detection kit provided by the invention has very excellent detection performances such as detection sensitivity, detection repeatability and anti-interference capability when being applied to detection of GFAP protein, also has good storage stability, can be well applied to rapid quantitative detection of GFAP, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a GFAP chemiluminescence detection kit, its application, and a method for detecting GFAP concentration. Background Art

[0002] Traumatic brain injury (TBI) is a brain injury caused by external forces. TBI can disrupt the normal functions of the brain, thereby leading to impairment of a person's cognitive ability or physical function. Currently, CT scan is the only objective, simple, and reliable option widely used to assist clinicians in evaluating TBI. However, compared with other detection methods, CT scan has problems such as strong subjectivity of results, low positive rate, and potential radiation risks. During the clinical diagnosis and treatment process of TBI, stroke-related blood biomarkers can be used as auxiliary diagnostic tools, and the detection results of blood biomarkers can provide rapid, objective, and effective basis for the early diagnosis, stroke classification, and prognosis evaluation of clinical TBI patients.

[0003] Astrocytes are mainly distributed in the central nervous system, and glial fibrillary acidic protein (GFAP) is a marker of astrocyte activation, participating in the formation of the cytoskeleton and maintaining its tensile strength. GFAP is also expressed in chondrocytes, fibroblasts, myoepithelial cells, lymphocytes, and hepatic stellate cells; however, the GFAP expressed in the peripheral system is different in structure from that expressed in the central nervous system, and GFAP has certain tissue specificity. During the development of TBI, GFAP will enter the blood through the blood-brain barrier within 1 hour, causing a significant increase in GFAP expressed in the central nervous system in the serum, which can be used as a biomarker for TBI diagnosis.

[0004] Therefore, obtaining a detection reagent with good detection effect for GFAP expressed in the central nervous system is of great significance for the early diagnosis, differential diagnosis, and prognosis judgment of TBI. Summary of the Invention

[0005] The first object of the present invention is to provide a GFAP chemiluminescence detection kit. When this detection kit is applied to the detection of GFAP protein, it has very excellent detection performance such as detection sensitivity, detection repeatability, and anti-interference ability. This detection kit also has good storage stability and can be well applied to the rapid quantitative detection of GFAP, having good application prospects.

[0006] The second object of the present invention is to provide a method for detecting GFAP concentration.

[0007] The third object of the present invention is to provide the application of the above GFAP chemiluminescence detection kit in the detection of GFAP for non-diagnostic purposes.

[0008] Specifically, the GFAP chemiluminescence detection kit provided by the present invention specifically includes: magnetic bead freeze-dried microspheres coated with anti-GFAP antibody 1, acridinium ester-labeled anti-GFAP antibody 2 freeze-dried microspheres, sample diluent, and optionally a freeze-dried microsphere reconstitution solution; the magnetic bead freeze-dried microspheres coated with anti-GFAP antibody 1 are prepared by mixing magnetic bead microspheres coated with anti-GFAP antibody 1 with a reagent storage solution and then freeze-drying; the acridinium ester-labeled anti-GFAP antibody 2 freeze-dried microspheres are prepared by mixing acridinium ester-labeled anti-GFAP antibody 2 with a reagent storage solution and then freeze-drying; the reagent storage solution includes 3-5 wt% mannitol, 3-5 wt% trehalose, 0.5-1 wt% casein, 0.05-0.1 wt% Tween 20, 0.05-0.1 wt% gelatin, and 88.8-92 wt% TBS buffer; the sample diluent includes 5-10 wt% newborn bovine serum, 0.05-0.075 wt% benzoate compound, and 89.95-94.95 wt% PBS buffer solution.

[0009] Further, the preparation of the magnetic bead freeze-dried microspheres coated with anti-GFAP antibody 1 includes: mixing the magnetic bead microspheres coated with anti-GFAP antibody 1 with a reagent storage solution to obtain a magnetic bead microsphere freeze-dried solution; subjecting the magnetic bead microsphere freeze-dried solution to liquid nitrogen droplet pre-cooling treatment to obtain frozen magnetic bead microspheres; subjecting the frozen magnetic bead microspheres to freeze-drying to obtain the magnetic bead freeze-dried microspheres coated with anti-GFAP antibody 1.

[0010] Further, in the magnetic bead microsphere freeze-dried solution, the concentration of the magnetic bead microspheres coated with anti-GFAP antibody 1 is 0.1-1 mg / mL.

[0011] Further, in the preparation of the magnetic bead freeze-dried microspheres coated with anti-GFAP antibody 1, in the liquid nitrogen droplet pre-cooling treatment, the spotting amount of the magnetic bead microsphere freeze-dried solution is 10-40 μL.

[0012] Further, the preparation of the acridinium ester-labeled anti-GFAP antibody 2 freeze-dried microspheres includes: mixing the acridinium ester-labeled anti-GFAP antibody 2 with a reagent storage solution to obtain an acridinium ester microsphere freeze-dried solution; subjecting the acridinium ester microsphere freeze-dried solution to liquid nitrogen droplet pre-cooling treatment to obtain frozen acridinium ester microspheres; subjecting the frozen acridinium ester microspheres to freeze-drying to obtain the acridinium ester-labeled anti-GFAP antibody 2 freeze-dried microspheres.

[0013] Further, in the acridinium ester microsphere freeze-dried solution, the concentration of the acridinium ester-labeled anti-GFAP antibody 2 is 0.1-1 μg / mL.

[0014] Further, in the preparation of the acridinium ester-labeled anti-GFAP antibody 2 freeze-dried microspheres, in the liquid nitrogen drop pre-cooling treatment, the sample application amount of the acridinium ester microsphere freeze-drying solution is 10-40 μL.

[0015] Further, in the reagent storage solution, the concentration of the TBS buffer solution is 25-50 mM, and the pH is 7-7.5.

[0016] Further, in the sample diluent, the benzoate compound is selected from one or more of methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, and butyl p-hydroxybenzoate.

[0017] Further, in the sample diluent, the concentration of the PBS buffer solution is 40-60 mM, and the pH is 7-7.5.

[0018] Further, the freeze-dried microsphere reconstitution solution includes 1-5 wt% sodium chloride and 30-50 mM EDTA.

[0019] The method for detecting the GFAP concentration provided by the present invention specifically includes: using the above-mentioned GFAP chemiluminescence detection kit to detect the sample.

[0020] Further, the detection method includes: taking the freeze-dried microsphere reconstitution solution to reconstitute the magnetic bead freeze-dried microspheres and the acridinium ester-labeled anti-GFAP antibody 2 freeze-dried microspheres to obtain a detection reagent; taking the sample diluent and mixing it with the sample to be tested to obtain a test solution; taking the detection reagent and the test solution for incubation treatment and chemiluminescence detection to obtain the GFAP concentration.

[0021] Further, based on the volume of the detection reagent, the concentration of the magnetic bead microspheres coated with anti-GFAP antibody 1 is 0.05-0.1 mg / mL, and the concentration of the acridinium ester-labeled anti-GFAP antibody 2 is 0.05-0.1 μg / mL.

[0022] Further, the mixing volume ratio of the sample diluent to the sample to be tested is (1-3):1.

[0023] Further, the mixing volume ratio of the detection reagent to the test solution is (1-5):1.

[0024] The present invention also provides the application of the above-mentioned GFAP chemiluminescence detection kit in the detection of GFAP for non-diagnostic purposes.

[0025] Beneficial effects:

[0026] In the GFAP chemiluminescence detection kit provided by the present invention, by using a reagent storage solution comprising mannitol, trehalose, casein, Tween 20, gelatin and TBS buffer at specific contents, and mixing it with magnetic bead microspheres coated with anti-GFAP antibody 1 or acridinium ester-labeled anti-GFAP antibody 2, it can not only effectively protect anti-GFAP antibody 1 and anti-GFAP antibody 2 during lyophilization and subsequent storage, but also act together with a sample diluent comprising specific components during the chemiluminescence detection process to optimize the existence state of GFAP in the solution, and cooperate synergistically to enhance the specific recognition and binding ability of anti-GFAP antibody 1 and 2 to GFAP. When this detection kit is applied to GFAP detection, it has a wider linear detection range, lower detection sensitivity, higher detection accuracy and anti-interference ability, and can be well applied to GFAP detection for non-diagnostic purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the standard curve of GFAP concentration and luminescence value of the GFAP chemiluminescence detection kit provided in Example 1 of the present invention;

[0028] Figure 2 It is the standard curve of GFAP concentration and luminescence value of the GFAP chemiluminescence detection kit provided in Example 2 of the present invention;

[0029] Figure 3 It is the standard curve of GFAP concentration and luminescence value of the GFAP chemiluminescence detection kit provided in Example 3 of the present invention;

[0030] Figure 4 It is the standard curve of GFAP concentration and luminescence value of the GFAP chemiluminescence detection kit provided in Example 4 of the present invention;

[0031] Figure 5 It is the standard curve of GFAP concentration and luminescence value of the GFAP chemiluminescence detection kit provided in Example 5 of the present invention;

[0032] Figure 6 It is the standard curve of GFAP concentration and luminescence value of the GFAP chemiluminescence detection kit provided in Comparative Example 1;

[0033] Figure 7 It is the standard curve of GFAP concentration and luminescence value of the GFAP chemiluminescence detection kit provided in Comparative Example 2;

[0034] Figure 8 It is the standard curve of GFAP concentration and luminescence value of the GFAP chemiluminescence detection kit provided in Comparative Example 3;

[0035] Figure 9Standard curve of GFAP concentration and luminescence value for the GFAP chemiluminescence detection kit provided in Comparative Example 4. Detailed implementation mode

[0036] The GFAP chemiluminescence detection kit provided by the present invention specifically includes: magnetic bead freeze-dried microspheres coated with anti-GFAP antibody 1, acridinium ester-labeled anti-GFAP antibody 2 freeze-dried microspheres, sample diluent, and optionally a freeze-dried microsphere reconstitution solution.

[0037] In the present invention, the magnetic bead freeze-dried microspheres coated with anti-GFAP antibody 1 specifically refer to those prepared by mixing magnetic bead microspheres coated with anti-GFAP antibody 1 with a reagent storage solution and then freeze-drying, which are reagents for capturing and immobilizing GFAP in chemiluminescence detection; the acridinium ester-labeled anti-GFAP antibody 2 freeze-dried microspheres specifically refer to those prepared by mixing acridinium ester-labeled anti-GFAP antibody 2 with a reagent storage solution and then freeze-drying, which are reagents for specifically recognizing GFAP in chemiluminescence detection.

[0038] In the present invention, the reagent storage solution specifically includes: 3-5 wt% mannitol, such as 3 wt%, 3.5 wt%, 4 wt%, 4.5, 5 wt% or any value between them; 3-5 wt% trehalose, such as 3 wt%, 3.1 wt%, 3.6 wt%, 3.9 wt%, 4 wt%, 4.6 wt%, 4.9 wt%, 5 wt% or any value between them; 0.5-1 wt% casein, such as 0.5 wt%, 0.6 wt%, 0.75 wt%, 0.8 wt%, 0.9 wt%, 1 wt% or any value between them; 0.05-0.1 wt% Tween 20, such as 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.075 wt%, 0.8 wt%, 0.1 wt% or any value between them; 0.05-0.1 wt% gelatin, such as 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.1 wt% or any value between them; and 88.8-92 wt% TBS buffer, such as 88.8 wt%, 89 wt%, 90 wt%, 91 wt%, 91.5 wt%, 92 wt% or any value between them.

[0039] In some specific implementation modes, the concentration of the TBS buffer is preferably 25-50 mM, such as 25 mM, 28 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM or any value between them; the pH is preferably 7-7.5, such as 7, 7.1, 7.3, 7.4, 7.5 or any value between them.

[0040] In the present invention, the sample diluent specifically comprises: 5-10 wt% of newborn bovine serum, such as 5 wt%, 6 wt%, 8 wt%, 10 wt% or any value therebetween; 0.05-0.075 wt% of a benzoate compound, such as 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.075 wt% or any value therebetween; and 89.95-94.95 wt% of a PBS buffer solution, such as 89.95 wt%, 90 wt%, 91.15 wt%, 92 wt%, 94 wt%, 94.95 wt% or any value therebetween.

[0041] In some specific embodiments, specific examples of the benzoate compound include but are not limited to: one or more of methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, and butyl p-hydroxybenzoate.

[0042] In some specific embodiments, the concentration of the PBS buffer solution is preferably 40-60 mM, such as 40 mM, 45 mM, 50 mM, 55 mM, 60 mM or any value therebetween; and the pH is preferably 7-7.5, such as 7, 7.1, 7.4, 7.5 or any value therebetween.

[0043] In the present invention, the magnetic bead microspheres coated with anti-GFAP antibody 1 and the acridinium ester-labeled anti-GFAP antibody 2 are a reagent commonly used in the conventional detection of GFAP; that is, the reagent storage solution of the present invention has relatively good universality for the existing anti-GFAP chemiluminescent detection antibodies and their detection effects. Those skilled in the art can select existing anti-GFAP antibodies and compound them with the reagent storage solution and the sample diluent according to actual needs to achieve the detection of GFAP. The present invention does not particularly limit the magnetic bead microspheres coated with anti-GFAP antibody 1 and the acridinium ester-labeled anti-GFAP antibody 2.

[0044] In some specific embodiments, the anti-GFAP antibody 1 is preferably the anti-GFAP antibody GFAP83cc purchased from Hytide Biotech, and the anti-GFAP antibody 2 is preferably the anti-GFAP antibody GFAP81cc purchased from Hytide Biotech; at this time, the reagent storage solution and the sample diluent have good optimization effects on the above antibody combination, effectively expanding the linear range of GFAP detection and reducing the detection sensitivity.

[0045] In the present invention, the preparation of the freeze-dried microspheres of magnetic beads coated with anti-GFAP antibody 1 specifically includes: mixing the magnetic bead microspheres coated with anti-GFAP antibody 1 with a reagent storage solution to obtain a freeze-drying solution of magnetic bead microspheres; subjecting the freeze-drying solution of magnetic bead microspheres to a pre-cooling treatment by dripping beads with liquid nitrogen to obtain frozen magnetic bead microspheres; and subjecting the frozen magnetic bead microspheres to freeze-drying to obtain the freeze-dried microspheres of magnetic beads coated with anti-GFAP antibody 1.

[0046] In some specific embodiments, in the freeze-drying solution of magnetic bead microspheres, the concentration of the magnetic bead microspheres coated with anti-GFAP antibody 1 is preferably 0.1 - 1 mg / mL, such as 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL or any value therebetween.

[0047] In some specific embodiments, in the preparation of the freeze-dried microspheres of magnetic beads coated with anti-GFAP antibody 1, the sample application amount of the freeze-drying solution of magnetic bead microspheres in the pre-cooling treatment by dripping beads with liquid nitrogen is preferably 10 - 40 μL, such as 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 40 μL or any value therebetween.

[0048] In the present invention, the preparation of the freeze-dried microspheres of acridinium-labeled anti-GFAP antibody 2 specifically includes: mixing the acridinium-labeled anti-GFAP antibody 2 with a reagent storage solution to obtain a freeze-drying solution of acridinium microspheres; subjecting the freeze-drying solution of acridinium microspheres to a pre-cooling treatment by dripping beads with liquid nitrogen to obtain frozen acridinium microspheres; and subjecting the frozen acridinium microspheres to freeze-drying to obtain the freeze-dried microspheres of acridinium-labeled anti-GFAP antibody 2.

[0049] In some specific embodiments, in the freeze-drying solution of acridinium microspheres, the concentration of the acridinium-labeled anti-GFAP antibody 2 is preferably 0.1 - 1 μg / mL, such as 0.1 μg / mL, 0.15 μg / mL, 0.3 μg / mL, 0.5 μg / mL, 0.8 μg / mL, 1 μg / mL or any value therebetween.

[0050] In some specific embodiments, in the preparation of the freeze-dried microspheres of acridinium-labeled anti-GFAP antibody 2, the sample application amount of the freeze-drying solution of acridinium microspheres in the pre-cooling treatment by dripping beads with liquid nitrogen is preferably 10 - 40 μL, such as 10 μL, 15 μL, 18 μL, 20 μL, 25 μL, 30 μL, 40 μL or any value therebetween.

[0051] In the invention, the freeze-dried microsphere reconstitution solution is used to dissolve the freeze-dried microspheres coated with anti-GFAP antibody 1 and / or the freeze-dried microspheres of acridinium ester-labeled anti-GFAP antibody 2, and specific examples thereof may include, but are not limited to, one or more of water, PBS buffer solution, and TBS buffer solution.

[0052] In some specific embodiments, the freeze-dried microsphere reconstitution solution preferably includes 1-5 wt% of sodium chloride, such as 1 wt%, 1.3 wt%, 1.8 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt% or any value therebetween; and 30-50 mM of EDTA, such as 30 mM, 35 mM, 38 mM, 40 mM, 45 mM, 50 mM or any value therebetween. At this time, compared with conventional solutions such as water, PBS buffer solution, and TBS buffer solution, the freeze-dried microsphere reconstitution solution can cooperate synergistically with the reagent storage solution and the sample dilution solution to achieve better GFAP detection effects.

[0053] In the present invention, the GFAP chemiluminescence detection kit further includes a series of calibration products for constructing a standard curve of GFAP concentration and luminescence value; wherein, the series of calibration products are prepared by mixing a sample dilution solution and a GFAP antigen.

[0054] Based on the effects of the above GFAP chemiluminescence detection kit, the present invention also provides a method for detecting GFAP concentration. This detection method uses the above GFAP chemiluminescence detection kit to detect a sample, and specifically includes: taking a freeze-dried microsphere reconstitution solution to reconstitute the magnetic bead freeze-dried microspheres and the acridinium ester-labeled anti-GFAP antibody 2 freeze-dried microspheres to obtain a detection reagent; taking a sample dilution solution and mixing it with the sample to be tested to obtain a test solution; taking the detection reagent and the test solution for incubation treatment and chemiluminescence detection to obtain the GFAP concentration.

[0055] In some specific embodiments, based on the volume of the detection reagent, the concentration of the magnetic bead microspheres coated with anti-GFAP antibody 1 is preferably 0.05-0.1 mg / mL, such as 0.05 mg / mL, 0.06 mg / mL, 0.075 mg / mL, 0.08 mg / mL, 0.9 mg / mL, 0.1 mg / mL or any value therebetween; the concentration of the acridinium ester-labeled anti-GFAP antibody 2 is preferably 0.05-0.1 μg / mL, such as 0.05 μg / mL, 0.08 μg / mL, 0.09 μg / mL, 0.1 μg / mL or any value therebetween.

[0056] In some specific embodiments, the mixing volume ratio of the sample diluent to the sample to be tested is preferably (1-3):1, such as 1:1, 1.3:1, 1.5:1, 2:1, 2.3:1, 2.5:1, 3:1 or any value therebetween.

[0057] In some specific embodiments, the mixing volume ratio of the detection reagent to the test solution is preferably (1-5):1, such as 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 4:1, 5:1 or any value therebetween.

[0058] In the present invention, the incubation treatment is a technical means commonly used in existing chemiluminescence detection techniques. Those skilled in the art can make adaptive selections according to actual needs, and the present invention does not particularly limit it.

[0059] In the present invention, the chemiluminescence detection is a technical means commonly used in existing chemiluminescence detections. Those skilled in the art can make adaptive selections for processes such as pre-excitation, excitation, and luminescence value detection according to actual needs, and the present invention does not particularly limit it.

[0060] Based on the effects of the above-mentioned GFAP chemiluminescence detection kit, the present invention also provides the application of the GFAP chemiluminescence detection kit in the detection of GFAP for non-diagnostic purposes.

[0061] The embodiments of the present invention are described in detail below. The examples of the embodiments are intended to explain the present invention and should not be construed as limiting the present invention. For those not specifying specific techniques or conditions in the examples, the techniques or conditions described in the literature in the art or according to the product specifications are followed. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchases.

[0062] Example 1

[0063] This example is used to illustrate a GFAP chemiluminescence detection kit. The preparation of the GFAP chemiluminescence detection kit specifically includes:

[0064] 1. Preparation of the magnetic bead microsphere freeze-dried solution: (1) The magnetic beads (carboxyl, 2.9 μm, manufactured by JSR Corporation, Japan) are washed and resuspended with MES buffer (0.2 M, pH = 7.4) containing 0.1 mL / L of Proclin300 (AbMole, product number M25386) to obtain a magnetic bead suspension with a concentration of 10 mg / mL.

[0065] (2) Take EDC (final concentration: 0.1 mg / mL) and NHS (final concentration: 0.2 mg / mL) and add them to the magnetic bead suspension. Activate at 25 °C and 250 rpm for 30 min, then centrifuge. Wash with MES buffer (0.2 M, pH = 7.4) containing 0.1 mL / L of Proclin 300 and resuspend to obtain an activated magnetic bead suspension with a concentration of 10 mg / mL.

[0066] (3) Take GFAP antibody 1 (from Hytide Bio, catalog number: 4G25 - GFAP83cc) at an addition amount with a final concentration of 100 μg / mL and add it to the activated magnetic bead suspension. Crosslink at 25 °C and 250 rpm for 3 h, then centrifuge to obtain magnetic beads coated with GFAP antibody 1.

[0067] (5) Take the magnetic beads coated with GFAP antibody 1 and mix it with the magnetic bead blocking solution at an addition amount with a final concentration of 0.1 mg / mL. Block at 25 °C for 60 min, then centrifuge. Wash with the reagent storage solution and resuspend to obtain a magnetic bead microsphere freeze-dried solution with a concentration of 0.5 mg / mL;

[0068] Among them, the magnetic bead blocking solution includes: Tris-HCl buffer (0.01 M, pH = 7.4), 5 g / L of BSA, and 0.1% (v / v) of Tween 20; the reagent storage solution includes: 3 wt% of mannitol, 5 wt% of trehalose, 0.5 wt% of casein, 0.1 wt% of Tween 20, 0.05 wt% of gelatin, and 91.35 wt% of TBS buffer (40 mM, pH = 7.4).

[0069] 2. Preparation of acridinium ester microsphere freeze-dried solution: Take GFAP antibody 2 (from Hytide Bio, catalog number: 4G25 - GFAP81cc) and mix it with acridinium ester at a molar ratio of 1:20. Add PBS buffer (0.02 M, pH = 7.4) to make the concentration of GFAP antibody 2 in the solution 1 mg / mL. Crosslink at 25 °C for 3 h; take lysine at an addition amount with a molar ratio of lysine to acridinium ester of 140:1 and add it to the solution. Block at 25 °C for 30 min. Then take the solution after blocking and PBS buffer (0.02 M, pH = 7.4) and add them to a 50 KD dialysis bag for dialysis 5 times, with each dialysis time being 3 h. Collect the filtrate and dilute it with the reagent storage solution to obtain an acridinium ester microsphere freeze-dried solution with a concentration of 0.5 μg / mL.

[0070] 3. Preparation of single - person - portion GFAP freeze - dried microsphere detection reagent: Respectively take the magnetic bead microsphere freeze - dried solution and the acridinium - ester microsphere freeze - dried solution and add them to a liquid nitrogen spot - sampler. Set the spot - sampling volume to 20 μL per drop, and perform spot - sampling to form frozen microspheres. Transfer the frozen microspheres to a freeze - dryer. After freeze - drying, obtain the microsphere freeze - dried preparation, fill it with a protective gas, package and store it to obtain the single - person - portion GFAP freeze - dried microsphere detection reagent.

[0071] 4. Preparation of freeze - dried microsphere reconstitution solution: Mix NaCl, EDTA and PBS buffer solution (50 mM, pH = 7.4) to obtain the freeze - dried microsphere reconstitution solution. The concentration of NaCl in the freeze - dried microsphere reconstitution solution is 4 wt%, and the concentration of EDTA is 40 mM.

[0072] 5. Preparation of sample diluent: Mix newborn bovine serum, methylparaben and PBS buffer solution (50 mM, pH = 7.4) to obtain the sample diluent. The concentration of newborn bovine serum in the sample diluent is 8 wt%, and the concentration of methylparaben is 0.06 wt%.

[0073] 6. Preparation of calibration quality control products: (1) Dilute GFAP antigen (Haitai Biotech, product number 8G45) with the sample diluent to obtain a series of calibrators with concentrations of 0.02 ng / mL, 0.19 ng / mL, 1.05 ng / mL, 4.90 ng / mL and 25.08 ng / mL respectively.

[0074] (2) Dilute GFAP antigen with the sample diluent to obtain a low - value calibrator and a high - value calibrator with GFAP antigen concentrations of 0.05 ng / mL and 14.39 ng / mL respectively.

[0075] Example 2

[0076] The GFAP chemiluminescence detection kit provided in this example is basically the same as that in Example 1. The difference is that the reagent storage solution includes: 5 wt% mannitol, 5 wt% trehalose, 1 wt% casein, 0.1 wt% Tween 20, 0.1 wt% gelatin and 88.8% TBS buffer solution (40 mM, pH = 7.4). Under the same other conditions, the GFAP chemiluminescence detection kit is obtained.

[0077] Example 3

[0078] The GFAP chemiluminescence detection kit provided in this example is basically the same as that in Example 1. The difference is that the sample diluent includes: 8 wt% newborn bovine serum, 0.075 wt% methylparaben and 91.925% PBS buffer solution (50 mM, pH = 7.4). Under the same other conditions, the GFAP chemiluminescence detection kit is obtained.

[0079] Example 4

[0080] The GFAP chemiluminescence detection kit provided in this example is basically the same as that in Example 1, except that the sample diluent includes: 8 wt% newborn bovine serum, 0.05 wt% methyl p-hydroxybenzoate, and 91.95 wt% PBS buffer solution (50 mM, pH = 7.4). Under the same other conditions, a GFAP chemiluminescence detection kit is obtained.

[0081] Example 5

[0082] The GFAP chemiluminescence detection kit provided in this example is basically the same as that in Example 1, except that butyl p-hydroxybenzoate is used to replace methyl p-hydroxybenzoate in the sample diluent in equal mass. Under the same other conditions, a GFAP chemiluminescence detection kit is obtained.

[0083] Comparative Example 1

[0084] The GFAP chemiluminescence detection kit provided in this comparative example is basically the same as that in Example 1, except that the reagent storage solution includes: 5 wt% mannitol, 10 wt% trehalose, 1 wt% casein, 0.1 wt% Tween 20, 0.1 wt% gelatin, and 83.8% TBS buffer solution (40 mM, pH = 7.4). Under the same other conditions, a GFAP chemiluminescence detection kit is obtained.

[0085] Comparative Example 2

[0086] The GFAP chemiluminescence detection kit provided in this comparative example is basically the same as that in Example 1, except that the reagent storage solution includes: 3 wt% mannitol, 5 wt% trehalose, 0.5 wt% casein, 0.1 wt% Tween 20, 0.05 wt% gelatin, 1 wt% proclin 300 (Yingxin, product number TXP32652), and 90.35 wt% TBS buffer solution (40 mM, pH = 7.4). Under the same other conditions, a GFAP chemiluminescence detection kit is obtained.

[0087] Comparative Example 3

[0088] The GFAP chemiluminescence detection kit provided in this comparative example is basically the same as that in Example 1, except that the reagent storage solution includes: 3 wt% mannitol, 5 wt% trehalose, 0.5 wt% casein, 0.1 wt% Tween 20, 0.05 wt% gelatin, 1 wt% methyl p-hydroxybenzoate, and 90.35 wt% TBS buffer solution (40 mM, pH = 7.4). Under the same other conditions, a GFAP chemiluminescence detection kit is obtained.

[0089] Comparative Example 4

[0090] The GFAP chemiluminescence detection kit provided in this embodiment is basically the same as that in Example 1, except that methyl paraben in the sample diluent is replaced by proclin300 with equal mass, and other conditions are the same, thus obtaining the GFAP chemiluminescence detection kit.

[0091] Test Example

[0092] This test example is used to illustrate the detection performance of the GFAP chemiluminescence detection kits provided in the above embodiments and comparative examples. The specific tests include:

[0093] 1. Establishment of GFAP standard curve: Take 125 μL / sample of the addition amount to dissolve the single-person portion of GFAP freeze-dried microsphere detection reagent with the freeze-dried microsphere reconstitution solution. Then, add 50 μL of a series of calibrators respectively. After incubating at 37 °C for 10 min, wash and detect the luminescence value. Use a fully automatic chemiluminescence immunoassay analyzer and refer to the instruction manual to test the luminescence values of each calibrator and establish a standard curve. The results are shown in Table 1 and Figures 1-9 as shown.

[0094] Table 1.

[0095]

[0096] From the test results shown in Table 1 and Figures 1-9 it can be seen that compared with Comparative Examples 1-3, in the standard curves measured by the GFAP chemiluminescence detection kits provided in Embodiments 1-5 of the present invention, there is an excellent linear correlation between the GFAP concentration and the luminescence value.

[0097] 2. GFAP detection sensitivity test: Take 125 μL / sample of the addition amount to dissolve the single-person portion of GFAP freeze-dried microsphere detection reagent with the freeze-dried microsphere reconstitution solution. Then, add 50 μL of each sample diluent as a blank sample respectively. After incubating at 37 °C for 10 min, use a fully automatic chemiluminescence immunoassay analyzer and refer to the instruction manual to test the luminescence value. Repeat the test 20 times, calculate the average value (X) and standard deviation (SD) of the luminescence value, calculate the luminescence value corresponding to the detection sensitivity of the reagent according to the following formula, and substitute it into each standard curve to calculate the detection sensitivity. The results are shown in Table 2.

[0098] Luminescence value corresponding to detection sensitivity = X + 2SD

[0099] Table 2.

[0100] Group Average luminescence value Standard deviation Detection sensitivity (ng / mL) Example 1 500.20 18.70 0.0023 Example 2 1231.25 34.21 0.0789 Example 3 429.45 22.62 0.0120 Example 4 455.35 24.90 0.0129 Example 5 2375.70 41.51 0.0984

[0101] As can be seen from the test results shown in Table 2, the GFAP chemiluminescence detection kits provided in Examples 1 to 5 of the present invention all have very excellent detection sensitivities, and the detection sensitivity of the GFAP chemiluminescence detection kit provided in Example 1 is as low as 0.0023 ng / mL.

[0102] 3. GFAP detection repeatability test: Take 125 μL per portion of the addition amount, dissolve the freeze-dried microsphere reconstitution solution to dissolve the single-person portion of the GFAP freeze-dried microsphere detection reagent. Then, add 50 μL of the low-value calibrator and the high-value calibrator respectively. After incubating at 37 °C for 10 min, use an automatic chemiluminescence immunoassay analyzer and refer to the instruction manual to measure the luminescence value. Substitute the luminescence value into the corresponding standard curve to calculate the corresponding GFAP concentration, and repeat the test 10 times. The calculated average concentration, standard deviation (SD), and coefficient of variation (CV) results of GFAP are shown in Table 3.

[0103] Table 3.

[0104]

[0105] As can be seen from the test results shown in Table 3, the GFAP chemiluminescence detection kits provided in Examples 1 to 5 of the present invention all have good detection repeatabilities, and the GFAP chemiluminescence detection kit provided in Example 1 has very excellent precision.

[0106] 4. GFAP detection anti-interference test: (1) Take the sample diluent and mix it with clinical sera I - III respectively according to a volume ratio of 1:2 to obtain the test blood samples I - III.

[0107] (2) Add pure triglyceride to pure water to prepare a triglyceride stock solution with a concentration of 200 g / L. Take the triglyceride stock solution and add it to test blood sample I according to a volume ratio of 1:19 and mix well to obtain a high-concentration triglyceride interference sample; take the triglyceride stock solution and add it to pure water according to a volume ratio of 1:19 and mix well, then take the mixed solution and add it to test blood sample I according to a volume ratio of 1:19 and mix well to obtain a low-concentration triglyceride interference sample.

[0108] (3) Add pure bilirubin to 0.1 mol / L NaOH aqueous solution to prepare a bilirubin stock solution with a concentration of 400 g / L. Take the bilirubin stock solution and add it to test blood sample II according to a volume ratio of 1:19 and mix well to obtain a high-concentration bilirubin interference sample; take the bilirubin stock solution and add it to pure water according to a volume ratio of 19:1 and mix well, then take the mixed solution and add it to test blood sample II according to a volume ratio of 1:19 and mix well to obtain a low-concentration bilirubin interference sample.

[0109] (4) Add the pure hemoglobin product to pure water to prepare a hemoglobin stock solution with a concentration of 100 g / L. Take the hemoglobin stock solution according to a volume ratio of 1:19 and add it to the test blood sample III and mix well to obtain a high-concentration hemoglobin interference sample; take the hemoglobin stock solution according to a volume ratio of 19:1, add it to pure water and mix well, then take the mixed solution according to a volume ratio of 1:19 and add it to the test blood sample III and mix well to obtain a low-concentration hemoglobin interference sample.

[0110] (5) Take 125 μL per portion of the addition amount, dissolve the single-person portion of the freeze-dried microsphere reconstitution solution of the GFAP freeze-dried microsphere detection reagent provided in Example 1, then add 50 μL of the above interference samples respectively, and use the test blood samples I, II, and III without added interference as controls. After incubating at 37 °C for 10 min, use an automatic chemiluminescence immunoassay analyzer and refer to the instruction manual to measure the luminescence value. Substitute the luminescence value into the corresponding standard curve to calculate the corresponding GFAP concentration, repeat the test 3 times, calculate the average GFAP concentration and the relative deviation, and the results are shown in Table 4.

[0111] Table 4.

[0112]

[0113] As can be seen from the test results shown in Table 4, the GFAP chemiluminescence detection kit provided in Example 1 of the present invention has very excellent anti-interference ability.

[0114] 5. Stability of the GFAP chemiluminescence detection kit: Take the single-person portion of the GFAP freeze-dried microsphere detection reagent provided in Example 1 and place it in an electrothermal constant temperature oven at 45 °C ± 2 °C for 0 d and 30 d respectively. Take 125 μL per portion of the addition amount, dissolve the single-person portion of the GFAP freeze-dried microsphere detection reagent with the freeze-dried microsphere reconstitution solution, then add 50 μL of the high-value calibrator respectively. After incubating at 37 °C for 10 min, use an automatic chemiluminescence immunoassay analyzer to measure the luminescence value, repeat the test 3 times, calculate the average value and the decline rate, and the results are shown in Table 5.

[0115] Table 5.

[0116]

[0117] As can be seen from the test results shown in Table 5, after storing the GFAP chemiluminescence detection kit provided in Example 1 of the present invention at 45 °C for 30 d, the detection decline rate is only 11.77%, indicating excellent stability.

[0118] 6. Determination of the level of GFAP in patients with craniocerebral injury: (1) Thirty patients with craniocerebral injury admitted to the emergency department of the hospital were selected, and sample diluents were taken at a volume ratio of 1:2 and mixed with the clinical serum samples collected from the patients with craniocerebral injury to obtain the blood samples to be tested.

[0119] (2) Take 125 μL per portion of the addition amount to dissolve the single - person - portion GFAP freeze - dried microsphere detection reagent with the freeze - dried microsphere reconstitution solution, then add 50 μL of the blood sample to be tested respectively. After incubating at 37 °C for 10 min, use an automatic chemiluminescence immunoassay analyzer and refer to the instruction manual to test the luminescence value, and substitute the luminescence value into the standard curve to calculate the corresponding GFAP concentration. The results are shown in Table 6.

[0120] Table 6.

[0121]

[0122]

[0123] As can be seen from the test results shown in Table 6, the CT / MRI results of 16 patients with craniocerebral injury mainly showed cerebral hemorrhage and glioblastoma, and the GFAP concentration detected in their blood was significantly higher than the range value of the GFAP concentration in normal human blood samples disclosed in the literature [1] ≤0.049 ng / mL.

[0124] [1] Determination of glial fibrillary acidic protein (GFAP) in the serum of patients with acute brain injury [J]. Foreign Medical Sciences (Clinical Biochemistry and Laboratory Medicine), 1999.

[0125] Although the embodiments of the present invention have been shown and described above, it can be understood that the above - mentioned embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above - mentioned embodiments within the scope of the present invention without departing from the principle and purpose of the present invention.

Claims

1. A GFAP chemiluminescence detection kit, characterized in that: The detection kit comprises: anti-GFAP antibody 1 coated magnetic bead freeze-dried microspheres, acridine lipid labeled anti-GFAP antibody 2 freeze-dried microspheres, sample diluent and optional freeze-dried microsphere reconstitution solution; The anti-GFAP antibody 1 coated magnetic bead lyophilized microspheres are prepared by mixing the anti-GFAP antibody 1 coated magnetic bead microspheres with a reagent storage solution and then freeze-drying; the acridinium lipid-labeled anti-GFAP antibody 2 lyophilized microspheres are prepared by mixing the acridinium lipid-labeled anti-GFAP antibody 2 with a reagent storage solution and then freeze-drying; the reagent storage solution comprises 3-5wt% mannitol, 3-5wt% trehalose, 0.5-1wt% casein, 0.05-0.1wt% Tween 20, 0.05-0.1wt% gelatin and 88.8-92wt% TBS buffer; The sample diluent comprises 5-10 wt% of newborn calf serum, 0.05-0.075 wt% of benzoate compound and 89.95-94.95 wt% of PBS buffer solution.

2. The GFAP chemiluminescence detection kit according to claim 1, characterized in that: The preparation of the anti-GFAP antibody 1 coated magnetic bead lyophilized microspheres comprises: mixing the anti-GFAP antibody 1 coated magnetic bead microspheres with a reagent storage solution to obtain a magnetic bead microsphere lyophilized solution; performing a liquid nitrogen drop bead precooling treatment on the magnetic bead microsphere lyophilized solution to obtain frozen magnetic bead microspheres; performing freeze drying on the frozen magnetic bead microspheres to obtain the anti-GFAP antibody 1 coated magnetic bead lyophilized microspheres; Optionally, in the lyophilized solution of magnetic beads, the concentration of the magnetic beads coated with the anti-GFAP antibody 1 is 0.1 to 1 mg / mL; Optionally, in the liquid nitrogen bead pre-cooling treatment, the spotting volume of the magnetic bead microsphere freeze-dried solution is 10 to 40 μL.

3. The GFAP chemiluminescence detection kit according to claim 1, characterized in that: The preparation of the acridinium lipid-labeled anti-GFAP antibody 2 freeze-dried microspheres comprises: mixing the acridinium lipid-labeled anti-GFAP antibody 2 with a reagent storage solution to obtain an acridinium lipid microsphere freeze-dried solution; performing a liquid nitrogen bead precooling treatment on the acridinium lipid microsphere freeze-dried solution to obtain frozen acridinium lipid microspheres; performing freeze drying on the frozen acridinium lipid microspheres to obtain the acridinium lipid-labeled anti-GFAP antibody 2 freeze-dried microspheres; Optionally, in the acridinium lipid microsphere freeze-dried solution, the concentration of the acridinium lipid-labeled anti-GFAP antibody 2 is 0.1 to 1 μg / mL; Optionally, during the liquid nitrogen bead pre-cooling treatment, the spotting volume of the acridinium lipid microsphere freeze-dried solution is 10 to 40 μL.

4. The GFAP chemiluminescence detection kit according to claim 1, characterized in that: In the reagent storage solution, the concentration of the TBS buffer is 25-50 mM, and the pH is 7-7.

5.

5. The GFAP chemiluminescence detection kit according to claim 1, characterized in that: In the sample diluent, the benzoate compound is selected from one or more of methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate and butyl parahydroxybenzoate; Optionally, the PBS buffer solution has a concentration of 40 to 60 mM and a pH of 7 to 7.

5.

6. The GFAP chemiluminescence detection kit according to claim 1, characterized in that: The freeze-dried microsphere reconstitution solution includes 1-5 wt % sodium chloride and 30-50 mM EDTA.

7. A method for detecting GFAP concentration, characterized in that: The detection method comprises: taking the GFAP chemiluminescence detection kit described in claims 1 to 6 to detect the sample.

8. The method for detecting GFAP concentration according to claim 7, characterized in that: The detection method comprises: taking a freeze-dried microsphere reconstitution solution to reconstitute the magnetic bead freeze-dried microspheres and the acridinium lipid-labeled anti-GFAP antibody 2 freeze-dried microspheres to obtain a detection reagent; taking a sample diluent to mix with a sample to be tested to obtain a test solution; taking the detection reagent and the test solution to perform incubation treatment and chemiluminescence detection to obtain a GFAP concentration.

9. The method for detecting GFAP concentration according to claim 8, characterized in that: Based on the volume of the detection reagent, the concentration of the magnetic beads coated with the anti-GFAP antibody 1 is 0.05-0.1 mg / mL, and the concentration of the acridinium lipid-labeled anti-GFAP antibody 2 is 0.05-0.1 μg / mL; Optionally, the mixing volume ratio of the sample diluent to the sample to be tested is (1-3):1; Optionally, the mixed volume ratio of the detection reagent and the test solution is (1-5):

1.

10. Use of the GFAP chemiluminescence detection kit according to any one of claims 1 to 6 in GFAP detection for non-diagnostic purposes.

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