A GFAP, NFL and UCH-L1 combined detection kit and its use in early detection of brain trauma

Abstract

The present invention provides a GFAP, NFL and UCH-L1 combined detection kit, comprising: an acridinium ester labeled antibody working solution and a magnetic bead coated antibody working solution; the acridinium ester labeled antibody working solution contains acridinium ester labeled GFAP, NFL and UCH-L1 antibodies, and the magnetic bead coated antibody working solution contains magnetic bead coated GFAP, NFL and UCH-L1 antibodies. By screening the components and the content of each component of the acridinium ester labeled working solution and the magnetic bead labeled working solution, a stable working environment can be provided for the acridinium ester labeled GFAP, NFL and UCH-L1 antibodies and the magnetic beads labeled with the GFAP, NFL and UCH-L1 antibodies, and specific recognition and detection of GFAP, NFL and UCH-L1 are achieved, thereby realizing the triple detection of GFAP, NFL and UCH-L1 for early diagnosis of patients with brain trauma.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a GFAP, NFL and UCH-L1 combined detection kit, which can be applied to the early detection of brain trauma. Background Art

[0002] Brain trauma-related diseases, including traumatic brain injury (TBI), involve complex pathological processes that affect various cellular components of the brain. Traditional diagnostic methods for brain trauma mainly rely on imaging examinations and clinical assessments, which may not be able to detect subtle injuries in the early stages.

[0003] GFAP (Glial Fibrillary Acidic Protein) is an intermediate filament protein that is mainly expressed in astrocytes of the central nervous system (CNS). It plays an important role in maintaining the structural integrity of astrocytes and in the repair process after CNS injury. In biological fluids such as blood and cerebrospinal fluid (CSF), increased levels of GFAP indicate damage or activation of astrocytes, which occurs when the CNS is injured. In brain trauma, increased levels of GFAP are associated with the degree of astrocyte activation and gliosis, reflecting the severity of the injury.

[0004] The NFL (Neurofilament Light) protein is a structural protein that makes up neurofilaments in neurons, providing structural support and maintaining the integrity of axons. It is essential for the proper function of axons, the long projections of neurons that transmit nerve impulses. In biological fluids, elevated NFL levels indicate axonal damage or degeneration, a hallmark of many neurodegenerative diseases and brain injuries. In brain trauma, elevated NFL levels correlate with the extent of axonal damage, providing insights into injury severity and progression.

[0005] UCH-L1 (Ubiquitin C-terminal Hydrolase-L1) is an enzyme found primarily in neurons and is involved in the ubiquitin-proteasome system that regulates protein degradation and turnover. It maintains the health and function of neurons by removing damaged or misfolded proteins. In biological fluids, increased levels of UCH-L1 are associated with neuronal cell body damage and neuronal death. In brain trauma, increased levels of UCH-L1 are associated with the degree of neuronal damage, providing relevant information on the severity of neuronal damage.

[0006] The correlation between GFAP, NFL, and UCH-L1 is that they represent different cellular components and injury mechanisms within the brain, providing a multifaceted perspective on brain trauma. In clinical applications, these biomarkers have significant early diagnostic capabilities, even before clinical symptoms appear. Combining measurements of GFAP, NFL, and UCH-L1 can help determine the severity of injury and predict long-term outcomes. Regular monitoring of these biomarkers can also track the progression of injury and the effectiveness of therapeutic interventions.

[0007] Chemiluminescent immunoassay (CLIA) is a highly sensitive and specific diagnostic technique used to detect and quantify disease biomarkers in biological samples. The target antigen is bound to a specific antibody, which then reacts with a chemiluminescent substrate to produce light. The measured luminescence intensity is proportional to the concentration of the target biomarker, thereby achieving accurate diagnosis of the target disease. In the prior art, traumatic brain injury detection usually uses a single indicator (such as GFAP) for detection, but using only a single indicator is not reliable. In addition, when detecting biomarkers (such as GFAP, NFL, and UCH-L1), the accuracy and stability of chemiluminescence may be affected by various factors. Therefore, when detecting multiple biomarkers at the same time, there is an urgent need for an immunoassay method with high precision, low detection background signal, high sensitivity, and a wide linear range. Summary of the invention

[0008] The object of the present invention is to provide a GFAP, NFL and UCH-L1 combined detection kit, comprising: an acridinium ester labeled antibody working solution and a magnetic bead coated antibody working solution; the acridinium ester labeled antibody working solution contains acridinium ester labeled GFAP, NFL and UCH-L1 antibodies, and the magnetic bead coated antibody working solution contains magnetic bead coated GFAP, NFL and UCH-L1 antibodies;

[0009] The magnetic bead-coated antibody working solution is composed of: 0.2-0.4 mg / mL magnetic bead-coated GFAP, NFL and UCH-L1 antibodies, 20-50 mM Tris, 100-200 mM NaCl, 20-50 mM MES, 1.5-2% BSA, 1.5-2% sucrose, 0.1-0.2% Tween-20, 0.5-1% bilirubin oxidase, 0.05-0.1% ascorbic acid oxidase and 0.005-0.01% Proclin300, and the pH of the working solution is 7.0-7.5;

[0010] The acridinium ester-labeled antibody working solution is composed of: 0.6-1.2 μg / mL acridinium ester-labeled GFAP, NFL and UCH-L1 antibodies, 20-50 mM Tris, 100-200 mM NaCl, 1.5-2% BSA, 1.5-2% sucrose, 0.1-0.2% Tween-20, 0.5-1% bilirubin oxidase, 0.05-0.1% ascorbic acid oxidase and 0.005-0.01% Proclin300, and the pH of the working solution is 6.0-6.5.

[0011] The present invention has conducted in-depth research on the working solution of magnetic beads coated with antibodies and the working solution of acridinium ester labeled antibodies. In the working solution, Tris buffer, NaCl, BSA, sucrose, Tween-20, bilirubin oxidase, ascorbic acid oxidase, and Proclin300 are used to effectively avoid mutual interference between GFAP, NFL, and UCH-L1 antigen detection, improve the detection sensitivity and accuracy of the three, and provide an effective, rapid, and accurate detection method for early screening of patients with brain trauma. More importantly, the applicant has found that by adding bilirubin oxidase and ascorbic acid oxidase to the working solution, the interference of bilirubin and ascorbic acid is effectively avoided, the reaction system is prevented from being turbid, and the substrate stability and the anti-interference ability of the reagent are enhanced. In particular, the sensitivity of NFL and UCH-L1 can be effectively improved. Preferably, the weight ratio of bilirubin oxidase and ascorbic acid oxidase is 5 to 10:1. More preferably, the weight ratio of the two is 10:1. In addition, adding 20-50 mM MES to the magnetic bead-coated antibody working solution is beneficial to maintaining the stability of the magnetic bead-coated antibody, and the detection kit has good long-term storage stability.

[0012] In addition, the applicant's research also found that the pH of the working solution will also affect antibody detection. The pH of the magnetic bead-coated antibody working solution is alkaline, which will affect the sensitivity, repeatability and recovery rate of antibody detection. Preferably, the pH of the magnetic bead-coated antibody working solution is 7.0-7.5, and the pH of the acridinium ester-labeled antibody working solution is 6.0-6.5.

[0013] In some embodiments, the kit further includes a treatment solution, which is composed of: 25-50 mM Tris, 100-200 mM NaCl, 2-3% urea, 1.5-2% BSA, 1.5-2% sucrose, 0.1-0.2% Tween-20, and 0.005-0.01% Proclin300; the pH of the treatment solution is 7.0-7.5. Pretreatment of the sample can effectively eliminate blood cells in plasma, avoid the possibility of blood cells swallowing magnetic beads, and thus improve the accuracy of the test. In particular, the addition of a small amount of urea can quickly eliminate red blood cells in plasma.

[0014] In some embodiments, the volume ratio of the treatment liquid to the sample to be tested is ≤ 3, preferably 1: 2. Studies have found that if the volume ratio of the treatment liquid to the sample to be tested is too large, the sample concentration is low, which affects the detection sensitivity of the sample.

[0015] In some embodiments, the sample-containing processing solution, GFAP, NFL and UCH-L1 magnetic bead-coated antibody working solution and acridinium ester-labeled GFAP, NFL and UCH-L1 antibody working solution are prepared in a volume ratio of 1:2:2.

[0016] In some embodiments, the combined detection kit further comprises a substrate solution, and the substrate solution is a chemiluminescent substrate solution.

[0017] The present invention also provides a preparation process of magnetic beads coated with GFAP, NFL and UCH-L1 antibodies, comprising the following steps:

[0018] 1) Dilute GFAP, NFL and UCH-L1 antibodies with dialysate, and then dialyze for 16-24 hours at 2-8°C to obtain a mixed coating antibody solution;

[0019] 2) Rinse the magnetic beads with coating buffer, add NHS and EDC solution to the washed magnetic beads, mix well and incubate for 0.5-1 hour to wash the magnetic beads;

[0020] 3) Add the magnetic beads from step 2) to the mixed coated antibody solution from step 1), rotate and mix well at 25°C, incubate for 1-3 hours, and wash the magnetic beads with magnetic bead washing solution 1;

[0021] 4) Add magnetic bead blocking solution, rotate and mix, incubate at 25°C for 2-4 hours, and wash the magnetic beads with magnetic bead washing solution 2 to obtain magnetic beads coated with GFAP, NFL and UCH-L1 antibodies.

[0022] In some embodiments, the dialysate in step 1) is 0.1 M PBS buffer with a pH of 7.8.

[0023] In some embodiments, the coating buffer in step 2) is 100 mM MES buffer at pH 5.0.

[0024] In some embodiments, the concentration of the NHS solution in step 2) is 0.02-0.1 mg / mL, and the concentration of the EDC solution is 0.02-0.1 mg / mL.

[0025] In some embodiments, in step 3), the magnetic beads: antibodies are mixed at a weight ratio of 50:1. The inventors of the present application have also tried different ratios during the experiment, among which a larger ratio resulted in poor antibody labeling effect, and a smaller ratio affected the substrate signal. In general, a weight ratio of 50:1 is preferred for best results.

[0026] In some embodiments, the magnetic bead washing solution 1 in step 3) consists of the following ingredients: 25 mM Tris, 150 mM NaCl, and 2% gelatin.

[0027] In some embodiments, the magnetic bead blocking solution in step 4) consists of the following components: 50 mM Tris, 1% BSA, 0.01% Triton X-100, and 0.09% NaN3.

[0028] In some embodiments, the pH of the magnetic bead blocking solution described in step 4) is 8.0.

[0029] In some embodiments, the magnetic bead washing solution 2 described in step 4) consists of the following components: 25 mM Tris, 150 mM NaCl.

[0030] In some embodiments, the magnetic beads are magnetic microparticles comprising carboxyl functional groups.

[0031] In some embodiments, the magnetic beads have a particle size of 20 to 60 nm. Preferably, the magnetic beads labeled with NFL and UCH-L1 antibodies are selected from two types of magnetic beads with a particle size of 20 to 40 nm and a particle size of 40 to 60 nm, and the weight ratio of the two is 3:1. This further improves the linear range and sensitivity of NFL and UCH-L1 detection.

[0032] The present invention also provides a preparation process of magnetic beads coated with GFAP, NFL and UCH-L1 antibodies, comprising the following steps:

[0033] (1) Dilute GFAP, NFL and UCH-L1 antibodies to 1 mg / mL with PBS buffer (0.1 M, pH 7.8), and then use PBS buffer at 2-8 °C to obtain a mixed coating antibody solution;

[0034] (2) Rinse the magnetic beads with pH 5.0 100 mM MES buffer, add NHS (concentration 0.1 mg / mL) and EDC (concentration 0.1 mg / mL) solutions to the washed magnetic beads, mix well, and incubate at 25°C for 30 minutes with rotation, then wash the magnetic beads again.

[0035] (3) Add the mixed coating antibody solution at a ratio of 50:1 (mass ratio) and rotate at 25°C to mix well and incubate for 2 hours. Wash the magnetic beads again using magnetic bead washing solution 1 (25 mM Tris, 150 mM NaCl, 2% gelatin).

[0036] (4) Add magnetic bead blocking solution and rotate to mix at 25°C for 3 hours. The magnetic bead blocking solution is 50 mM Tris, 1% BSA, 0.01% Triton X-100, 0.09% NaN3, pH 8.0. After incubation, the magnetic beads are washed three times with magnetic bead washing solution (25 mM Tris pH 7.8, 150 mM NaCl). The washed magnetic beads are resuspended to a concentration of 10 mg / mL and stored at 2-8°C until use. The magnetic beads are coated with GFAP, NFL and UCH-L1 antibodies.

[0037] The present invention also provides a process for preparing acridinium ester-labeled GFAP, NFL and UCH-L1 antibodies, comprising the following steps:

[0038] A) Dilute the GFAP, NFL and UCH-L1 labeled antibodies with dialysate, and then dialyze for 16-24 hours at 2-8°C to obtain a mixed labeled antibody solution;

[0039] B) diluting the acridinium ester DMSO solution with labeling buffer, mixing the diluted acridinium ester solution with the mixed antibody solution to be labeled, and reacting at 25°C with shaking for 2-3 hours;

[0040] C) Add magnetic bead blocking solution and block the reaction at 25°C for 20-50 min;

[0041] D) Purification by chromatography column to obtain acridinium ester labeled GFAP, NFL and UCH-L1 antibodies.

[0042] In some embodiments, the dialysate in step A) is 0.1 M PBS buffer, and the pH of the buffer is 7.8.

[0043] In some embodiments, the labeling buffer in step B) is 0.1 M PBS buffer, and the pH of the buffer is 7.8.

[0044] In some embodiments, in step B), the weight ratio of antibody to be labeled: acridinium ester is 5:1. The inventors of the present application have also tried different ratios during the experiment. A larger weight ratio resulted in poor antibody labeling effect, while a smaller weight ratio affected the substrate signal. Generally speaking, a weight ratio of 5:1 is preferred for better results.

[0045] In some embodiments, the blocking solution in step C) is a PBS solution containing 1% lysine, with a pH of 7.8. The inventors of the present application have also tried PBS solutions containing different proportions of lysine in the blocking agent during the experiment, and the lysine content was too high, such as 2%, and the signal value was generally not as good as the effect of low concentration.

[0046] In some embodiments, the purification buffer in step D) is 0.1 M PBS buffer, pH 7.8.

[0047] Furthermore, the present invention also provides a process for preparing acridinium ester-labeled GFAP, NFL and UCH-L1 antibodies, comprising the following steps:

[0048] (1) Dilute the GFAP, NFL and UCH-L1 labeled antibodies to 0.5 mg / mL with PBS buffer (0.1 M, pH 7.8), and then dialyze against PBS buffer (0.1 M, pH 7.8) at 2-8°C for 16-24 hours, changing the solution at least 4 times with an interval of at least 1 hour between each change, to obtain a mixed labeled antibody solution.

[0049] (2) Dilute the acridinium ester DMSO solution with PBS buffer (0.1 M, pH 7.8) and use it within 5 minutes after preparation. The mass ratio of labeled antibody: acridinium ester is 5:1. Mix the diluted acridinium ester solution with the mixed antibody solution to be labeled, and react at 25°C with shaking for 2 hours. Protect from light throughout the process.

[0050] (3) Add PBS solution (0.1 M, pH 7.8) containing 1% lysine to a final lysine concentration of 0.25% and incubate the reaction at 25°C for 30 min.

[0051] (4) Purification was performed by chromatography column using PBS buffer (0.1 M, pH 7.8). The purified acridinium ester-labeled antibody was resuspended to a concentration of 25 ug / mL and stored at 2-8°C until use. Thus, acridinium ester-labeled GFAP, NFL and UCH-L1 antibodies were obtained.

[0052] Furthermore, the present invention also provides a method for using a GFAP, NFL and UCH-L1 combined detection kit, comprising the following steps:

[0053] (1) Add the treatment solution to the sample to be tested and treat at 25°C for 5 min;

[0054] (2) The treatment solution containing the sample to be tested, the working solution of GFAP, NFL and UCH-L1 magnetic bead-coated antibodies and the working solution of GFAP, NFL and UCH-L1 acridinium ester-labeled antibodies were mixed in a volume ratio of 1:2:2 and reacted at 25°C for 5 min;

[0055] (3) subjecting the reaction solution to magnetic separation and collecting magnetic beads;

[0056] (4) Wash the magnetic beads, then add the chemiluminescent substrate solution, mix well, and detect the luminescence value.

[0057] In some embodiments, the treatment solution is composed of: 25-50 mM Tris, 100-200 mM NaCl, 2-3% urea, 1.5-2% BSA, 1.5-2% sucrose, 0.1-0.2% Tween-20, and 0.005-0.01% Proclin300, and the pH of the treatment solution is 7.0-7.5.

[0058] Furthermore, the present invention also provides a use of the above-mentioned GFAP, NFL and UCH-L1 combined detection kit in the preparation of a product for detecting GFAP, NFL and UCH-L1 antigens in early brain trauma.

[0059] Beneficial effects:

[0060] 1. The present invention provides a GFAP, NFL and UCH-L1 detection kit. By screening the components and contents of the acridinium ester labeled working solution and the magnetic bead labeled working solution, a stable working environment can be provided for the acridinium ester labeled GFAP, NFL and UCH-L1 antibodies and the magnetic beads labeled with the GFAP, NFL and UCH-L1 antibodies, and the specific identification and detection of GFAP, NFL and UCH-L1 can be achieved. The detection sensitivity is high, the detection range is wide, and the linear range is good. The minimum detection line of GFAP can reach 0.545 pg / mL, the minimum detection line of NFL can reach 0.476 pg / mL, and the minimum detection line of UCH-L1 can reach 1.568 pg / mL; at the same time, the repeatability and recovery rate of the test results of the kit meet the requirements. The GFAP, NFL and UCH-L1 detection kit provided by the present invention realizes the simultaneous detection of GFAP, NFL and UCH-L1, and the accuracy and detection efficiency are greatly improved.

[0061] 2. The GFAP, NFL and UCH-L1 detection kits provided by the present invention preferably pre-treat the samples to quickly eliminate blood cells in the plasma and effectively avoid the possibility of blood cells swallowing magnetic beads.

[0062] 3. The present invention provides a method for preparing a working solution of GFAP, NFL and UCH-L1 magnetic beads coated with antibodies. In the preparation process, NHS and EDC are first used to activate magnetic beads, thereby improving the coupling efficiency of GFAP, NFL and UCH-L1 antibodies with magnetic beads; MES buffer is used to dissolve magnetic beads to ensure the dispersion stability of magnetic beads, effectively preventing the detection signal from being affected by the coagulation of magnetic beads, thereby ensuring the accuracy of the detection results; when the kit is used for plasma detection, the components in the blood are complex. The preparation method provided by the present invention adds magnetic bead blocking solution after coupling GFAP, NFL and UCH-L1 antibodies with magnetic beads, thereby avoiding non-specific binding that may be generated when the samples are detected later, thereby further ensuring the accuracy of the detection results. The use of magnetic bead blocking solution to simultaneously achieve effective blocking of magnetic beads can ensure the activity of GFAP, NFL and UCH-L1 antibodies and improve the detection performance of the kit.

[0063] 4. The present invention also provides a method for preparing a working solution of GFAP, NFL and UCH-L1 acridinium ester-labeled antibodies. During the preparation process, a PBS solution containing lysine is added to terminate the activation reaction, thereby effectively avoiding the occurrence of nonspecific reactions and ensuring the stability of the conjugates of GFAP, NFL and UCH-L1 antibodies and acridinium esters as well as the activity of the antibodies.

[0064] 5. The present invention also provides a method for using the GFAP, NFL and UCH-L1 detection kit, which has simple operation, uniform reaction system and high stability. DETAILED DESCRIPTION

[0065] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0066] Unless otherwise specified, the reagents or consumables used in the present invention are commercially available.

[0067] Unless otherwise specified herein, % refers to mass percentage.

[0068] Example 1: Preparation of magnetic bead-coated antibody working solution and acridinium ester-labeled antibody working solution

[0069] (1) Preparation of magnetic beads coated with GFAP, NFL and UCH-L1 antibody working solution:

[0070] Dilute GFAP, NFL and UCH-L1 antibodies to 1 mg / mL with PBS buffer (0.1 M, pH 7.8), and then dialyze with PBS buffer (0.1 M, pH 7.8) at 2-8 °C for 16-24 hours (change the solution at least 4 times during this period, with an interval of at least 1 hour between each change to ensure that the dialysis ratio meets the requirements) to obtain a mixed coating antibody solution.

[0071] The magnetic beads were washed three times with pH 5.0 100 mM MES buffer. NHS (N-hydroxysuccinimide, concentration 0.1 mg / mL) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, concentration 0.1 mg / mL) solutions were added to the washed magnetic beads, mixed and incubated at 25°C for 30 minutes, and the magnetic beads were washed again. Then, the mixed coating antibody solution was added at a ratio of magnetic beads: antibody = 50:1 (mass ratio), incubated at 25°C for 2 hours, and the magnetic beads were washed again with magnetic bead washing solution (25 mM Tris, 150 mM NaCl, 2% gelatin). Then, add magnetic bead blocking solution and rotate and mix at 25°C for 3 hours. The magnetic bead blocking solution is 50 mM Tris, 1% BSA, 0.01% Triton X-100, 0.09% NaN3, pH8.0. After incubation, the magnetic beads are washed three times with magnetic bead washing solution (25 mM Tris pH 7.8, 150 mM NaCl). The washed magnetic beads are resuspended to a concentration of 10 mg / mL and stored at 2-8°C for use. The magnetic beads are coated with GFAP, NFL and UCH-L1 antibodies.

[0072] The magnetic beads coated with GFAP, NFL and UCH-L1 antibody working solution is composed of: 0.2 mg / mL magnetic beads coated with GFAP, NFL and UCH-L1 antibodies, 50 mM Tris, 150 mM NaCl, 50 mM MES, 1.5% BSA, 1.5% sucrose, 0.1% Tween-20, 0.5% bilirubin oxidase, 0.1% ascorbic acid oxidase and 0.005% Proclin300, and the pH of the working solution is 7.0-7.5.

[0073] (2) Preparation of acridinium ester labeled GFAP, NFL and UCH-L1 antibodies:

[0074] The GFAP, NFL and UCH-L1 antibodies to be labeled were diluted to 0.5 mg / mL with PBS buffer (0.1 M, pH 7.8), and then dialyzed with PBS buffer (0.1 M, pH 7.8) at 2-8°C for 16-24 hours (during which the solution was changed at least 4 times, with an interval of at least 1 hour between each change to ensure that the dialysis ratio meets the requirements) to obtain a mixed solution of the antibodies to be labeled.

[0075] Take 2 mg / mL acridinium ester DMSO solution, dilute the acridinium ester DMSO solution with PBS buffer (0.1 M, pH 7.8) (use within 5 minutes after preparation, light will reduce the performance of acridinium ester), label the antibody at a mass ratio of 5:1, mix the diluted acridinium ester solution with the mixed antibody solution to be labeled, shake and react at 25℃ for 2 hours, and protect from light throughout the process. Add PBS solution containing 1% lysine (0.1 M, pH 7.8) to make the final concentration of lysine reach 0.25%, and block at room temperature (25℃) for 30 min. Finally, use a chromatography column filled with G-25 for purification, and the purification buffer is PBS buffer (0.1 M, pH 7.8). The purified acridinium ester-labeled antibody is resuspended to a concentration of 25 ug / mL and stored at 2-8℃ for use, and acridinium ester-labeled GFAP, NFL and UCH-L1 antibodies are obtained.

[0076] The acridinium ester labeled GFAP, NFL and UCH-L1 antibody working solution is composed of: 0.6 μg / mL GFAP, NFL and UCH-L1 acridinium ester labeled antibodies, 50 mM Tris, 150 mM NaCl, 1.5% BSA, 1.5% sucrose, 0.1% Tween-20, 0.5% bilirubin oxidase, 0.1% ascorbic acid oxidase and 0.005% Proclin300, and the pH of the working solution is 6.0-6.5.

[0077] Implementation 2

[0078] (1) Preparation of magnetic beads coated with GFAP, NFL and UCH-L1 antibody working solution:

[0079] Dilute GFAP, NFL and UCH-L1 antibodies to 1 mg / mL with PBS buffer (0.1 M, pH 7.8), and then dialyze with PBS buffer (0.1 M, pH 7.8) at 2-8 °C for 16-24 hours (change the solution at least 4 times during this period, with an interval of at least 1 hour between each change to ensure that the dialysis ratio meets the requirements) to obtain a mixed coating antibody solution.

[0080] The magnetic beads were washed three times with pH 5.0 100 mM MES buffer. NHS (N-hydroxysuccinimide, concentration 0.1 mg / mL) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, concentration 0.1 mg / mL) solutions were added to the washed magnetic beads, mixed and incubated at 25°C for 30 minutes, and the magnetic beads were washed again. Then, the mixed coating antibody solution was added at a ratio of magnetic beads: antibody = 50:1 (mass ratio), incubated at 25°C for 2 hours, and the magnetic beads were washed again with magnetic bead washing solution (25 mM Tris, 150 mM NaCl, 2% gelatin). Then, add magnetic bead blocking solution and rotate and mix at 25°C for 3 hours. The magnetic bead blocking solution is 50 mM Tris, 1% BSA, 0.01% Triton X-100, 0.09% NaN3, pH8.0. After incubation, the magnetic beads are washed three times with magnetic bead washing solution (25 mM Tris pH 7.8, 150 mM NaCl). The washed magnetic beads are resuspended to a concentration of 10 mg / mL and stored at 2-8°C for use. The magnetic beads are coated with GFAP, NFL and UCH-L1 antibodies.

[0081] The magnetic bead-coated GFAP, NFL and UCH-L1 antibody working solution is composed of: 0.4 mg / mL GFAP, NFL and UCH-L1 magnetic bead-coated antibodies, 20 mM Tris, 150 mM NaCl, 20 mM MES, 2% BSA, 2% sucrose, 0.1% Tween-20, 1% bilirubin oxidase, 0.1% ascorbic acid oxidase and 0.01% Proclin300, and the pH of the working solution is 7.0-7.5.

[0082] (2) Preparation of acridinium ester labeled GFAP, NFL and UCH-L1 antibodies:

[0083] The GFAP, NFL and UCH-L1 antibodies to be labeled were diluted to 0.5 mg / mL with PBS buffer (0.1 M, pH 7.8), and then dialyzed with PBS buffer (0.1 M, pH 7.8) at 2-8°C for 16-24 hours (during which the solution was changed at least 4 times, with an interval of at least 1 hour between each change to ensure that the dialysis ratio meets the requirements) to obtain a mixed solution of the antibodies to be labeled.

[0084] Take 2 mg / mL acridinium ester DMSO solution, dilute the acridinium ester DMSO solution with PBS buffer (0.1 M, pH 7.8) (use within 5 minutes after preparation, light will reduce the performance of acridinium ester), label the antibody at a mass ratio of 5:1, mix the diluted acridinium ester solution with the mixed antibody solution to be labeled, shake and react at 25℃ for 120 minutes, and protect from light throughout the process. Add PBS solution containing 1% lysine (0.1 M, pH 7.8) to make the final concentration of lysine reach 0.25%, and block at room temperature (25℃) for 30 min. Finally, use a chromatography column filled with G-25 for purification, and the purification buffer is PBS buffer (0.1 M, pH 7.8). The purified acridinium ester-labeled antibody is resuspended to a concentration of 25 ug / mL and stored at 2-8℃ for use, and acridinium ester-labeled GFAP, NFL and UCH-L1 antibodies are obtained.

[0085] The acridinium ester labeled GFAP, NFL and UCH-L1 antibody working solution is composed of: 1.2 μg / mL GFAP, NFL and UCH-L1 acridinium ester labeled antibodies, 20 mM Tris, 150 mM NaCl, 2% BSA, 2% sucrose, 0.1% Tween-20, 1% bilirubin oxidase, 0.1% ascorbic acid oxidase and 0.01% Proclin300, and the pH of the working solution is 6.0-6.5.

[0086] Comparative Example 1

[0087] The only difference from Example 1 is that the pH of the working solution of the magnetic beads coated with GFAP, NFL and UCH-L1 antibodies is 8.0-9.0.

[0088] The only difference from Example 1 is that the pH of the acridinium ester labeled GFAP, NFL and UCH-L1 antibody working solution is 7.0-7.5.

[0089] Comparative Example 2

[0090] The only difference from Example 1 is that the magnetic bead-coated GFAP, NFL and UCH-L1 antibody working solution is composed of: 0.2 mg / mL GFAP, NFL and UCH-L1 magnetic bead-coated antibodies, 50 mM Tris, 150 mM NaCl, 1.5% BSA, 1.5% sucrose, 0.1% Tween-20 and 0.005% Proclin300, and the pH of the working solution is 7.0-7.5.

[0091] The acridinium ester labeled GFAP, NFL and UCH-L1 antibody working solution is composed of: 0.6 μg / mL GFAP, NFL and UCH-L1 acridinium ester labeled antibodies, 50 mM Tris, 150 mM NaCl, 1.5% BSA, 1.5% sucrose, 0.1% Tween-20 and 0.005% Proclin300, and the pH of the working solution is 6.0-6.5.

[0092] Test Example 1: Determination of samples

[0093] The method for using the GFAP, NFL and UCH-L1 detection kits comprises the following steps:

[0094] (1) Add a treatment solution to the sample to be tested and treat it at a temperature of 25°C for 5 min; wherein the volume ratio of the treatment solution to the sample to be tested is ≤3, preferably, the volume ratio of the treatment solution to the sample to be tested is 1:2. The treatment solution is composed of: 20 mM Tris, 150 mM NaCl, 3% urea, 2% BSA, 2% sucrose, 0.1% Tween-20, and 0.01% Proclin300, and the pH of the treatment solution is 7.0-7.5.

[0095] (2) The treatment solution containing the sample to be tested, the working solution of GFAP, NFL and UCH-L1 magnetic bead-coated antibodies and the working solution of GFAP, NFL and UCH-L1 acridinium ester-labeled antibodies were mixed in a volume ratio of 1:2:2 and reacted at 25°C for 5 min;

[0096] (3) subjecting the reaction solution to magnetic separation and collecting magnetic beads;

[0097] (4) Wash the magnetic beads, then add the chemiluminescent substrate solution, mix well, and detect the luminescence value.

[0098] Test Example 2: Performance Evaluation

[0099] (1) Linear detection

[0100] GFAP, NFL and UCH-L1 were diluted with standard diluent and prepared into calibration solutions of different concentrations. Then, the kit in the embodiment was used to detect the GFAP, NFL and UCH-L1 calibrators, and the luminescence intensity values ​​corresponding to each calibrator were read respectively. The standard curve was fitted with the concentration as the horizontal axis and the luminescence intensity as the vertical axis. The results showed that the linear correlation coefficients of GFAP, NFL and UCH-L1 were all >0.99.

[0101] (2) Minimum detection limit detection

[0102] The zero-concentration calibrator was used as a sample for detection, and the measurement was repeated 20 times to obtain the relative luminescence intensity (RLU) value of the 20 measurement results, and the mean value (M) and standard deviation (SD) were calculated to obtain M+2SD. A linear equation was obtained by performing a two-point regression fitting based on the concentration-RLU between the zero-concentration calibrator and the adjacent calibrator. The RLU value of M+2SD (positive reaction) or M-2SD (negative reaction) was substituted into the above equation to obtain the corresponding concentration value, which is the minimum detection limit. The test results are shown in Table 1.

[0103] Table 1 Test results

[0104] Minimum detection limit GFAP (pg / mL) NFL (pg / mL) UCH-L1 (pg / mL) Example 1 0.657 0.889 2.054 Example 2 0.545 0.476 1.568 Comparative Example 1 1.043 1.864 6.234 Comparative Example 2 0.943 4.142 5.562

[0105] (3) Repeatability test

[0106] Two samples with different concentration levels (high value and low value) were used, and each concentration sample was tested 10 times. The mean (M) and standard deviation (SD) of the 10 measurement results were calculated, and then the coefficient of variation (CV) was obtained according to the formula CV = SD / M × 100%. Among them, CV is the coefficient of variation, SD is the standard deviation of the 10 measurement results, and M is the mean of the 10 measurement results. The results show that the CV is within ≤8%.

[0107] Table 2 NFL repeatability test results

[0108]

[0109] Table 3 GFAP repeatability test results

[0110]

[0111] Table 4 UCH-L1 repeatability test results

[0112]

[0113] (4) Accuracy test

[0114] The samples of GFAP, NFL and UCH-L1 standards at high and low concentration levels were measured three times, and the relative deviation between the average value (denoted as M) and the labeled value was calculated. The relative deviation B of the measured concentration was calculated according to the formula B = (MT) / T × 100%, where B is the relative deviation, M is the average value of the measured concentration, and T is the labeled value. The results showed that the relative deviation was ≤10%.

[0115] Table 5 NFL accuracy test results

[0116]

[0117] Table 6 GFAP accuracy test results

[0118]

[0119] Table 7 UCH-L1 accuracy test results

[0120]

[0121] (5) Recovery rate test

[0122] High-concentration sample A is added to low-concentration sample B. (The volume ratio between added sample A and sample B is 1:9, and the total concentration after addition must be within the linear range of the kit). Mixed sample A+B, sample A and sample B are measured separately, and each is repeated 3 times. The average value is calculated, and the recovery rate is calculated according to the formula. R is the recovery rate, V is the volume of sample A liquid, V0 is the volume of sample B liquid, C is the average detection concentration of sample B liquid after adding A liquid, C0 is the average detection concentration of sample B liquid, and C s is the average detection concentration of sample A. The results show that the recovery rate is in the range of 85% to 115%.

[0123]

[0124] Table 8 NFL recovery rate determination results

[0125]

[0126] Table 9 GFAP recovery rate determination results

[0127]

[0128] Table 10 UCH-L1 recovery rate determination results

[0129]

[0130] Test case 3: Early diagnosis of patients with brain trauma

[0131] The samples were analyzed using the assay method of Test Example 1, and the biomarkers GFAP, NFL, and UCH-L1 with an area under the curve (AUC) greater than 0.8 were identified by receiver operating characteristic (ROC) curve analysis. The samples were placed in a luminescent immunoassay analyzer. The analyzer added each sample to each biomarker reagent component in turn, ensuring adequate mixing, incubation, and washing. Subsequently, the analyzer automatically measured and recorded the concentration of each biomarker. In patients with traumatic brain injury (TBI), early diagnosis and timely treatment are essential to improve prognosis. The levels of GFAP, NFL, and UCH-L1 in 25 patients with TBI and 25 healthy controls were analyzed through clinical studies.

[0132] A logistic regression model was used to predict the occurrence of TBI. The model formula is as follows:

[0133]

[0134] Among them, p is the probability of TBI, GFAP, NFL and UCH-L1 are the detected concentration values, β0 is the natural constant obtained by regression, β1, β2 and β3 are the regression coefficients of GFAP, NFL and UCH-L1. β0, β1, β2 and β3 are 0.981, 0.442, 0.579 and 0.398 respectively.

[0135] The experiment showed that the average GFAP level in patients with brain trauma was 715.38 pg / mL, the average NFL level was 97.43 pg / mL, and the average UCH-L1 level was 358.52 pg / mL. In contrast, the GFAP, NFL, and UCH-L1 levels in healthy controls were significantly lower, at 95.93 pg / mL, 10.27 pg / mL, and 19.21 pg / mL, respectively. The AUC of the combined diagnosis was 0.937, the specificity was 96.15%, and the sensitivity was 92.31%, which was better than the performance of single indicator detection.

[0136] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and substitutions can be made without departing from the technical principles of the present invention. These improvements and substitutions should also be regarded as the scope of protection of the present invention.

Claims

1. A GFAP, NFL and UCH-L1 combined detection kit, include: Acridinium ester labeled antibody working solution and magnetic bead coated antibody working solution; The acridinium ester labeled antibody working solution contains acridinium ester labeled GFAP, NFL and UCH-L1 antibodies, and the magnetic bead coated antibody working solution contains magnetic bead coated GFAP, NFL and UCH-L1 antibodies; The magnetic bead-coated antibody working solution is composed of: 0.2-0.4 mg / mL magnetic bead-coated GFAP, NFL and UCH-L1 antibodies, 20-50 mM Tris, 100-200 mM NaCl, 20-50 mM MES, 1.5-2% BSA, 1.5-2% sucrose, 0.1-0.2% Tween-20, 0.5-1% bilirubin oxidase, 0.05-0.1% ascorbic acid oxidase and 0.005-0.01% Proclin300, and the pH of the working solution is 7.0-7.5; The acridinium ester-labeled antibody working solution is composed of: 0.6-1.2 μg / mL acridinium ester-labeled GFAP, NFL and UCH-L1 antibodies, 20-50 mM Tris, 100-200 mM NaCl, 1.5-2% BSA, 1.5-2% sucrose, 0.1-0.2% Tween-20, 0.5-1% bilirubin oxidase, 0.05-0.1% ascorbic acid oxidase and 0.005-0.01% Proclin300, and the pH of the working solution is 6.0-6.5; The preparation process of the magnetic beads coated with GFAP, NFL and UCH-L1 antibodies comprises the following steps: 1) Dilute GFAP, NFL and UCH-L1 antibodies with dialysate, and then dialyze at 2-8°C for 16-24 hours to obtain a mixed coated antibody solution; the dialysate is 0.1 M PBS buffer with a pH of 7.8; 2) The magnetic beads are washed with a coating buffer, and NHS and EDC solutions are added to the washed magnetic beads, mixed and incubated for 0.5-1 hour to wash the magnetic beads; the coating buffer is a pH 5.0 100 mM MES buffer; 3) adding the magnetic beads from step 2) to the mixed coated antibody solution from step 1), rotating and mixing at 25° C. for 1-3 hours, and washing the magnetic beads with magnetic bead washing solution 1; the magnetic bead washing solution 1 is composed of the following components: 25 mM Tris, 150 mM NaCl, and 2% gelatin; 4) Add magnetic bead blocking solution and rotate and mix at 25°C for 2-4 hours, and wash the magnetic beads with magnetic bead washing solution 2 to obtain magnetic beads coated with GFAP, NFL and UCH-L1 antibodies; the magnetic bead blocking solution is composed of the following components: 50 mM Tris, 1% BSA, 0.01% Triton X-100, 0.09% NaN 3 ; The magnetic bead cleaning solution 2 is composed of the following components: 25 mM Tris, 150 mM NaCl; The preparation process of the acridinium ester-labeled GFAP, NFL and UCH-L1 antibodies comprises the following steps: A) diluting the GFAP, NFL and UCH-L1 labeled antibodies with a dialysate, and then dialyzing for 16-24 hours at 2-8° C. to obtain a mixed labeled antibody solution; the dialysate is 0.1 M PBS buffer, and the pH of the buffer is 7.8; B) diluting the acridinium ester DMSO solution with a labeling buffer, mixing the diluted acridinium ester solution with the mixed antibody solution to be labeled, and reacting at 25° C. with shaking for 2-3 hours; the labeling buffer is 0.1 M PBS buffer, and the pH of the buffer is 7.8; C) adding magnetic bead blocking solution and performing blocking reaction at 25°C for 20-50 min; the blocking solution is a PBS solution containing 1% lysine, with a pH of 7.8; D) Purification by chromatography column to obtain acridinium ester labeled GFAP, NFL and UCH-L1 antibodies.

2. The GFAP, NFL and UCH-L1 combined detection kit according to claim 1, Features: The kit further comprises a treatment solution, which is composed of 25-50 mM Tris, 100-200 mM NaCl, 2-3% urea, 1.5-2% BSA, 1.5-2% sucrose, 0.1-0.2% Tween-20, and 0.005-0.01% Proclin300.

3. The GFAP, NFL and UCH-L1 combined detection kit according to claim 2, Features: The volume ratio of the treatment liquid to the sample to be tested is 1:

2.

4. The GFAP, NFL and UCH-L1 combined detection kit according to claim 1, Features: In step 3), the beads were mixed at a mass ratio of 50:1.

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