A neurofilament light chain protein detection kit and a detection method thereof

Abstract

This application discloses a detection kit and method for neurofilament light chain protein, relating to the field of biodetection technology. The kit comprises 10-30 parts of magnetic bead working solution and 40-60 parts of luminescent label. The raw materials for preparing the magnetic bead working solution include N-hydroxysuccinimide-biotin, neurofilament light chain protein monoclonal antibody A, streptavidin magnetic microspheres, blocking solution, and magnetic bead buffer. The raw materials for preparing the luminescent label include neurofilament light chain protein monoclonal antibody B, aminated graphene oxide, dopamine hydrochloride, acridine succinimide ester, glutaraldehyde, bovine serum albumin, and luminescent label buffer. This application uses aminated graphene oxide as a signal amplification carrier, combined with a polydopamine film, which can provide abundant covalent binding sites for subsequent acridine ester-labeled antibodies, thereby significantly improving the detection sensitivity.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, and in particular to a neurofilament light chain protein detection kit and its detection method. Background Technology

[0002] Neurofilament light chain protein (NSCLT) is a neuronal cytoplasmic protein mainly distributed in the cell bodies and axons of central and peripheral neurons. It participates in the formation of the neuronal cytoskeleton and plays a crucial role in maintaining axonal diameter, radial growth, and signal transduction velocity. When nerve axons are damaged or degenerate, NSCLT is released from the site of injury into the intercellular matrix and further enters the cerebrospinal fluid and peripheral blood. The amount released is significantly positively correlated with the degree of axonal damage. Therefore, NSCLT is widely recognized as an important biomarker reflecting the process of nerve axonal injury and neurodegeneration. In recent years, numerous studies have shown that NSCLT levels have extremely high clinical value in the early diagnosis, differential diagnosis, disease progression monitoring, and efficacy evaluation of various neurological diseases, including Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, and traumatic brain injury. It can even reflect the presence of nerve damage before the appearance of clinical symptoms.

[0003] Currently, the detection of neurofilament light chain proteins mainly relies on immunological methods, including enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay, single-molecule array technology, and magnetic microparticle chemiluminescence immunoassay. However, due to the low concentration of neurofilament light chain proteins in blood, the detection sensitivity of traditional ELISA and conventional chemiluminescence immunoassay is limited, and there is a risk of missed diagnoses.

[0004] Magnetic microparticle chemiluminescence immunoassay has demonstrated significant application value in the field of bioanalysis due to its combination of high sensitivity and high specificity of immunoassay. However, in conventional magnetic microparticle chemiluminescence kits, the active sites of antibodies are often masked after coupling with the magnetic beads during the magnetic bead coating process, resulting in insufficient effective antibody capture density and thus unsatisfactory actual detection sensitivity of the kit. Summary of the Invention

[0005] To improve the sensitivity of detection, this application provides a neurofilament light chain protein detection kit and a detection method thereof.

[0006] In a first aspect, this application provides a neurofilament light chain protein detection kit, which adopts the following technical solution: A neurofilament light chain protein detection kit includes 10-30 parts of magnetic bead working solution and 40-60 parts of luminescent label. The raw materials for preparing the magnetic bead working solution include N-hydroxysuccinimide-biotin, neurofilament light chain protein monoclonal antibody A, streptavidin magnetic microspheres, blocking solution, and magnetic bead buffer. The raw materials for preparing the luminescent label include neurofilament light chain protein monoclonal antibody B, aminated graphene oxide, dopamine hydrochloride, acridine succinimide ester, glutaraldehyde, bovine serum albumin, and luminescent label buffer.

[0007] By adopting the above technical solution, this application achieves core optimization of the luminescent label from the signal end and, combined with the avidin-biotin amplification system at the capture end, a significant improvement in detection sensitivity is achieved. Specifically, the luminescent label uses aminated graphene oxide as the signal amplification carrier. First, dopamine hydrochloride self-polymerizes on the surface of aminated graphene oxide under weakly alkaline conditions to form a polydopamine film. This film is rich in catechol and amino functional groups, which can synergistically interact with the glutaraldehyde crosslinking agent to provide abundant and uniform covalent binding sites for the subsequent acridinium ester-labeled antibody B. Based on this, utilizing the high specific surface area of ​​graphene oxide, a large number of antibody B molecules can be efficiently coupled onto a single nanocarrier, thereby significantly increasing the effective luminescent molecule loading on a single luminescent label and achieving exponential signal amplification. In addition, the blocking treatment with bovine serum albumin effectively blocks the residual non-specific binding sites on the carrier, significantly reducing the detection background signal. At the capture end, streptavidin magnetic microspheres efficiently capture antibody A through the biotin-streptavidin system, further ensuring the effective enrichment of the target antigen. This synergistic improvement in signal-to-noise ratio from both the capture and detection ends significantly enhances the sensitivity of the detection.

[0008] Optionally, the preparation steps of the aminated graphene oxide include: mixing graphene oxide and deionized water, ultrasonically dispersing, adding ethylenediamine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, reacting at room temperature, centrifuging to remove the supernatant, washing, and drying to obtain aminated graphene oxide.

[0009] By employing the above-described technical solution, this application activates the carboxyl groups on the surface of graphene oxide with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and then performs an amidation reaction with ethylenediamine under the catalysis of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, thereby introducing a large number of amino functional groups onto the surface of graphene oxide. These amino groups enhance the dispersion stability of graphene oxide in the aqueous phase and provide sufficient active anchor sites for the subsequent uniform deposition of polydopamine films and glutaraldehyde crosslinking reaction, thus laying the foundation for the efficient immobilization of antibody B. Simultaneously, this reaction is carried out at room temperature without introducing strong acids, strong alkalis, or high-temperature treatment, effectively maintaining the structural integrity of the graphene oxide sheets and avoiding sheet stacking and aggregation, which is beneficial for maintaining the large specific surface area advantage of the nanocarrier.

[0010] Optionally, the graphene oxide sheet diameter is 100-300 nm.

[0011] By adopting the above technical solution, this application uses graphene oxide with a sheet diameter of 100-300 nm. While ensuring a large specific surface area, it optimizes the spatial accessibility of edge carboxyl and amino functional groups, which is beneficial for the uniform modification of subsequent polydopamine films and the efficient coupling of antibody B. This ensures that a single nanocarrier can load enough luminescent molecules to achieve signal amplification. At the same time, this sheet diameter range avoids the problems of accelerated material sedimentation rate and sheet stacking and agglomeration caused by excessively large particle size, which is conducive to maintaining the long-term uniform dispersion of luminescent markers in solution.

[0012] Optionally, the preparation steps of the luminescent label include: mixing aminated graphene oxide and tris(hydroxymethyl)aminomethane hydrochloride buffer, ultrasonically dispersing, adding dopamine hydrochloride, stirring evenly, adding glutaraldehyde solution, oscillating for activation, centrifuging, and resuspending in phosphate buffer to obtain a dispersion; mixing neurofilament light chain protein monoclonal antibody B with phosphate buffer to obtain antibody B mixture; mixing acridine succinimide ester and N,N-dimethylformamide, adding to antibody B mixture, reacting in the dark, separating and purifying through a dialysis bag, then adding the dispersion, stirring evenly, adding bovine serum albumin, blocking reaction, centrifuging, and adding luminescent label buffer to obtain the luminescent label.

[0013] Furthermore, the raw materials for preparing the luminescent labeling buffer include disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, bovine serum albumin, Proclin 300 antibacterial agent, and polysorbate-20.

[0014] Optionally, the preparation steps of the magnetic bead working solution include: adding N-hydroxysuccinimide-biotin to neurofilament light chain protein monoclonal antibody A, incubating, adding phosphate buffer, centrifuging, removing the supernatant, adding phosphate buffer to obtain biotin-labeled neurofilament light chain protein monoclonal antibody A; washing streptavidin magnetic microspheres with phosphate buffer, magnetically separating to remove the supernatant, adding the biotin-labeled neurofilament light chain protein monoclonal antibody A, mixing, then adding blocking buffer for blocking reaction, after the reaction is completed, magnetically separating again to remove the supernatant, adding magnetic bead buffer to obtain the magnetic bead working solution.

[0015] Furthermore, the raw materials for preparing the magnetic bead buffer solution include tris(hydroxymethyl)aminomethane, sodium chloride, Proclin 300 antibacterial agent, polysorbate-20, and bovine serum albumin.

[0016] Furthermore, the raw materials for preparing the blocking solution include bovine serum albumin and tris(hydroxymethyl)aminomethane hydrochloride buffer.

[0017] Optionally, the concentration of streptavidin magnetic microspheres in the magnetic bead working solution is 0.1-1 mg / mL, and the concentration of neurofilament light chain protein monoclonal antibody B in the luminescent label is 0.01-5 μg / mL.

[0018] By adopting the above technical solution, this application controls the concentration of streptavidin magnetic microspheres in the magnetic bead working solution to 0.1-1 mg / mL and the concentration of neurofilament light chain protein monoclonal antibody B in the luminescent label to 0.01-5 μg / mL. This allows the capture efficiency to match the signal amplification efficiency, avoiding both the signal dilution effect caused by excessive capture and insufficient signal, and the background increase caused by excessive signal and insufficient capture, thereby achieving the optimal signal-to-noise ratio.

[0019] Optionally, the calibrator may also include a calibrator in which the concentration of neurofilament light chain protein antigen is 5-2000 pg / mL.

[0020] By adopting the above technical solution, the calibrator of this application can provide a reliable external standard reference for the accurate quantification of the sample to be tested, thereby ensuring that the neurofilament light chain protein at different concentration levels in clinical samples can be accurately quantified.

[0021] Optionally, it may also include quality control materials, wherein the concentration of neurofilament light chain protein antigen in the quality control materials is 50-1000 pg / mL.

[0022] By adopting the above technical solution, the quality control products of this application can ensure the accuracy and repeatability of test results by monitoring the effectiveness of the entire testing process.

[0023] Secondly, this application provides a detection method for neurofilament light chain protein using a neurofilament light chain protein detection kit, employing the following technical solution: A method for detecting neurofilament light chain proteins using a neurofilament light chain protein detection kit, comprising the following steps: S1. Take the sample to be tested, add the magnetic bead working solution and the luminescent marker, mix well, incubate, magnetically separate, wash, and obtain the complex. S2. Detect the intensity of the luminescent signal of the complex and calculate the concentration of neurofilament light chain protein in the sample to be tested.

[0024] In summary, this application includes at least one of the following beneficial technical effects: 1. This application focuses on optimizing the luminescent label at the signal end and combines it with an avidin-biotin amplification system at the capture end, achieving a significant improvement in detection sensitivity. Specifically, the luminescent label uses aminated graphene oxide as the signal amplification carrier. First, dopamine hydrochloride self-polymerizes on the surface of aminated graphene oxide under weakly alkaline conditions to form a polydopamine film. This film is rich in catechol and amino functional groups, which can synergistically interact with the glutaraldehyde crosslinking agent to provide abundant and uniform covalent binding sites for the subsequent acridinium ester-labeled antibody B. Based on this, utilizing the high specific surface area of ​​graphene oxide, a large number of antibody B molecules can be efficiently coupled onto a single nanocarrier, thereby significantly increasing the effective luminescent molecule loading on a single luminescent label and achieving exponential signal amplification. In addition, the blocking treatment with bovine serum albumin effectively blocks the residual non-specific binding sites on the carrier, significantly reducing the detection background signal. At the capture end, streptavidin magnetic microspheres efficiently capture antibody A through the biotin-streptavidin system, further ensuring the effective enrichment of the target antigen. This synergistically improves the signal-to-noise ratio from both the capture and detection ends, thereby significantly enhancing the detection sensitivity. 2. This application activates the carboxyl groups on the surface of graphene oxide with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and then performs an amidation reaction with ethylenediamine under the catalysis of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, thereby introducing a large number of amino functional groups on the surface of graphene oxide. These amino groups can enhance the dispersion stability of graphene oxide in the aqueous phase, and provide sufficient active anchor sites for the subsequent uniform deposition of polydopamine films and glutaraldehyde crosslinking reaction, thus laying the foundation for the efficient immobilization of antibody B. At the same time, the reaction is carried out at room temperature without the introduction of strong acids, strong bases or high temperature treatment, effectively maintaining the structural integrity of graphene oxide sheets, avoiding sheet stacking and agglomeration, and helping to maintain the large specific surface area advantage of the nanocarrier. Detailed Implementation

[0025] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0026] This application discloses a neurofilament light chain protein detection kit, comprising 10-30 parts of magnetic bead working solution and 40-60 parts of luminescent label. The raw materials for preparing the magnetic bead working solution include N-hydroxysuccinimide-biotin, neurofilament light chain protein monoclonal antibody A, streptavidin magnetic microspheres, blocking solution, and magnetic bead buffer. The raw materials for preparing the luminescent label include neurofilament light chain protein monoclonal antibody B, aminated graphene oxide, dopamine hydrochloride, acridine succinimide ester, glutaraldehyde, bovine serum albumin, and luminescent label buffer.

[0027] A method for detecting neurofilament light chain proteins using a neurofilament light chain protein detection kit, comprising the following steps: S1. Take the sample to be tested, add the magnetic bead working solution and the luminescent marker, mix well, incubate, magnetically separate, wash, and obtain the complex. S2. Detect the intensity of the luminescent signal of the complex and calculate the concentration of neurofilament light chain protein in the sample to be tested.

[0028] All raw materials used in the embodiments of this application are commercially available, wherein: Tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 8.5, 10 mM, Dalian Meilun Biotechnology Co., Ltd. Phosphate buffer, pH 7.4, Shanghai Kanglang Biotechnology Co., Ltd. Bovine serum albumin, Shanghai Aladdin Biochemical Technology Co., Ltd.; Proclin 300 antibacterial agent, Beijing Wokai Biotechnology Co., Ltd.; Polysorbate-20, Beijing Wokai Biotechnology Co., Ltd.; Acridine succinimide ester, Hubei Xindesheng Materials Technology Co., Ltd.; D-Trehalose, Shanghai Aladdin Biochemical Technology Co., Ltd.; Ethylenediaminetetraacetic acid (EDTA), Shanghai Aladdin Biochemical Technology Co., Ltd. Anhydrous N,N-dimethylformamide, Shanghai Aladdin Biochemical Technology Co., Ltd.; Graphene oxide, sheet diameter 100-300nm, Xi'an Qiyue Biotechnology Co., Ltd. Dopamine hydrochloride, Shanghai Aladdin Biochemical Technology Co., Ltd. Ethylenediamine, Shanghai Aladdin Biochemical Technology Co., Ltd.; 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, Shanghai Aladdin Biochemical Technology Co., Ltd.; N-Hydroxysuccinimide-Biotin, Shanghai Enzyme-Linked Biotechnology Co., Ltd.; Neurofilament light chain protein monoclonal antibody A, Shanghai Medis Medical Technology Co., Ltd.; Neurofilament light chain protein monoclonal antibody B, Shanghai Medis Medical Technology Co., Ltd.

[0029] Preparation Example 1 Preparation of antigen buffer: 2.9 g of disodium hydrogen phosphate dodecahydrate, 0.29 g of sodium dihydrogen phosphate dihydrate, 8.77 g of sodium chloride, 20 g of D-trehalose, and 3.72 g of EDTA were added to 100 mL of ultrapure water and stirred until homogeneous. 1.03 mL of Proclin 300 antibacterial agent and 1 mL of polysorbate-20 were added and stirred until completely dissolved. The pH was adjusted to 7.4, and then 30 g of bovine serum albumin was added. The mixture was stirred until homogeneous and the volume was brought to 1 L to obtain the antigen buffer.

[0030] Preparation Example 2 Preparation of magnetic bead buffer: Add 6.06g of tris(hydroxymethyl)aminomethane and 8.18g of sodium chloride to 100mL of ultrapure water and stir until homogeneous. Add 1.03g of Proclin300 antibacterial agent and 1g of polysorbate-20 and stir until completely dissolved. Adjust the pH to 7.4, then add 10g of bovine serum albumin and stir until completely dissolved. Make up the volume to 1L to obtain magnetic bead buffer.

[0031] Preparation Example 3 Preparation of luminescent labeling buffer: 2.9 g of disodium hydrogen phosphate dodecahydrate, 0.29 g of sodium dihydrogen phosphate dihydrate, and 8.77 g of sodium chloride were added to 100 mL of ultrapure water and stirred until homogeneous. 1.03 g of Proclin 300 antibacterial agent and 1 g of polysorbate-20 were added and stirred until completely dissolved. The pH was adjusted to 7.4, and then 10 g of bovine serum albumin was added and stirred until completely dissolved. The volume was adjusted to 1 L to obtain the luminescent labeling buffer.

[0032] Preparation Example 4 Preparation of blocking solution: Mix 3g of bovine serum albumin and 100mL of tris(hydroxymethyl)aminomethane hydrochloride buffer and stir until completely dissolved to obtain the blocking solution.

[0033] Preparation Example 5 The preparation steps of aminated graphene oxide include: mixing 5 mg of graphene oxide with 20 mL of deionized water, ultrasonically dispersing at 300 W for 1 h, adding 1 mg of ethylenediamine and 2 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, reacting at room temperature for 3 h, centrifuging at 1000 rpm for 10 min, removing the supernatant, washing 3 times with deionized water, and drying at 80 °C for 6 h to obtain aminated graphene oxide.

[0034] Example 1: Preparation method of neurofilament light chain protein detection kit Preparation of magnetic bead working solution: 1 mg N-hydroxysuccinimide-biotin was added to 20 mg neurofilament light chain protein monoclonal antibody A, incubated at room temperature for 30 min, phosphate buffer was added, and centrifuged at 12000 rpm for 5 min. The supernatant was removed, and phosphate buffer was added until the concentration of neurofilament light chain protein monoclonal antibody A was 1 mg / mL to obtain biotin-labeled neurofilament light chain protein monoclonal antibody A. 10 mg of streptavidin magnetic microspheres were washed with phosphate buffer, magnetically separated to remove the supernatant, 0.1 mg of biotin-labeled neurofilament light chain protein monoclonal antibody A was added, and the mixture was stirred at room temperature for 1 h. Then, 20 μL of the blocking solution obtained in Preparation Example 4 was added, and the blocking reaction was carried out for 1 h. The supernatant was removed again by magnetic separation, and the magnetic bead buffer obtained in Preparation Example 2 was added to adjust the concentration of streptavidin magnetic microspheres to 0.2 mg / mL to obtain the magnetic bead working solution.

[0035] Preparation of luminescent markers: 0.2 mg of aminated graphene oxide obtained in Preparation Example 5 was mixed with 10 mL of tris(hydroxymethyl)aminomethane hydrochloride buffer, and ultrasonically dispersed at 300 W for 30 min. 0.2 mg of dopamine hydrochloride was added, and the mixture was stirred at 200 rpm for 3 h. 2 μ L of 25% (v / v) glutaraldehyde solution (25 mL of glutaraldehyde was diluted to 100 mL with deionized water) was added, and the mixture was activated by shaking at 200 rpm for 1.5 h. After activation, centrifuge at 10,000 rpm for 10 min, remove the supernatant, resuspend and wash three times with phosphate buffer, and finally resuspend in 5 mL of phosphate buffer to obtain a dispersion. Dissolve 1 mg of neurofilament light chain protein monoclonal antibody B in 1 mL of phosphate buffer, add acridinium ester solution (0.1 mg acridinium succinimide ester and 50 μL of anhydrous N,N-dimethylformamide), react at 4 °C in the dark for 1.5 h, separate and purify through a dialysis bag with a molecular weight cutoff of 10 kDa, add the dispersion, shake at 200 rpm for 2 h, add 1 mg of bovine serum albumin, block reaction at room temperature for 1 h, centrifuge at 10,000 rpm for 10 min, remove the supernatant, resuspend the precipitate in the luminescent labeling buffer obtained in Preparation Example 3, adjust the concentration of neurofilament light chain protein monoclonal antibody B to 0.2 μg / mL, and obtain the luminescent label.

[0036] Preparation of calibrators: The neurofilament light chain protein antigen was diluted to concentrations of 5 pg / mL, 10 pg / mL, 20 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, 500 pg / mL, 1000 pg / mL, and 2000 pg / mL using the antigen buffer obtained in Preparation Example 1, to obtain calibrators with different concentrations of neurofilament light chain protein antigen; the antigen buffer obtained in Preparation Example 1 was used as the zero-concentration calibrator.

[0037] Preparation of quality control samples: The neurofilament light chain protein antigen was diluted to 50 pg / mL quality control sample 1 and 1000 pg / mL quality control sample 2 using the antigen buffer obtained in Preparation Example 1.

[0038] Example 2: Detection method using a neurofilament light chain protein detection kit Take 25 μL of the sample to be tested, add 20 μL of magnetic bead working solution and 50 μL of luminescent label, mix well, and incubate at 37℃ for 8 min to obtain a magnetic bead-coated antibody-antigen-luminescently labeled antibody complex. Wash the magnetic bead-coated antibody-antigen-luminescently labeled antibody complex with phosphate buffer, detect the luminescence value, and calculate the concentration of neurofilament light chain protein in the sample based on the standard curve plotted by the calibrator and the sample luminescence value.

[0039] Example 3: Stability Testing The neurofilament light chain protein detection kit obtained in Example 1 was stored at 2-8°C. Calibrators with a neurofilament light chain protein antigen concentration of 100 pg / mL were tested at storage times of 1, 4, 8, 12, 16, 20, 24, 28, and 32 months, with each sample tested three times. The detection stability was analyzed. The results showed that the neurofilament light chain protein detection kit has a shelf life of 18 months at 2-8°C.

[0040] Example 4: Specificity Detection A calibrator with a neurofilament light chain protein antigen concentration of 100 pg / mL was supplemented with 100 pg / mL of glial fibrillary acidic protein (GFAP), 200 pg / mL of brain-specific protein product 9.5 (PGP9.5), and 200 pg / mL of S100 calcium-binding protein (S100), respectively. The neurofilament light chain protein detection kit obtained in Example 1 was then used for detection. Experimental results showed that the presence of interfering substances had no effect on the detection of neurofilament light chain proteins, and no cross-reactivity was observed.

[0041] Example 5: Sensitivity Detection Using a zero-concentration calibrator as a sample, the neurofilament light chain protein detection kit obtained in Example 1 was used for repeated measurements 20 times. The luminescence values ​​of the 20 measurements were obtained, and the mean (M) and standard deviation (SD) were calculated to obtain the luminescence value corresponding to M+2SD. Separately, luminescence values ​​of 20 pg / mL neurofilament light chain protein were detected using the same kit, and the average luminescence value was calculated after two measurements. The average luminescence value of the zero-concentration calibrator was fitted to the average luminescence value of the calibrator with a neurofilament light chain protein antigen concentration of 20 pg / mL to obtain a linear equation. The luminescence value corresponding to M+2SD was then substituted into the linear equation to calculate the corresponding concentration value, which is the limit of detection (LOD), as shown in Table 1.

[0042] Table 1. Blank Limit Detection of Neurofilament Light Chain Protein Detection Kit

[0043] As shown in Table 1, by fitting the average luminescence value of the zero-concentration calibrator to the average luminescence value of the 20 pg / mL calibrator, a linear equation was obtained: y = 747.88x + 618.40, where y is the luminescence value and x is the sample concentration (in pg / mL). Based on the mean and standard deviation of the blank limits, the limit of detection for the neurofilament light chain protein assay kit was determined to be 0.09 pg / mL.

[0044] Example 6: Linear Range Detection The 2000 pg / mL neurofilament light chain protein was diluted with the antigen buffer obtained in Preparation Example 1, and then the concentration of neurofilament light chain protein was detected using the neurofilament light chain protein detection kit obtained in Example 1. The dilution ratios were 1, 1:2, 1:4, 1:20, 1:100, 1:200, 1:400, and 1:999. Each sample at each dilution was tested twice to obtain the luminescence value and detection concentration. The average of the two measurements for each sample was calculated, and the detection concentration of neurofilament light chain protein was also calculated. A linear regression analysis was performed with the dilution concentration as the independent variable and the mean of the measurement results (i.e., the detection concentration of neurofilament light chain protein) as the dependent variable. The correlation coefficients were obtained, and the results are shown in Table 2.

[0045] Table 2 Linear range of the neurofilament light chain protein detection kit

[0046] As shown in Table 2, the correlation coefficient is 0.9992 within the linear range of 2.24-2000.97 pg / mL, indicating that the neurofilament light chain protein detection kit of this application has good linearity in the range of 2.3-2000 pg / mL.

[0047] Example 7: Repeatability Detection The concentration of neurofilament light chain protein was detected using the neurofilament light chain protein detection kit obtained in Example 1 for quality control samples 1 and 2. Each sample was tested 10 times, and the mean and standard deviation of the 10 test results were calculated. The coefficient of variation was also calculated, and the results are shown in Table 3. Wherein, the coefficient of variation = (standard deviation / mean) × 100%.

[0048] Table 3. Reproducibility of the neurofilament light chain protein detection kit

[0049] As shown in Table 3, the coefficients of variation for quality control sample 1 and quality control sample 2 were within 2.43-3.09% after 10 tests, indicating that the neurofilament light chain protein detection kit of this application has good reproducibility.

[0050] Example 8: Accuracy Testing The concentrations of neurofilament light chain protein antigen at concentrations of 100 pg / mL, 200 pg / mL, and 1000 pg / mL were measured using the kit obtained in Example 1. Each sample was tested three times, and the average of the three test results was calculated. The relative deviation was also calculated, and the results are shown in Table 4. Relative deviation = (average value - standard value) / standard value × 100%.

[0051] Table 4. Accuracy of the Neurofilament Light Chain Protein Detection Kit

[0052] As shown in Table 4, the relative deviations of the calibrators with neurofilament light chain protein antigen concentrations of 100 pg / mL, 200 pg / mL, and 1000 pg / mL were within -3.22% to 0.68% when tested three times, indicating that the accuracy of the neurofilament light chain protein detection kit of this application is good.

[0053] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A neurofilament light chain protein detection kit, characterized by, The product comprises 10-30 parts of magnetic bead working solution and 40-60 parts of luminescent label. The raw materials for preparing the magnetic bead working solution include N-hydroxysuccinimide-biotin, neurofilament light chain protein monoclonal antibody A, streptavidin magnetic microspheres, blocking solution, and magnetic bead buffer. The raw materials for preparing the luminescent label include neurofilament light chain protein monoclonal antibody B, aminated graphene oxide, dopamine hydrochloride, acridine succinimide ester, glutaraldehyde, bovine serum albumin, and luminescent label buffer.

2. The neurofilament light chain protein detection kit according to claim 1, characterized in that, The preparation steps of the aminated graphene oxide include: mixing graphene oxide and deionized water, ultrasonically dispersing, adding ethylenediamine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, reacting at room temperature, centrifuging to remove the supernatant, washing, and drying to obtain aminated graphene oxide.

3. The neurofilament light chain protein detection kit according to claim 2, characterized in that, The graphene oxide has a sheet diameter of 100-300 nm.

4. The neurofilament light chain protein detection kit according to claim 1, characterized in that, The preparation steps of the luminescent label include: mixing aminated graphene oxide and tris(hydroxymethyl)aminomethane hydrochloride buffer, ultrasonically dispersing, adding dopamine hydrochloride, stirring evenly, adding glutaraldehyde solution, oscillating for activation, centrifuging, and resuspending in phosphate buffer to obtain a dispersion; mixing neurofilament light chain protein monoclonal antibody B with phosphate buffer to obtain antibody B mixture; mixing acridine succinimide ester and N,N-dimethylformamide, adding to antibody B mixture, reacting in the dark, separating and purifying through a dialysis bag, then adding the dispersion, stirring evenly, adding bovine serum albumin, blocking reaction, centrifuging, and adding luminescent label buffer to obtain the luminescent label.

5. The neurofilament light chain protein detection kit according to claim 4, characterized in that, The raw materials for preparing the luminescent labeling buffer include disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, bovine serum albumin, Proclin 300 antibacterial agent, and polysorbate-20.

6. The neurofilament light chain protein detection kit according to claim 1, characterized in that, The preparation steps of the magnetic bead working solution include: adding N-hydroxysuccinimide-biotin to neurofilament light chain protein monoclonal antibody A, incubating, adding phosphate buffer, centrifuging, removing the supernatant, adding phosphate buffer to obtain biotin-labeled neurofilament light chain protein monoclonal antibody A; washing streptavidin magnetic microspheres with phosphate buffer, magnetically separating to remove the supernatant, adding the biotin-labeled neurofilament light chain protein monoclonal antibody A, mixing, then adding blocking solution for blocking reaction, after the reaction is completed, magnetically separating again to remove the supernatant, adding magnetic bead buffer to obtain the magnetic bead working solution.

7. The neurofilament light chain protein detection kit according to claim 6, characterized in that, The raw materials for preparing the magnetic bead buffer solution include tris(hydroxymethyl)aminomethane, sodium chloride, Proclin 300 antibacterial agent, polysorbate-20, and bovine serum albumin.

8. The neurofilament light chain protein detection kit according to claim 6, characterized in that, The raw materials for preparing the blocking solution include bovine serum albumin and tris(hydroxymethyl)aminomethane hydrochloride buffer.

9. The neurofilament light chain protein detection kit according to claim 1, characterized in that, The concentration of streptavidin magnetic microspheres in the working solution of the magnetic beads is 0.1-1 mg / mL, and the concentration of neurofilament light chain protein monoclonal antibody B in the luminescent label is 0.01-5 μg / mL.

10. A method for detecting neurofilament light chain protein using the neurofilament light chain protein detection kit according to any one of claims 1 to 9, characterized in that, Includes the following steps: S1. Take the sample to be tested, add the magnetic bead working solution and the luminescent marker, mix well, incubate, magnetically separate, wash, and obtain the complex. S2. Detect the intensity of the luminescent signal of the complex and calculate the concentration of neurofilament light chain protein in the sample to be tested.

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