Vascular endothelial growth factor chemiluminescence detection kit and preparation method thereof

Abstract

The invention relates to the field of in vitro diagnostic reagents. The invention discloses a chemiluminescence detection kit for a vascular endothelial growth factor (VEGF). A reagent R1 comprises magnetic beads coated with a VEGF antibody 1, wherein the surfaces of the magnetic beads are covalently grafted with poly-zwitterionic polymers; and the R2 reagent comprises a VEGF antibody 2 marked by a silicon shell acridinium ester nano-cluster. The kit further comprises a calibration product and a quality control product, wherein the calibration product comprises solutions with VEGF antigen concentrations of 0, 20, 100, 400, 800 and 2000 pg / mL; the quality control product is a solution with the VEGF antigen concentration of 50pg / mL and a solution with the VEGF antigen concentration of 2,200 pg / mL respectively. The invention also provides a preparation method of the chemiluminiscence detection kit for the vascular endothelial growth factor. The invention also provides a detection method of the chemiluminiscence detection kit for the vascular endothelial growth factor. The invention also provides application of the chemiluminiscence detection kit for the vascular endothelial growth factor to detection of the concentration of the vascular endothelial growth factor. The chemiluminiscence detection kit for the vascular endothelial growth factor, provided by the invention, is high in anti-interference capability and good in long-term stability.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagents, specifically to a chemiluminescent detection kit for vascular endothelial growth factor and its preparation method. Background Technology

[0002] Vascular endothelial growth factor (VEGF) is a core cytokine regulating angiogenesis and plays a crucial role in tumor growth, metastasis, ischemic diseases, and inflammation. Quantitative detection of VEGF concentrations in serum or plasma is of significant clinical value for early screening of malignant tumors, efficacy evaluation, prognosis assessment, and guidance on the use of anti-angiogenic targeted drugs.

[0003] Currently, in vitro detection methods for VEGF mainly include enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLIA). Among them, magnetic particle chemiluminescence immunoassay has become the mainstream development direction due to its high degree of automation, good sensitivity, and wide detection range. However, existing technologies still have many problems: serum samples have complex compositions and contain various interfering substances such as hemoglobin, triglycerides, and bilirubin, which may lead to false positive or false negative results; traditional blocking and washing strategies are sometimes insufficient to completely eliminate high background and non-specific binding; and the overall stability of the kits is poor, with short shelf life after opening and short storage life.

[0004] Therefore, there is an urgent need to develop a chemiluminescent assay kit for vascular endothelial growth factor that can improve the stability of the kit and effectively resist interference from complex sample matrices, thereby providing more accurate, reliable and stable test results for clinical use. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a chemiluminescence immunoassay kit for vascular endothelial growth factor (VEGF) and its preparation method. The VEGF chemiluminescence immunoassay kit provided by this invention exhibits strong anti-interference capabilities and good long-term stability.

[0006] To address the above problems, the present invention provides the following technical solution: A chemiluminescent assay kit for vascular endothelial growth factor includes reagent R1 and reagent R2; wherein: The R1 reagent comprises magnetic beads coated with VEGF antibody 1 and covalently grafted with zwitterionic polymer. The R2 reagent comprises VEGF antibody 2 labeled with silicone acridine ester nanoclusters.

[0007] The vascular endothelial growth factor chemiluminescence detection kit described above also includes calibrators and quality control products. The calibrators include solutions with VEGF antigen concentrations of 0, 20, 100, 400, 800, and 2000 pg / mL, respectively; the quality control products are solutions with VEGF antigen concentrations of 50 and 2000 pg / mL, respectively.

[0008] Based on the same inventive concept, this invention provides a method for preparing a chemiluminescent detection kit for vascular endothelial growth factor, including the preparation of reagent R1, specifically comprising the following steps: S11. Prepare magnetic beads with surface covalently grafted with a zwitterionic polymer; preferably, the zwitterionic polymer is polymethacrylate sulfobetaine; S12. Mix the magnetic beads prepared in S11 with VEGF antibody 1 in the conjugation buffer, resuspend the magnetic beads and incubate. S13. Then, add blocking buffer for incubation, magnetic separation, washing, and resuspending in storage buffer to obtain VEGF antibody 1 coated surface covalently grafted zwitterionic polymer magnetic beads.

[0009] In the preparation method of the vascular endothelial growth factor chemiluminescence detection kit described above, in step S12, the mass ratio of the magnetic beads to VEGF antibody 1 is 1:(0.5-5). Preferably, in step S12, the mass ratio of the magnetic beads to VEGF antibody 1 is 1:1.

[0010] In the preparation method of the vascular endothelial growth factor chemiluminescence detection kit as described above, in step S12, the coupling buffer is a borate or phosphate buffer with a pH of 7.4–8.5, and contains 0.1%–1% (w / v) BSA. Preferably, in step S12, the coupling buffer is a borate buffer with a pH of 7.4, and contains 1% (w / v) BSA.

[0011] As described above, in the preparation method of the vascular endothelial growth factor chemiluminescence detection kit, in step S13, the blocking buffer is a 0.01 M to 0.05 M phosphate buffer with pH 7.4 to 8.5, containing 10 mM propargylamine, 5% (w / v) trehalose, 1% (w / v) BSA, and 1% (w / v) casein. Preferably, in step S13, the blocking buffer is a 0.02 M phosphate buffer with pH 7.8, containing 10 mM propargylamine, 5% (w / v) trehalose, 1% (w / v) BSA, and 1% (w / v) casein.

[0012] The preparation method of the vascular endothelial growth factor chemiluminescence detection kit described above also includes the preparation of reagent R2, specifically including the following steps: S21. Preparation of silicone-shell acridine ester nanoclusters; Specifically, the preparation of silicone-shell acridine ester nanoclusters includes the following steps: Take aminosilicone spheres into a centrifuge tube, centrifuge and discard the supernatant, wash with borate buffer, resuspend in borate buffer containing acridine ester, mix by rotation at room temperature in the dark, resuspend in anhydrous ethanol, add tetraethyl orthosilicate and ammonia water solution, stir at room temperature for 3-5 h, centrifuge and discard the supernatant, wash with ethanol-water solution with a volume ratio of 1:1, resuspend in PBS buffer, place in a dialysis bag and dialyze for 3-5 h to remove unreacted small molecule reagents and byproducts, collect the product, and dilute to volume with PBS buffer to obtain silicone-shell acridine ester nanocluster stock solution, and store at 4℃ in the dark; S22. The silica-shell acridinium ester nanoclusters prepared in step S21 are uniformly mixed with VEGF antibody 2 and incubated at room temperature for 0.5-2 hours to obtain a mixture. The incubated mixture is then transferred to a dialysis bag and dialyzed with dialysis solution for 3-5 days. S23. After dialysis, collect the solution in the dialysis bag to obtain purified VEGF antibody 2 labeled with silicone acridine nanoclusters.

[0013] This invention is based on the double-antibody sandwich principle. VEGF antibody 1 (immobilized on magnetic beads) and VEGF antibody 2 (labeled signal) respectively recognize different epitopes of the VEGF antigen, forming a stable complex of "magnetic beads-VEGF antibody 1-antigen-VEGF antibody 2-signal nanoclusters". This design ensures high detection specificity. Utilizing the superparamagnetism of the magnetic beads, efficient and rapid separation and washing of the target complex from the sample matrix and unreacted reagents can be achieved through a simple magnetic field, greatly reducing interference from non-specific binding. In an alkaline hydrogen peroxide environment, the ester bonds of the acridine ester structure of the silica-shell acridine ester nanoclusters break, forming an unstable dioxone intermediate. Upon decomposition, it emits photons. The relative intensity (RLU) of the chemiluminescence signal is positively correlated with the concentration of VEGF antigen in the sample within the effective range. By establishing a standard curve using a series of calibrators, accurate quantification of the sample can be achieved.

[0014] In this invention, reagent R1 comprises magnetic beads coated with VEGF antibody 1 and covalently grafted with a zwitterionic polymer. The zwitterionic polymer, such as polymethacrylate sulfobetaine, forms a dense "hydration layer" on the surface of the magnetic beads through the strong hydration of its intramolecular positive and negative charged groups. This hydration layer effectively repels the non-specific adsorption of biomolecules such as proteins and cell debris, significantly reducing background signals and improving the signal-to-noise ratio from a physicochemical perspective. The zwitterionic polymer covalently and orients VEGF antibody 1 through chemical coupling, forming a brush-like structure that provides three-dimensional space and flexibility, increases antibody loading, and facilitates the orientation of its antigen-binding domains towards the solution, maintaining high antibody bioactivity and orientation consistency.

[0015] The R2 reagent comprises VEGF antibody 2 labeled with silica-shelled acridine ester nanoclusters. Because hundreds to thousands of acridine ester molecules are embedded or covalently bonded within a single silica nanoshell of the silica-shelled acridine ester nanocluster, the formation of a single immune complex triggers synchronous luminescence of all acridine ester molecules within the nanocluster in an alkaline hydrogen peroxide system. This achieves a geometric amplification from a single binding event to a clustered chemiluminescent signal, resulting in ultra-high sensitivity. The silica shell provides a robust physical barrier for the internal acridine ester molecules, isolating them from environmental factors such as moisture, oxygen, and quenchers. This significantly improves the storage stability of the label and the uniformity of luminescence efficiency, overcoming the drawback of easy hydrolysis of the ester bonds of small-molecule acridine esters, and enhancing the stability of the reagent kit.

[0016] Based on the same inventive concept, this invention provides a detection method for the vascular endothelial growth factor chemiluminescence detection kit as described above or the vascular endothelial growth factor chemiluminescence detection kit prepared by the preparation method as described above, comprising the following steps: S41. Sample addition and first incubation: Add the sample to be tested to the reaction vessel, then add the R1 reagent, mix well, and incubate at 37°C for 10-30 min to form VEGF antigen-antibody 1 complex; S42. Magnetic separation and washing: Place the reaction vessel in a magnetic field and let it stand for 1 to 3 minutes to enrich the magnetic beads. Discard the supernatant. Add washing solution, mix well, and perform magnetic separation again and discard the supernatant. Repeat washing 1 to 3 times. S43. Labeling and second incubation: Add the R2 reagent to the washed magnetic bead complex, mix well, and incubate at 37°C for 5–20 min to form a double antibody sandwich complex; S44. Repeat magnetic separation and washing: Repeat step S42 to remove unbound R2 reagent; S45. Chemiluminescence detection: The washed magnetic beads are resuspended in a chemiluminescence substrate solution, which includes solution A and solution B, wherein solution A is a citrate buffer solution containing 0.1% to 1.0% (w / v) hydrogen peroxide, and solution B is a sodium hydroxide solution with a pH of 9.0 to 11.0; after mixing, the relative luminescence intensity value is immediately read on the chemiluminescence immunoassay analyzer. S46. Result Calculation: Plot a standard curve with the calibrator concentration on the x-axis and the corresponding luminescence intensity value on the y-axis; calculate the VEGF antigen concentration of the sample to be tested using the standard curve based on the luminescence intensity value of the sample.

[0017] In the detection method of the vascular endothelial growth factor chemiluminescence assay kit described above, the volume ratio of the test sample to reagent R1 and reagent R2 is 1:(0.5-2):(0.5-2). Preferably, the volume ratio of the test sample to reagent R1 and reagent R2 is 1:2:2.

[0018] Based on the same inventive concept, this invention provides the application of the vascular endothelial growth factor chemiluminescence detection kit as described above or the vascular endothelial growth factor chemiluminescence detection kit prepared by the preparation method as described above in detecting the concentration of vascular endothelial growth factor.

[0019] Compared with existing technologies, the effects and advantages of this invention are: 1. This invention provides a chemiluminescent detection kit for vascular endothelial growth factor (VEGF), based on a double-antibody sandwich method. VEGF antibody 1 is immobilized on the surface of magnetic beads as a capture antibody; VEGF antibody 2 is bound to a silica-based acridine ester nanocluster to form a labeled antibody. During detection, the VEG antigen first binds to the capture antibody, then reacts with the labeled antibody, ultimately forming an immune complex of "magnetic beads-VEGF antibody 1-antigen-VEGF antibody 2-signal nanocluster". This structure significantly improves the specificity of detection through dual recognition at spatial sites.

[0020] 2. This invention provides a chemiluminescent detection kit for vascular endothelial growth factor. The silica shell provides an "armor" for the chemically active acridine ester, effectively preventing its hydrolysis, oxidation and fluorescence quenching, and extending the shelf life of the labeled antibody. The kit can be stored stably at 4°C for a long time (≥12 months), with high intra- and inter-batch precision (low CV value) and reliable reproducibility of results.

[0021] 3. This invention provides a chemiluminescence detection kit for vascular endothelial growth factor. The strong hydration layer formed by the zwitterionic polymer strongly repels the non-specific adsorption of biomolecules such as proteins from a physicochemical perspective, and has strong anti-interference ability. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated listed items.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0026] Amino magnetic beads, purchased from Suzhou Beaver Biomedical Engineering Co., Ltd., product model BeaverBeads TM MagNH270201-50; Polymethacrylate sulfobetaine (or simply "PSBMA"), purchased from Xiamen Sainuobang Biotechnology Co., Ltd., with a molecular weight of 309.42; VEGF antibody 1 is a mouse anti-human VEGF monoclonal antibody, purchased from Beijing Sinocare Co., Ltd., catalog number: 101465-MM12; VEGF antibody 2 is a mouse anti-human VEGF monoclonal antibody, purchased from Beijing Sinocare Co., Ltd., catalog number: 101465-MM02; Aminosilicone spheres, purchased from Nanjing Xianfeng Nanomaterials Technology Co., Ltd. Acridinium ester, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., CAS No.: 115853-74-2; VEGF antigen, purchased from Suzhou Ruitebai Biotechnology Co., Ltd., product number: QRE-102B; The blocking agent, mouse IgG, was purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number: AD1450.

[0027] Example 1: Preparation method of chemiluminescent detection kit for vascular endothelial growth factor The chemiluminescent detection kit for vascular endothelial growth factor provided in this embodiment includes the following components: R1 reagent: VEGF antibody-coated surface covalently grafted zwitterionic polymer magnetic beads; R2 reagent: VEGF antibody labeled with silicone acridine nanoclusters; Calibrators: Solutions with VEGF antigen concentrations of 0, 20, 100, 400, 800, and 2000 pg / mL; Quality control products: solutions with VEGF antigen concentrations of 50 and 2000 pg / mL.

[0028] The preparation method of the vascular endothelial growth factor chemiluminescence detection kit provided in this embodiment includes the following steps: Preparation of S1.R1 reagent S11. Weigh 50 mg of polymethacrylic acid sulfobetaine and dissolve it in 5 mL of 2-(N-morpholino)ethanesulfonic acid buffer (or simply "MES buffer"). Add 9.6 mg of EDC·HCl and 5.8 mg of NHS, and activate the polymer solution by shaking at room temperature for 30 min. Take 10 mg of amino magnetic beads, wash them with MES buffer, and mix them with the activated polymer solution. Rotate the mixture at 25°C in the dark for 3 h. After the reaction, magnetically separate the supernatant and add 1 mL of 1 M Tris-HCl solution (pH 8.0) to block the mixture for 10 min. Then wash the magnetic beads twice with PBS buffer and finally resuspend them in 2 mL of PBS buffer containing 1% (w / v) BSA. Filter the mixture through a 0.22 μm filter membrane to obtain magnetic beads with surface covalently grafted zwitterionic polymer. Store the beads at 4°C for later use. S12. Take 5 mg of the magnetic beads prepared in step S11, magnetically separate and discard the supernatant, wash once with 0.02 M borate buffer containing 1% (w / v) BSA at pH 7.4, resuspend the magnetic beads in 1.0 mL of the buffer, add 5 mg of VEGF antibody 1, and then add the same BSA-containing borate buffer to make the total reaction volume 6 mL. Place the mixture at 25 °C and mix at 20 rpm for 3 h. S13. After the antibody conjugation incubation in S12 is completed, magnetic separation is performed, the supernatant is discarded, 3.0 mL of blocking buffer is added to the magnetic beads, the magnetic beads are resuspended, and incubated at 25 °C for 1 h. After incubation, magnetic separation is performed again, the supernatant is discarded, the magnetic bead complex is washed once with PBS buffer, and finally, the magnetic beads are resuspended in storage buffer and the volume is adjusted to a final concentration of 2.0 mg / mL, thus obtaining the VEGF antibody 1-coated surface covalently grafted zwitterionic polymer magnetic beads, which are used as reagent R1 and stored at 4 °C.

[0029] In step S13 above, the blocking buffer is a 0.02 M phosphate buffer at pH 7.8, containing 10 mM propargylamine, 5% (w / v) trehalose, 1% (w / v) BSA and 1% (w / v) casein; the preservation buffer is a 0.01 M phosphate buffer at pH 7.4, containing 1% (w / v) BSA and 5% (w / v) trehalose.

[0030] Preparation of S2.R2 reagent S21. Take 10 mg of aminosilicone beads into a centrifuge tube, centrifuge and discard the supernatant, wash once with pH 9.0 borate buffer, resuspend in 5 mL of borate buffer containing 0.1 mg / mL acridine ester, mix by rotation at room temperature in the dark for 2 h, resuspend in 10 mL of anhydrous ethanol, add 100 μL of tetraethyl orthosilicate and 50 μL of 28% ammonia solution, stir at room temperature for 3 h, centrifuge and discard the supernatant, wash three times with ethanol-water solution (1:1 volume ratio), resuspend in 5 mL of PBS buffer, place in a dialysis bag and dialyze for 3 h to remove unreacted small molecule reagents and byproducts, collect the product, and adjust the volume to 10.0 mL with PBS buffer to obtain the stock solution of silicone-shell acridine ester nanoclusters, and store at 4 °C in the dark. S22. Take 0.5 mL of silicone acridinium ester nanocluster stock solution into a 1.5 mL centrifuge tube, add 0.25 mL of VEGF antibody 2 solution, and add 0.25 mL of borate buffer to a total volume of 1 mL. Vortex to mix well, and incubate at room temperature in the dark for 1.5 h. Transfer the incubated mixture into a dialysis bag and dialyze the dialysate at 4 °C in the dark. Change the dialysate every 12 h for a total of 3 days. S23. After dialysis, collect the solution in the dialysis bag and filter it through a 0.22 μm filter membrane to obtain VEGF antibody 2 labeled with silicone acridine ester nanoclusters.

[0031] S3. Preparation of calibrators and quality control samples Take 0.6g HEPES, 5g BSA, 0.8g sodium chloride, 0.1mL 1mol / L magnesium chloride aqueous solution, 0.1mL 0.1mol / L zinc chloride aqueous solution, 0.1g sodium azide, 5g trehalose and 10mg mouse IgG, add them to a container, add 100mL purified water and stir well. Adjust the pH to 8.0±0.1 with 1mol / L sodium hydroxide aqueous solution to obtain the basic buffer solution.

[0032] Using this basic buffer, VEGF antigen was prepared into calibrators with concentrations of 0, 20, 100, 400, 800, and 2000 pg / mL.

[0033] The VEGF antigen was prepared into quality control samples with concentrations of 50 and 2000 pg / mL using this basic buffer solution.

[0034] Comparative Example 1: The only difference from Example 1 is that the magnetic beads with surface covalently grafted zwitterionic polymer are replaced with epoxy magnetic beads.

[0035] Comparative Example 2: The only difference from Example 1 is that the silica-shell acridine ester nanoclusters are replaced with acridine esters.

[0036] Example 2: Detection method of vascular endothelial growth factor chemiluminescence assay kit The detection method of the vascular endothelial growth factor chemiluminescence detection kit prepared in Example 1, taking the Jiujia Biochemiluminescence Immunoassay Analyzer J-1000 as an example, specifically includes the following steps: S41. Sample addition and first incubation: Add 50 μL of the sample to be tested to the reaction vessel, then add 100 μL of L1 reagent, mix well, and incubate at 37°C for 15 min to form VEGF antigen-antibody 1 complex; S42. Magnetic separation and washing: Place the reaction vessel in a magnetic field and let it stand for 2 minutes to enrich the magnetic beads. Discard the supernatant. Add 300 μL of washing solution, mix well, and perform magnetic separation again and discard the supernatant. Repeat the washing 3 times. The washing solution is PBS buffer containing 0.05% (w / v) Tween-20. S43. Labeling and second incubation: Add 100 μL L2 reagent to the washed magnetic bead complex, mix well, and incubate at 37°C for 10 min to form a double antibody sandwich complex. S44. Repeat magnetic separation and washing: Repeat step S42 to remove unbound R2 reagent; S45. Chemiluminescence detection: The washed magnetic beads are resuspended in a chemiluminescence substrate solution containing 100 μL A solution and 100 μL B solution, wherein solution A is a citrate buffer solution containing 0.5% (w / v) hydrogen peroxide (pH 4.0), and solution B is a 0.1 M sodium hydroxide solution (pH 10.5); after mixing, the relative luminescence intensity (RLU) value is immediately read on the chemiluminescence immunoassay analyzer. S46. Result Calculation: Plot a calibration curve by analyzing the set concentration of the calibrator and the measured luminescence value with the corresponding luminescence value on the x-axis; and calculate the VEGF antigen concentration corresponding to the sample based on the luminescence intensity value of the sample to be tested using the standard curve.

[0037] Example 3 Performance Evaluation of the Chemiluminescent Detection Kit for Vascular Endothelial Growth Factor 3.1 Sensitivity Measurement (1) Experimental methods Using phosphate-buffered saline (PBS, pH 7.4) containing 5% (w / v) BSA as a blank sample, the vascular endothelial growth factor chemiluminescence detection kits prepared according to the methods of Example 1, Comparative Example 1, and Comparative Example 2 were repeatedly measured 20 times according to the chemiluminescence immunoassay analyzer detection method described above. The limit of detection was defined as the blank mean plus twice the standard deviation. The results are shown in Table 1.

[0038] (2) Experimental results and analysis Table 1. Sensitivity test results of the kit

[0039] The results, as shown in Table 1, indicate that the limit of detection (LOD) of the kit prepared in Example 1 was 3.8 pg / mL, which was superior to Comparative Examples 1 and 2. This is because the extremely low nonspecific adsorption background provided by the zwitterionic polymer magnetic beads, combined with the strong signal amplification capability provided by the silica-shell acridine ester nanoclusters, resulted in higher sensitivity in Example 1. In contrast, Comparative Example 1 used hydrophobic epoxy-based magnetic beads, which physically adsorbed a large amount of BSA, leading to an extremely high blank mean. Comparative Example 2 used direct acridine ester labeling; however, small-molecule acridine esters are easily quenched and hydrolyzed, resulting in an unstable number of active molecules, weak signal, and inability to detect lower concentrations of antigen.

[0040] 3.2 Precision determination (1) Experimental methods Several chemiluminescent assay kits for vascular endothelial growth factor (VEGF) were prepared using the methods described in Example 1, Comparative Example 1, and Comparative Example 2. Three samples (50 pg / mL, low value; 400 pg / mL, medium value; 2000 pg / mL, high value) were then tested using kits from the same batch and different batches to examine intra-batch and inter-batch differences. Precision was expressed as the coefficient of variation (CV) of the measured values, where CV(%) = SD / mean × 100. Intra-batch precision was defined as 20 consecutive measurements of the same sample under the same conditions from the same batch. Inter-batch precision was defined as 4 measurements of the sample by different operators using different batches of reagents over 5 consecutive working days. The results are shown in Table 2.

[0041] (2) Experimental results and analysis Table 2 Precision test results of the kit

[0042] The results, as shown in Table 2, indicate that the intra-batch CV of the kit prepared in Example 1 was <5% and the inter-batch CV was <7% for all concentration levels, significantly outperforming industry standards (intra-batch CV <10%, inter-batch CV <15%). This demonstrates that the kit's preparation process is highly stable, with minimal impact from operation, time, and batch size, exhibiting excellent reproducibility and reliability. In contrast, the kit prepared in Comparative Example 1 showed an intra-batch CV of 6.5-7.8% and an inter-batch CV as high as 10.3-12.5%. This is mainly attributed to the non-uniform surface of the epoxy magnetic beads, resulting in random antibody conjugation. Furthermore, traditional physical adsorption blocking struggles to form a uniform and stable interface, leading to significant differences in capture efficiency between different batches and even between different magnetic beads within the same batch, thus introducing substantial random errors. The kit prepared in Comparative Example 2 has better precision than Comparative Example 1 but worse precision than Example 1. It uses the zwitterionic brush magnetic beads of the present invention, which ensures the stability and low background of the capture process. Therefore, its precision is worse than that of Comparative Example 1, which uses epoxy magnetic beads. However, the small molecule acridine ester is easy to hydrolyze and quench, and its labeling efficiency decays with fluctuations, resulting in the stability of the signal generation process being inferior to that of the silicon-shell acridine ester nanoclusters in Example 1.

[0043] 3.3 Determination of Anti-interference Ability (1) Experimental methods The chemiluminescent immunoassay kits for vascular endothelial growth factor (VEGF) prepared in Example 1, Comparative Example 1, and Comparative Example 2 were used respectively. For each kit, a quality control sample with a target value of 50 pg / mL was used as the baseline sample, and clinically common endogenous interfering substances were added respectively: hemoglobin (final concentration 5 g / L, simulating hemolysis), triglycerides (final concentration 20 mmol / L, simulating lipemia), and bilirubin (final concentration 0.5 g / L, simulating jaundice). The VEGF concentration of each interfering sample was measured and compared with the target value, and the relative deviation was calculated. The results are shown in Table 3.

[0044] Deviation (%) = (Measured value of interfering sample - 50) / 50 × 100% (2) Experimental results and analysis Table 3. Results of interference resistance test of the reagent kit

[0045] The results, as shown in Table 3, indicate that the kit prepared in Example 1 exhibits better anti-interference capabilities: under all three interference conditions, the absolute value of the relative deviation is <5%. In contrast, the absolute value of the relative deviation of the kit prepared in Comparative Example 1 significantly exceeds 10%, and the absolute value of the relative deviation of the kit prepared in Comparative Example 2 is >5%.

[0046] 3.4 Stability test after opening (1) Experimental methods The R1 and R2 reagents, calibrators, and quality control samples of the vascular endothelial growth factor chemiluminescence detection kits prepared in Example 1, Comparative Example 1, and Comparative Example 2 were stored at 4°C after opening. The quality control samples at two concentration levels of 50 and 2000 pg / mL were measured on days 0, 7, 14, and 28, and the recovery rates were calculated. The results are shown in Table 4.

[0047] Recovery rate (%) = (Measured concentration / Theoretical concentration) × 100% (2) Experimental results and analysis Table 4 Results of the open-bottle stability test of the reagent kit

[0048] The results, as shown in Table 4, indicate that the kit prepared in Example 1 maintained excellent open-bottle stability, with the recovery rates of both high and low value control samples remaining within a narrow range of 97.0% - 102.4% throughout the 28-day opening period. The kit prepared in Comparative Example 1 exhibited significantly divergent recovery rates with opening time; the low-value recovery rate dropped to 84.0% on day 28, while the high-value recovery rate increased to 118.2%, indicating that its sealing layer failed and its performance was unreliable. The kit prepared in Comparative Example 2 showed significant fluctuations in recovery rate on day 28 (87.6% - 112.9%), indicating insufficient stability of the direct label in solution. Therefore, the kit prepared in Example 1 of this invention maintains stable performance after 28 days of storage at 4°C, demonstrating excellent and significantly better open-bottle stability than Comparative Example 1 and Comparative Example 2.

[0049] 3.5 Accelerated Stability Determination (1) Experimental methods The vascular endothelial growth factor chemiluminescence detection kits prepared in Example 1, Comparative Example 1, and Comparative Example 2 were subjected to accelerated degradation in a 37°C incubator. The kits were removed on days 0, 3, 7, and 14, and after being allowed to return to room temperature, the recoveries of the 50 pg / mL and 2000 pg / mL quality control samples were measured. The results are shown in Table 5.

[0050] (2) Experimental results and analysis Table 5. Results of accelerated stability testing of the reagent kit

[0051] The results, as shown in Table 5, indicate that the kit prepared in Example 1 maintained an excellent recovery rate of 95.2%–104.1% after 14 days of accelerated treatment at 37°C, with minimal variation. The kit prepared in Comparative Example 1 exhibited a sharp deterioration in performance, with significant discrepancies between high and low recovery rates on day 14 (75.3% and 129.4%), rendering it ineffective. The kit prepared in Comparative Example 2 showed significant stability degradation, with a recovery rate ranging from 83.5% to 120.8% on day 14, demonstrating that direct labels lacking silicone shell protection are not heat-resistant. This indicates that the kit of the present invention exhibits excellent thermal stability, providing a basis for predicting its long shelf life.

[0052] 3.6 Long-term stability test (1) Experimental methods The chemiluminescent immunoassay kits for vascular endothelial growth factor prepared in Example 1, Comparative Example 1, and Comparative Example 2 were stored at 4°C. At 12 months post-production, their sensitivity and precision were assessed, and the rate of change of each indicator relative to month 0 was calculated. The results are shown in Table 6.

[0053] (2) Experimental results and analysis Table 6. Results of long-term stability testing of the reagent kit

[0054] The results, as shown in Table 6, indicate that the kit prepared in Example 1 has an actual shelf life of at least 12 months at 4°C. The kit prepared in Comparative Example 1 exhibits poor performance after long-term storage and lacks storage stability. The kit prepared in Comparative Example 2 shows performance degradation mainly in sensitivity and high-value precision, exhibiting typical characteristics of small molecule acridine ester degradation, and thus poor long-term stability.

[0055] In summary, the reagent kit prepared in Example 1 of this invention exhibits comprehensive and significant advantages in the three stability tests of opening, accelerated testing, and long-term stability, demonstrating its reliability.

[0056] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A chemiluminescent assay kit for vascular endothelial growth factor, characterized in that, Including reagents R1 and R2; where: The R1 reagent comprises magnetic beads coated with VEGF antibody 1 and covalently grafted with zwitterionic polymer. The R2 reagent comprises VEGF antibody 2 labeled with silicone acridine ester nanoclusters.

2. The chemiluminescent detection kit for vascular endothelial growth factor according to claim 1, characterized in that, It also includes calibrators and quality control products, wherein the calibrators include solutions with VEGF antigen concentrations of 0, 20, 100, 400, 800, and 2000 pg / mL; and the quality control products are solutions with VEGF antigen concentrations of 50 and 2000 pg / mL.

3. The preparation method of the vascular endothelial growth factor chemiluminescence detection kit as described in claim 2, characterized in that, The preparation of reagent R1 includes the following steps: S11. Preparation of magnetic beads with surface covalently grafted zwitterionic polymer; S12. Mix the magnetic beads prepared in S11 with VEGF antibody 1 in the coupling buffer, resuspend the magnetic beads and incubate. S13. Then, add blocking buffer for incubation, magnetic separation, washing, and resuspending in storage buffer to obtain VEGF antibody 1 coated surface covalently grafted zwitterionic polymer magnetic beads.

4. The preparation method of the vascular endothelial growth factor chemiluminescence detection kit according to claim 3, characterized in that, In step S12, the mass ratio of the magnetic beads to VEGF antibody 1 is 1:(0.5-5).

5. The preparation method of the vascular endothelial growth factor chemiluminescence detection kit according to claim 3, characterized in that, In step S12, the coupling buffer is a borate or phosphate buffer with a pH of 7.4 to 8.5, and contains 0.1% to 1% (w / v) BSA.

6. The preparation method of the vascular endothelial growth factor chemiluminescence detection kit according to claim 3, characterized in that, In step S13, the blocking buffer is a 0.01 M to 0.05 M phosphate buffer with pH 7.4 to 8.5, containing 10 mM propargylamine, 5% (w / v) trehalose, 1% (w / v) BSA and 1% (w / v) casein.

7. The preparation method of the vascular endothelial growth factor chemiluminescence detection kit according to claim 3, characterized in that, It also includes the preparation of reagent R2, specifically including the following steps: S21. Preparation of silica-shell acridine ester nanoclusters; S22. The silica-shell acridinium ester nanoclusters prepared in step S21 are uniformly mixed with VEGF antibody 2 and incubated at room temperature for 0.5-2 hours to obtain a mixture. The incubated mixture is then transferred to a dialysis bag and dialyzed with dialysis solution for 3-5 days. S23. After dialysis, collect the solution in the dialysis bag to obtain purified VEGF antibody 2 labeled with silicone acridine nanoclusters.

8. A detection method for a vascular endothelial growth factor chemiluminescence detection kit as described in any one of claims 1 to 2, or a vascular endothelial growth factor chemiluminescence detection kit prepared by the preparation method as described in any one of claims 3 to 7, characterized in that, Includes the following steps: S41. Sample addition and first incubation: Add the sample to be tested to the reaction vessel, then add the R1 reagent, mix well, and incubate at 37°C for 10-30 min to form VEGF antigen-antibody 1 complex; S42. Magnetic separation and washing: Place the reaction vessel in a magnetic field and let it stand for 1 to 3 minutes to enrich the magnetic beads. Discard the supernatant. Add washing solution, mix well, and perform magnetic separation again and discard the supernatant. Repeat washing 1 to 3 times. S43. Labeling and second incubation: Add the R2 reagent to the washed magnetic bead complex, mix well, and incubate at 37°C for 5–20 min to form a double antibody sandwich complex; S44. Repeat magnetic separation and washing: Repeat step S42 to remove unbound R2 reagent; S45. Chemiluminescence detection: The washed magnetic beads are resuspended in a chemiluminescence substrate solution, which includes solution A and solution B, wherein solution A is a citrate buffer solution containing 0.1% to 1.0% (w / v) hydrogen peroxide, and solution B is a sodium hydroxide solution with a pH of 9.0 to 11.0; after mixing, the relative luminescence intensity value is immediately read on the chemiluminescence immunoassay analyzer. S46. Result Calculation: Plot a standard curve with the calibrator concentration as the x-axis and the corresponding luminous intensity value as the y-axis; The VEGF antigen concentration is calculated using a standard curve based on the luminescence intensity value of the sample to be tested.

9. The detection method of the vascular endothelial growth factor chemiluminescence detection kit according to claim 8, characterized in that, The volume ratio of the sample to be tested to reagent R1 and reagent R2 is 1:(0.5-2):(0.5-2).

10. The application of the vascular endothelial growth factor chemiluminescence detection kit according to any one of claims 1 to 2 or the vascular endothelial growth factor chemiluminescence detection kit prepared by the preparation method according to any one of claims 3 to 7 in the detection of vascular endothelial growth factor concentration.

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