A vascular endothelial growth factor (VEGF) detection kit and its preparation and use method

Abstract

The present invention belongs to the technical field of kits, and in particular to a vascular endothelial growth factor (VEGF) detection kit and its preparation and use methods. The present invention uses humanized antibodies to directly coat epoxy magnetic beads, avoiding the interference of the biotin-avidin system on the human body. At the same time, due to the simple coating technology, small batch differences, better curve linearity, greatly improved blank limit, repeatability can reach within 4%, greatly improving the accuracy of VEGF detection, and using a fully automatic chemiluminescence immunoassay instrument, the sensitivity and linearity are much higher than the horseradish peroxidase system.

Description

Technical Field

[0001] The invention belongs to the technical field of kits, and in particular relates to a vascular endothelial growth factor (VEGF) detection kit and a preparation and use method thereof. Background Art

[0002] Since Folkman first proposed the hypothesis that tumor growth depends on angiogenesis in 1971, the study of angiogenesis has developed rapidly. It has been confirmed that tumor cells can secrete a variety of angiogenic factors, thereby inducing local angiogenesis. Vascular endothelial growth factor is one of the most effective angiogenic factors, and has a specific division ability for endothelial cells. Studies on VEGF expression in recent years have shown that high expression of tumor VEGF is significantly associated with advanced tumors or poor prognosis. In many malignant tumors, VEGF expression levels have become a prognostic factor for monitoring tumor recurrence or patient survival, and are a very valuable tumor marker.

[0003] Although high expression of serum VEGF is non-specific for malignant tumors, it has the following advantages as a marker of vascular proliferation in distinguishing benign and malignant tumors, predicting staging and metastasis, monitoring efficacy and evaluating prognosis: 1) No need for surgical specimens or biopsy materials, suitable for all patients with malignant tumors, especially for patients with difficult to obtain biopsy materials. 2) Non-invasive and can be repeated continuously. 3) Inexpensive and time-saving. In short, serum VEGF detection is convenient and non-invasive, and can provide prognostic information that traditional clinical pathological prognostic indicators cannot achieve. It is a new means to judge prognosis and distinguish benign and malignant tumors. Dynamic detection of changes in serum VEGF in patients with malignant tumors helps to understand the progression of the disease in patients with malignant tumors.

[0004] VEGF has 6 isoforms, and different shearing methods will also have at least 5 protein forms, which will lead to many different types of VEGF. Most of the domestic VEGF reagent manufacturers are still enzyme-immune and plate-type luminescence, and the raw materials used are also recombinant and mouse monoclonal antibodies. For example, the Chinese patent application document (publication number: CN104711279A) discloses a vascular endothelial growth factor detection kit and its raw material preparation, which relates to a protein immunoassay method, and a monoclonal antibody preparation technology, as well as a vascular endothelial growth factor (VEGF) detection kit using the solution and its use method, which uses recombinant antigens and mouse monoclonal antibodies, streptavidin-biotin system, horseradish peroxidase, and a plate-type chemiluminescence system. However, there are still defects such as poor detection specificity, insufficient linearity, and insufficient automation. It can be seen that improving the accuracy of serum VEGF detection and realizing automation have become the primary issues of VEGF detection. Summary of the invention

[0005] The object of the present invention is to provide a highly automated, highly sensitive and linear vascular endothelial growth factor (VEGF) detection kit in view of the above problems existing in the prior art.

[0006] The purpose of the present invention can be achieved by the following technical scheme: a vascular endothelial growth factor (VEGF) detection kit, the kit comprising R1 reagent and R2 reagent;

[0007] The R1 reagent includes epoxy-based magnetic particles coated with humanized VEGF antibody;

[0008] The R2 reagent includes alkaline phosphatase-labeled VEGF human antibody.

[0009] The present invention uses natural antigens to prepare calibration products, and uses humanized antibodies to directly couple magnetic beads using a direct packaging system, and uses alkaline phosphatase and fully automatic chemiluminescence to specifically detect different types of VEGF, thereby avoiding the interference of the biotin-avidin system on the human body and greatly improving the accuracy of detection. Compared with conventional monoclonal antibodies, the humanized VEGF antibody used in the present invention has better specificity and can avoid the HAMA effect caused by the use of monoclonal antibodies.

[0010] In the above-mentioned vascular endothelial growth factor (VEGF) detection kit, the preparation method of epoxy-based magnetic particles coated with VEGF humanized antibody comprises the following steps:

[0011] S1, respectively adding coupling buffer to epoxy magnetic particles and VEGF human antibody to mix to obtain antibody solution and activated epoxy magnetic particles;

[0012] S2, adding the antibody solution to the activated epoxy magnetic particles, resuspending the magnetic beads and incubating;

[0013] S3. After washing, add blocking buffer for incubation, and finally dilute and store.

[0014] In the above-mentioned vascular endothelial growth factor (VEGF) detection kit, the mass ratio of epoxy magnetic particles to VEGF human antibody in step S1 is (45-55):1.

[0015] Epoxy magnetic beads are magnetic particles with epoxy functional groups modified on the surface and having superparamagnetism. Epoxy magnetic beads are monodisperse, non-agglomerated, regular spherical, with stable particle size, superparamagnetism, fast magnetic responsiveness, rich amino functional groups, monodispersity and submicron particle size. Epoxy groups can be directly covalently coupled with amino groups and hydroxyl groups in a variety of biological ligands (protein ligands, peptides, oligonucleotide probes, drug molecules, etc.).

[0016] Compared with traditional polydisperse magnetic beads, epoxy magnetic beads have uniform particle size and excellent dispersion performance. The thickness of the silicon layer is about 1 / 20 of the size of the magnetic beads, so the magnetic content of the magnetic beads is greater than 95%. It has faster magnetic responsiveness while maintaining good dispersion of the microspheres, extremely low nonspecific adsorption and more abundant binding sites. It is very easy to achieve rapid separation under the magnetic field and rapid resuspension and dispersion after withdrawing from the magnetic field. It is especially suitable for automated coupling operations. The uniform size of monodisperse amino magnetic beads can significantly reduce the batch-to-batch differences in experiments. Epoxy magnetic beads are 100% covalently bonded and can be used for rapid coupling and binding of target substances.

[0017] Therefore, the use of epoxy magnetic beads can greatly improve the activity of the conjugate, making the curve closer to a straight line, with a high linear range and a significantly improved blank limit. At the same time, the epoxy magnetic beads have excellent dispersibility and can improve repeatability.

[0018] In the above-mentioned vascular endothelial growth factor (VEGF) detection kit, the pH of the blocking buffer in step S3 is 7.4, and includes: 0.01-0.05 M Tris, 2-5 wt % BSA.

[0019] Preferably, the coupling buffer is 0.1-0.5 M Hepes buffer at pH 8.0.

[0020] In the above-mentioned vascular endothelial growth factor (VEGF) detection kit, the preparation method of the alkaline phosphatase-labeled VEGF humanized antibody comprises the following steps:

[0021] S1. Add VEGF human antibody to amino-free and thiol-free buffer, then add DTT or TCEP to react, and then add glycine to react to obtain an activated antibody solution;

[0022] S2, adding alkaline phosphatase to an amino-free and thiol-free buffer, then adding a DMF solution containing SMCC, and then adding glycine to react to obtain an alkaline phosphatase solution;

[0023] S3, uniformly mixing the activated antibody solution and the alkaline phosphatase solution, then adding the magnesium chloride solution to react, and finally purifying.

[0024] Preferably, after the VEGF human antibody is added to the amino-free and thiol-free buffer, the concentration of the VEGF human antibody is 2-4 mg / ml.

[0025] Preferably, the amino-free and thiol-free buffer is a 0.01 M PBS buffer at pH 7.4.

[0026] In the above-mentioned vascular endothelial growth factor (VEGF) detection kit, the mass ratio of VEGF human antibody to alkaline phosphatase is 1:(0.8-1.2).

[0027] In the above-mentioned vascular endothelial growth factor (VEGF) detection kit, the amount of DTT or TCEP added in step S1 is 0.5‰ to 2.5‰ microliters per milliliter of VEGF human antibody.

[0028] Preferably, the concentration of the DMF solution containing SMCC is 5-8 mg / ml.

[0029] The above-mentioned vascular endothelial growth factor (VEGF) detection kit also contains a calibrator, which includes a protein solution with a VEGF natural antigen concentration of 10, 30, 100, 500, and 1600 pg / mL respectively.

[0030] Preferably, the pH of the calibrator is 7.0-9.0, and includes at least one of BSA, casein, trehalose, and a blocking agent.

[0031] Preferably, the test kit further comprises a quality control product, which comprises a protein solution with a VEGF natural antigen concentration of 30 and 500 pg / mL, respectively.

[0032] The present invention also provides a method for using the above-mentioned vascular endothelial growth factor (VEGF) detection kit, which comprises the following steps: adding R1 reagent and R2 reagent to the original solution of the sample to be tested in sequence, incubating at 36-38°C for 6-8 minutes, placing in a magnetic field for separation, adding chemiluminescent substrate solution after washing, and detecting the chemiluminescent photon intensity.

[0033] Preferably, the chemiluminescent substrate solution is APS-5 substrate.

[0034] In the above-mentioned method for using the vascular endothelial growth factor (VEGF) detection kit, the volume ratio of the sample solution to be tested to the R1 reagent and the R2 reagent is 1:(0.9-1.1):(0.9-1.1).

[0035] Compared with the prior art, the kit of the present invention has the following advantages:

[0036] 1. The present invention uses humanized antibodies to directly coat epoxy magnetic beads, avoiding the interference of the biotin-avidin system on the human body. At the same time, due to the simple coating technology, small batch differences, better linear relationship of the curve, greatly improved blank limit, repeatability can be within 4%, greatly improving the accuracy of VEGF detection, and using a fully automatic chemiluminescence immunoassay analyzer, the sensitivity and linearity are much higher than the horseradish peroxidase system.

[0037] 2. The epoxy magnetic beads of the present invention are superparamagnetic nanoparticles with a GMA coating on the surface of the Fe3O4 magnetic core, and have a high density of epoxy groups on the surface. They are efficiently covalently coupled with the humanized antibodies of the present invention, are stable and will not fall off, have low nonspecific binding, and greatly enhance the stability of the reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the standard curve for the epoxy magnetic beads measured in Example 1.

[0039] Figure 2 A standard curve was set for the avidin magnetic beads in Comparative Example 1.

[0040] Figure 3 A standard curve was set for the carboxyl magnetic beads in Comparative Example 2.

[0041] Figure 4 A standard curve was set for the tosyl magnetic beads in comparative example 3.

[0042] Figure 5 A standard curve was set for the amino magnetic beads in comparative example 4.

[0043] Figure 6 This is a graph showing the correlation results of simultaneously measuring 200 samples with values ​​using the detection reagent prepared in Example 1 and the vascular endothelial growth factor detection kit (magnetic particle chemiluminescence method) produced by Kanghua Biotechnology Co., Ltd. DETAILED DESCRIPTION

[0044] The following are specific embodiments of the present invention, and the technical solutions of the present invention are further described in conjunction with the accompanying drawings, but the present invention is not limited to these embodiments.

[0045] The raw materials used in the following examples are as follows:

[0046] Epoxy magnetic beads were purchased from Premag Biotech;

[0047] Avidin magnetic beads were purchased from JSR Company;

[0048] Carboxyl magnetic beads were purchased from JSR Company;

[0049] Tosyl magnetic beads were purchased from JSR;

[0050] Amino magnetic beads were purchased from JSR Company;

[0051] VEGF human antibody was purchased from Abcam;

[0052] The coupling buffer was Hepes buffer at pH 8.0;

[0053] The blocking buffer was pH 7.4 and included: 0.05 M Tris, 2 wt % BSA;

[0054] PBS buffer 7.4;

[0055] The amino- and thiol-free buffer was 0.01 M PBS buffer at pH 7.4;

[0056] A DMF solution containing SMCC at a concentration of 6 mg / ml;

[0057] The blocking agent was purchased from Roche's mark33.

[0058] Embodiment 1:

[0059] Preparation of R1 reagent:

[0060] S1. Transfer 10 mg of epoxy magnetic beads to an EP tube;

[0061] Place S2 and EP tubes on a magnetic stand for 1 min, discard the supernatant, and resuspend the beads with 1 ml of coupling buffer;

[0062] S3, repeat step S2 three times to obtain activated epoxy-based magnetic particles for use;

[0063] S4, taking 0.1 mg of VEGF human antibody and adding it to 1 ml of coupling buffer to obtain an antibody solution;

[0064] S5. Add the antibody solution to the activated epoxy magnetic particles, couple at 25°C for 24 h, and wash the magnetic beads three times with 1 ml of PBS buffer;

[0065] S6. Add 1 ml of blocking buffer to the magnetic beads and incubate overnight at 4°C, then wash the magnetic beads 5 times with 1 ml of PBS buffer;

[0066] S7. Resuspend the magnetic beads to the desired concentration using PBS buffer (containing 0.1% azide) and store at 2-8°C.

[0067] Preparation of R2 reagent:

[0068] S1. Take 1 mg of antibody and replace it with amino-free and thiol-free buffer by dialysis, and concentrate it to 3 mg / ml using a concentrator tube;

[0069] S2, add DTT (dithiothreitol), react at room temperature for 20 minutes, then add 1M glycine at pH 7.3 and react at room temperature for 8 minutes, use PD-10 desalting column to replace the buffer, concentrate to 3 mg / ml to obtain activated antibody solution for standby use; the amount of DTT (dithiothreitol) and glycine added is 1‰ microliter per milliliter of antibody;

[0070] S3, take 1 mg of alkaline phosphatase and replace it with amino-free and thiol-free buffer by dialysis, and concentrate it to 3 mg / ml using a concentrator tube;

[0071] S4. Add DMF solution containing SMCC and react at room temperature for 20 minutes, then add 1M glycine at pH 7.3 and react at room temperature for 15 minutes, use PD-10 desalting column to replace the buffer, concentrate to 3 mg / ml to obtain alkaline phosphatase solution for use; the amount of DMF solution containing SMCC and glycine added is 5‰ of the volume of alkaline phosphatase.

[0072] S5. Mix the activated antibody solution and alkaline phosphatase in a mass ratio of 1:1, add 0.3 M magnesium chloride solution in a volume of 3‰ ml of the total reaction volume, react at 5°C for 12 hours, and finally purify and store at 2-8°C.

[0073] Preparation of calibrators and quality control products:

[0074] Take 0.6g HEPES, 5g BSA, 0.8g sodium chloride, 0.1ml 1mol magnesium chloride, 0.1ml 0.1mol zinc chloride, and 0.2g Proclin300, add them to a container, add 100ml purified water and stir evenly to obtain a pH 8.0 buffer solution, wherein the buffer solution contains 2% BSA, 5% trehalose, and 10mg / ml blocking agent.

[0075] The VEGF native antigen was prepared into calibrators with concentrations of 10, 30, 100, 500, and 1600 pg / mL using a buffer.

[0076] The VEGF native antigen was prepared into quality control products with concentrations of 30 and 500 pg / mL using a buffer solution.

[0077] Application Example 1:

[0078] The R1 reagent and R2 reagent prepared in Example 1 were added to the original solution of the sample to be tested in sequence, incubated at 37°C for 8 minutes, placed in a magnetic field for separation, and after washing, 150 μl of APS-5 substrate was added to detect the chemiluminescent photon intensity. The volume ratio of the original solution of the sample to be tested to the R1 reagent and the R2 reagent was 1:1:1.

[0079] Comparative Example 1:

[0080] The only difference from Example 1 is that the epoxy magnetic beads are replaced with avidin magnetic beads.

[0081] Comparative Example 2:

[0082] The only difference from Example 1 is that the epoxy magnetic beads are replaced by carboxyl magnetic beads.

[0083] Comparative Example 3:

[0084] The only difference from Example 1 is that the epoxy magnetic beads are replaced by tosyl magnetic beads.

[0085] Comparative Example 4:

[0086] The only difference from Example 1 is that the epoxy magnetic beads are replaced by amino magnetic beads.

[0087] Application Comparative Examples 1-4:

[0088] Prepare 4 test sample stock solutions, add R1 reagent and R2 reagent prepared in Comparative Example 1, R1 reagent and R2 reagent prepared in Comparative Example 2, R1 reagent and R2 reagent prepared in Comparative Example 3, and R1 reagent and R2 reagent prepared in Comparative Example 4 to the test sample stock solutions, incubate them together at 37°C for 8 min, place them in a magnetic field for separation, add 150 μl APS-5 substrate after washing, and detect the chemiluminescent photon intensity, wherein the volume ratio of the test sample stock solution to the R1 reagent and R2 reagent is 1:1:1.

[0089] Table 1: Luminescence values ​​of calibrants measured by epoxy magnetic beads in Example 1

[0090]

[0091] Table 2: Comparative Example 1 Avidin Magnetic Beads Determination of Calibrator Luminescence Values

[0092]

[0093]

[0094] Table 3: Comparative Example 2 Carboxyl Magnetic Beads for Calibrator Luminescence Values

[0095]

[0096] Table 4: Comparative Example 3 Tosyl Magnetic Beads Determination of Calibrator Luminescence Values

[0097]

[0098] Table 5: Comparative Example 4 Amino Magnetic Beads for Calibration Luminescence Values

[0099]

[0100] Figure 1-Figure 5 The standard curves of epoxy magnetic beads, avidin magnetic beads, carboxyl magnetic beads, tosyl magnetic beads, and amino magnetic beads are respectively measured in Example 1 and Comparative Examples 1-4. It can be seen from the calibration data and curve information that the luminescence value of the epoxy magnetic bead system is the highest, and the curve shape is closest to a straight line, and R2 is also the best at 0.99997.

[0101] Repeatability experiment: The system constructed with the magnetic beads of Example 1 and Comparative Examples 1-4 was used to test samples with concentrations of (30±6) pg / ml and (500±100) pg / ml in parallel, and the determination was repeated 10 times. The mean (M) and standard deviation (SD) of the 10 determination results were calculated. The coefficient of variation (CV) was calculated, CV = (SD / M) × 100%, and CV should be no greater than 8%.

[0102] Table 6: Repeatability results of the epoxy magnetic bead system in Example 1

[0103]

[0104] Table 7: Comparative Example 1 Avidin Magnetic Bead System Determination Repeatability Results

[0105]

[0106]

[0107] Table 8: Comparative Example 2 Carboxyl Magnetic Bead System Determination Repeatability Results

[0108]

[0109] Table 9: Comparative Example 3 Tosyl Magnetic Bead System Determination Repeatability Results

[0110]

[0111] Table 10: Comparative Example 4 Amino Magnetic Bead System Determination Repeatability Results

[0112]

[0113]

[0114] Through the repeatability test of the magnetic bead system prepared in the above Example 1 and Comparative Examples 1-4, the requirement of CV < 8% is met, but the CV of the epoxy magnetic bead system is within 2%, while the CV of other magnetic bead system tests is above 5%. It can be seen that the epoxy magnetic beads and VEGF human antibody system of the present invention can greatly improve the repeatability results.

[0115] Blank limit test: The magnetic bead system prepared in Example 1 and Comparative Examples 1-4 was used to simultaneously determine the blank limit, and the zero-concentration calibrator was used as a sample for detection. The measurement was repeated 20 times to obtain the luminescence value (RLU) of the 20 measurement results. The mean value (M) and standard deviation (SD) were calculated to obtain the luminescence value corresponding to M+2SD. A linear equation was obtained by performing a two-point regression fitting based on the concentration-RLU value results between the zero-concentration calibrator and the adjacent calibrator. The M+2SD luminescence value was substituted into the above equation to obtain the corresponding concentration value, which was the blank limit. The result was not higher than the blank detection limit (0.5 pg / mL).

[0116] Table 11: Blank limit results of epoxy magnetic bead system in Example 1

[0117]

[0118]

[0119] Table 12: Blank limit results of the avidin magnetic bead system in comparative example 1

[0120]

[0121]

[0122] Table 13: Blank limit results of carboxyl magnetic bead system in comparative example 2

[0123]

[0124] Table 14: Comparative Example 3 Tosyl Magnetic Bead System Determination Blank Limit Results

[0125]

[0126]

[0127] Table 15: Comparative Example 4 Amino Magnetic Bead System Determination Blank Limit Results

[0128]

[0129]

[0130] By testing the blank limits of the reagents prepared in Example 1 and Comparative Examples 1-4, the epoxy magnetic beads, carboxyl and avidin magnetic beads systems all meet the requirement of 0.5 pg / mL, but the blank limit of the epoxy magnetic beads system is 0.0493 pg / mL, while the blank limit of the avidin system is 0.3845 pg / mL, and the blank limit of the carboxyl system is 0.427 pg / mL. It can be seen that the epoxy magnetic bead system in the present invention can greatly improve the blank limit index of the test kit.

[0131] Accuracy test: The accuracy was simultaneously determined using the magnetic bead system prepared in Example 1 and Comparative Examples 1-4. Sample A with a concentration of (1000±100) pg / ml was added to sample B. The volume of sample A added did not exceed 10% of the total volume of sample A and sample B. The test was repeated 3 times, the average value was taken, and the recovery rate R was calculated according to the formula. The recovery rate should be between 85% and 115%.

[0132]

[0133] Where R: recovery rate; V: volume of sample A; V0: volume of sample B; C: detection concentration of sample B after adding A; C0: concentration of sample B; Cs: concentration of sample A.

[0134] Table 16: Accuracy results of epoxy magnetic bead system in Example 1

[0135]

[0136]

[0137] Table 17: Comparative Example 1 Avidin Magnetic Bead System Determination Accuracy Results

[0138]

[0139] Table 18: Comparative Example 2 Carboxyl Magnetic Bead System Determination Accuracy Results

[0140]

[0141] Table 19: Comparative Example 3 Tosyl Magnetic Bead System Determination Accuracy Results

[0142]

[0143] Table 20: Comparative Example 4 Amino Magnetic Bead System Determination Accuracy Results

[0144]

[0145] By testing the accuracy of the reagents prepared in the above-mentioned Example 1 and Comparative Examples 1-4, the recovery rate of the determination accuracy of the epoxy-based magnetic bead system was 102%, which met the requirements, while the determination accuracy of the other four magnetic bead systems was not within 85%-115%, which did not meet the requirements. It can be seen that the epoxy-based magnetic bead system can greatly improve the accuracy.

[0146] Linearity test: The magnetic bead systems prepared in Example 1 and Comparative Examples 1-4 were simultaneously tested for linearity. The high-value samples close to the upper limit of the linear range were diluted to at least 5 concentrations according to a certain ratio, and the low-value concentration samples must be close to the lower limit of the linear range. The samples of each concentration were tested 3 times, and the average value was calculated. The average value of the result and the dilution ratio were fitted with a least squares method to calculate the linear correlation coefficient r, which should be greater than 0.9900.

[0147] Table 21: Linearity results of epoxy magnetic bead system in Example 1

[0148]

[0149] Table 22: Comparative Example 1 Avidin Magnetic Bead System Determination Linearity Results

[0150]

[0151]

[0152] Table 23: Comparative Example 2 Carboxyl Magnetic Bead System Determination Linearity Results

[0153]

[0154] Table 24: Comparative Example 3 Tosyl Magnetic Bead System Determination Linearity Results

[0155]

[0156]

[0157] Table 25: Comparative Example 4 Amino Magnetic Bead System Determination Linearity Results

[0158]

[0159] Through the linearity test of the reagents prepared in the above Example 1 and Comparative Examples 1-4, the linearity R is greater than 0.9900, but the linearity result of the epoxy magnetic bead system is better than the results of other magnetic bead systems.

[0160] Figure 6 The correlation result diagram of the detection reagent prepared in Example 1 and the vascular endothelial growth factor detection kit (magnetic particle chemiluminescence method) of Kanghua Biotechnology Co., Ltd. for simultaneously measuring 200 samples with values. As can be seen from the figure, 200 samples were tested at the same time, and the correlation R2 between the detection kit of the present invention and the vascular endothelial growth factor detection kit (magnetic particle chemiluminescence method) of Kanghua Biotechnology Co., Ltd. was 0.9854, and the coefficient A was 1.0156, which shows that the detection kit of the present invention fully meets clinical needs.

[0161] In summary, by measuring the repeatability, linearity, accuracy, and blank limit of the reagents prepared in the above Example 1 and Comparative Examples 1-4, it was found that the various performance indicators of the epoxy magnetic bead system were better than those of other systems. It can be seen that the repeatability CV, accuracy, and blank limit of the kit of the present invention are greatly improved.

[0162] The parts of the technical scope of the present invention that are not exhausted by the midpoint value of the embodiment and the new technical solutions formed by the equivalent replacement of single or multiple technical features in the technical solutions of the embodiments are also within the scope of protection claimed by the present invention, and unless otherwise specified, there is no unique and irreplaceable combination between all the parameters involved in the solutions of the present invention.

[0163] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

[0164] Although the present invention has been described in detail and some specific embodiments have been cited, it is obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention.

Claims

1. A vascular endothelial growth factor detection kit, characterized in that: The kit includes R1 reagent, R2 reagent, calibrators and quality control products; The R1 reagent includes epoxy-based magnetic particles coated with humanized VEGF antibody; The R2 reagent includes an alkaline phosphatase-labeled VEGF human antibody; The preparation method of epoxy-based magnetic particles coated with humanized VEGF antibody comprises the following steps: S1. Adding coupling buffer to epoxy magnetic particles and VEGF human antibody respectively to mix them to obtain antibody solution and activated epoxy magnetic particles; S2, adding the antibody solution to the activated epoxy magnetic particles, resuspending the magnetic beads and incubating; S3, then after washing, add blocking buffer for incubation, and finally dilute and store; Step S1: the mass ratio of epoxy magnetic particles to VEGF human antibody is (45-55):1; The coupling buffer was 0.1-0.5 M Hepes buffer at pH 8.0; In step S3, the blocking buffer has a pH of 7.4 and includes: 0.01-0.05 M Tris, 2-5 wt % BSA; The alkaline phosphatase labeled VEGF humanized antibody comprises the following steps: (1) Add VEGF human antibody to amino-free and thiol-free buffer, then add DTT or TCEP to react, and then add glycine to react to obtain an activated antibody solution; (2) Add alkaline phosphatase to an amino-free and thiol-free buffer, then add a DMF solution containing SMCC, and then add glycine to react to obtain an alkaline phosphatase solution; (3) Evenly mix the activated antibody solution and the alkaline phosphatase solution, then add the magnesium chloride solution to react, and finally purify it; The mass ratio of VEGF human antibody to alkaline phosphatase is 1:(0.8-1.2); Step (1) The amount of DTT or TCEP added is 0.5‰ to 2.5‰ μl per ml of VEGF human antibody; The preparation methods of calibrators and quality control products include: Take 0.6g HEPES, 5g BSA, 0.8g sodium chloride, 0.1ml 1mol magnesium chloride, 0.1ml 0.1mol zinc chloride, and 0.2g Proclin300 and add them to a container, add 100ml purified water and stir evenly to obtain pH 8.0 buffer solution, which contains 2% BSA, 5% trehalose, and 10mg / ml blocking agent.

2. A vascular endothelial growth factor detection kit according to claim 1, characterized in that: The calibrator includes protein solutions with VEGF native antigen concentrations of 10, 30, 100, 500, and 1600 pg / mL, respectively.

Images