Method for preparing netrin-1 antibody-coated magnetic microparticles, the magnetic microparticles prepared by the method and applications thereof

Abstract

The present application relates to the field of immunoassay technology, and more particularly to a preparation method of Netrin-1 antibody coated magnetic microparticles, the magnetic microparticles prepared by the method and application thereof. The present application provides a preparation method of Netrin-1 antibody coated magnetic microparticles, the magnetic microparticles prepared by the method and application thereof. The method steps of the present application support each other, interact with each other, and together achieve a good magnetic bead coating effect. The prepared magnetic beads have high specificity, high sensitivity, good stability and higher accuracy. The kit of the present application realizes low background and high signal of the immune response, has good specificity, and makes the antigen and antibody have high detection sensitivity. The kit realizes automatic determination. The kit of the present application can be used with a full-automatic chemiluminescence instrument, reduces the influence of human operation on the test results, makes the detection results more reliable, more accurate, faster and more repeatable, and therefore has a good clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay technology, in particular to a preparation method of Netrin-1 antibody coated magnetic microparticles, the magnetic microparticles prepared by the method and the application thereof. BACKGROUND

[0002] Netrin-1 is an important pro-angiogenic substance, a laminin-related secreted protein, which plays multiple biological functions by binding to immunoglobulin-like transmembrane receptors. It can guide the regenerating axon to the distal nerve stump by interacting with the colorectal cancer deletion protein on the growth cone of the regenerating axon, the neural cell adhesion protein NGN antigen, the cell adhesion molecule of Down's syndrome and the melanoma cell adhesion molecule, and promote the extension of the axon. It has the effects of axon guidance, promoting axon growth and branching, promoting stem cell migration and promoting survival, and has a chemoattractive effect on the growth of nerve sprouts.

[0003] Angiogenesis is an important process of peripheral nerve regeneration after transection injury. The newly formed blood vessels not only provide oxygen and nutrients for the cells in the nerve bridge, but also serve as a substrate for Schwann cells to migrate to the nerve gap. A large number of studies have shown that the tumor marker CD146 is highly expressed on endothelial cells, and Netrin-1 can promote angiogenesis through CD146. Netrin-1 physiological concentration between 50 ng / mL and 150 ng / mL can promote the proliferation and migration of Schwann cells and endothelial cells through neogenin receptor and CD146 receptor, respectively. Unc5A-D is expressed by adult motor and sensory neurons, which can interact with Netrin-1 to slow down the extension speed of axons during the regeneration process. The tumor suppressor Unc5B is highly expressed in Schwann cells and endothelial cells, and exogenous Netrin-1 with a concentration higher than 500 ng / mL can inhibit the proliferation and migration of Schwann cells and endothelial cells mediated by Unc5B receptors.

[0004] Netrin-1 is involved in multiple links, can inhibit the aggregation of inflammatory cells in damaged tissues, organs or cells, inhibit the migration of inflammatory cells, promote the differentiation of suppressor macrophages to produce anti-inflammatory effect, and promote the differentiation of regulatory T lymphocytes. Netrin-1 is a monitoring agent for neural axon guidance factor, which is used for clinical diagnosis of acute kidney injury, self-repair process of damaged nerves after cerebral ischemic injury, chemotaxis of vascular endothelial cells and vascular smooth muscle cells, and induction of microvessel neogenesis. Therefore, the development of a neural axon guidance factor detection kit has important clinical value.

[0005] Netrin-1, a laminin-related protein, is an important angiogenic agent that inducing neurite outgrowth. It plays a role in the clinical diagnosis of acute kidney injury and in monitoring the self-repair process of damaged nerves after cerebral ischemia. Therefore, the development of a kit for detecting netrin-1 has important clinical value.

[0006] Currently, the only known method for measuring axon guidance factors in China is the enzyme-linked immunosorbent assay (ELISA) kit. The principle of ELISA is as follows: 1. Netrin-1 antibodies are bound to a solid-phase support surface while maintaining their immunoreactivity; 2. Netrin-1 antibodies are labeled with an enzyme that retains both immunoreactivity and enzymatic activity; 3. During the assay, the specimen and the enzyme-labeled Netrin-1 antibody are bound to the solid-phase support surface in separate steps; 4. Unreacted specimen and excess enzyme-labeled Netrin-1 antibody are washed away, leaving the amount of enzyme bound to the solid-phase support in a ratio proportional to the amount of the substance being tested in the specimen. Upon addition of the enzyme substrate, the substrate is catalyzed by the enzyme to produce a colored product. The amount of the product is directly related to the amount of the substance being tested in the specimen, allowing qualitative or quantitative analysis based on the intensity of the color reaction. The main disadvantages of ELISA include complex reaction kinetics, numerous influencing factors, unstable results, and a long reaction cycle.

[0007] Currently, there are few known methods for measuring axon guidance factors in China, mainly enzyme-linked immunosorbent assay (ELISA) kits. However, ELISA has the disadvantages of poor repeatability, poor linear gradient, complex operation, and poor overall sensitivity and specificity, making it unsuitable for the needs of timely diagnosis in emergency and clinical patients. Therefore, there is a need to develop detection kits for axon guidance factors with high accuracy, high sensitivity, wide linear range, and the ability to reduce detection costs. Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing Netrin-1 antibody-coated magnetic particles and the magnetic particles prepared therefrom and their applications.

[0009] The present invention provides a method for preparing magnetic microparticles coated with a Netrin-1 antibody, comprising activating carboxyl magnetic beads, mixing the beads with a Netrin-1 antibody solution, and incubating and blocking the beads to obtain magnetic microparticles coated with the Netrin-1 antibody.

[0010] The reagent for activating the carboxyl magnetic beads comprises: Tween 80, CHAPS, sodium fatty acid methyl ester sulfonate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and sodium N-hydroxybutanediamine sulfonate.

[0011] The preparation steps of the Netrin-1 antibody coated magnetic microparticles provided by the application include mixing magnetic beads with a Netrin-1 antibody solution, incubation and blocking. The parameters of the steps are optimized, such as the selection of a surfactant, the concentration of the surfactant, the conditions of incubation, the conditions of blocking, the experimental temperature, the ratio of materials used in the experiment, etc. The steps and parameters support each other and interact with each other to achieve good magnetic bead coating effect. Compared with magnetic beads obtained under inappropriate preparation parameters, the magnetic beads prepared by the application have higher specificity, higher sensitivity, better stability, higher accuracy and wider linear range, and therefore the detection cost can be reduced.

[0012] In the preparation method, the concentration of Tween 80 in the activating agent is 2 mg / ml, the concentration of CHAPS is 0.05 mg / ml, the concentration of sodium fatty acid methyl ester sulfonate is 0.3-1.0 M, the concentration of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride is 0.5-1.5 M, and the concentration of N-hydroxybutanediamine sodium salt is 0.2-0.8 M. In some embodiments, the concentration of sodium fatty acid methyl ester sulfonate in the activating agent is 0.8 M, the concentration of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride is 1.0 M, and the concentration of N-hydroxybutanediamine sodium salt is 0.5 M, and the prepared magnetic beads have better performance.

[0013] In some embodiments, in the preparation method, the Netrin-1 antibody solution includes 1-3 mg / ml Tween 80, 0.01-0.07 mg / ml CHAPS, 0.5-1.0 M sodium fatty acid methyl ester sulfonate, phosphate buffer and 0.5-1.5 times the volume of the Netrin-1 antibody solution.

[0014] In some embodiments, in the preparation method, the Netrin-1 antibody solution includes 2 mg / ml Tween 80, 0.05 mg / ml CHAPS, 0.8 M sodium fatty acid methyl ester sulfonate, phosphate buffer and 1 times the volume of the Netrin-1 antibody solution.

[0015] In the application, the concentration of the Netrin-1 antibody in the Netrin-1 antibody solution is 5 mg / ml.

[0016] In the preparation method, the conditions of incubation include 750 rpm shaking at 23℃ for 60 min. Experiments show that under the conditions, the incubation effect is better, which can better cooperate with other experimental parameters to obtain good magnetic bead coating effect.

[0017] In the preparation method of the present invention, ethanolamine is used for blocking the activity of the carboxyl groups on the magnetic beads. Experiments have shown that ethanolamine blocking has a better effect and can more effectively prevent the interference of residual carboxyl groups on subsequent reactions.

[0018] The present invention also provides Netrin-1 antibody-coated magnetic particles prepared by the preparation method.

[0019] The present invention also provides a reagent combination, which includes: reagent 1, reagent 2 and reagent 3, wherein,

[0020] Reagent 1 consists of buffer A and magnetic microparticles coated with Netrin-1 antibody prepared as described above;

[0021] Reagent 2 consists of buffer B and horseradish peroxidase-labeled streptavidin;

[0022] Reagent 3 consists of buffer C and biotinylated Netrin-1 antibody.

[0023] In reagent 1, the concentration of the magnetic particles coated with the Netrin-1 antibody is 1 mg / mL; buffer A comprises water, 1 wt‰ aminopyrine, 2 mol / L sucrose, 1 wt% BSA, and 2 wt‰ P300;

[0024] In reagent 2, the concentration of the horseradish peroxidase-labeled streptavidin is 0.02 wt% to 0.2 wt%; the buffer B is a 0.1 mol / L PB buffer containing 3 wt% BSA, 0.3 wt% ADP protein protective agent, 0.5 M NaCl, 0.2 wt% P300, 0.06 wt% Bro, and 1 wt‰ Triton-X 100;

[0025] In reagent 3, the concentration of the biotin-labeled Netrin-1 antibody is 0.02wt% to 0.2wt%; the buffer C includes 0.1mol / L PB buffer containing 3wt% BSA, 0.3wt% ADP protein protectant, 0.5M NaCl, 0.2wt% P300, 0.06wt% Bro, and 1wt‰ Triton-X 100. The reagent combination described in the present invention also includes a calibrator solution, which includes water, Netrin-1 and animal serum albumin. In the calibrator solution, the concentration of Netrin-1 is 50 to 50,000ng / mL. The animal serum protein is bovine serum albumin or sheep serum albumin. In some specific embodiments, bovine serum albumin is used for verification. The concentration of Netrin-1 is a 10-fold gradient. For example, in a specific embodiment, the standard has a total of 5 gradients, and the concentrations of Netrin-1 are 5 ng / mL, 50 ng / mL, 500 ng / mL, 5000 ng / mL and 50000 ng / mL, respectively.

[0026] The present invention also provides the use of the reagent combination in preparing a Netrin-1 detection reagent.

[0027] The Netrin-1 detection reagent of the present invention includes a Netrin-1 detection kit.

[0028] The present invention also provides a method for detecting nerve axon guidance factors, which comprises using the reagent to detect an object to be detected.

[0029] The present invention provides a method for preparing Netrin-1 antibody-coated magnetic microparticles, and the magnetic microparticles prepared therefrom and their applications. The steps of the method of the present invention support each other and interact with each other to achieve a good magnetic bead coating effect. The magnetic beads prepared have strong specificity, high sensitivity, good stability, and higher accuracy. The kit of the present invention achieves low background and high signal of immune response. Therefore, the kit not only has good specificity, but also enables both antigens and antibodies to achieve a high detection sensitivity. Fully automatic determination is achieved. Traditional enzyme-linked immunosorbent assays have many manual operation steps, a complex process, a low degree of automation, a long time consumption, and are prone to introducing contamination at various links, resulting in problems with the accuracy and reliability of the results. The kit of the present invention can be used in conjunction with a fully automatic chemiluminescence analyzer, reducing the impact of manual operation on the test results, making the test results more reliable, more accurate, faster, and more repeatable, and therefore has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive work:

[0031] Figure 1 1 shows the linear analysis diagram of Example 1;

[0032] Figure 2 4 shows the linear analysis diagram of Example 4;

[0033] Figure 3 4 shows the linear analysis diagram of Example 5. DETAILED DESCRIPTION

[0034] The present invention provides methods for preparing Netrin-1 antibody-coated magnetic microparticles, as well as the magnetic microparticles prepared and their applications. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications obvious to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can readily modify or appropriately alter and combine the methods and applications herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0035] The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will appreciate that the following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0036] Among them, axon guidance factor antibodies, bovine serum albumin, PB buffer, horseradish peroxidase, Na2HPO4, NaH2PO4, sodium chloride, Tween20, and Proclin300 are all products of Sigma, and sodium chloride is a Beijing Huameihuli Biochemical Reagent.

[0037] Example 1

[0038] Prepare reagent 1, reagent 2, reagent 3 and standard solution, then assemble the above reagents into a box and store at 2-8°C. Wherein:

[0039] Preparation of Reagent 1:

[0040] The first step is to mix and suspend the magnetic beads, including: taking 1 volume of carboxyl-modified magnetic beads, washing them with 100 volumes of PB buffer (pH = 7.0-8.5), and placing them on a magnet to separate the magnetic beads;

[0041] In the second step, the separated magnetic beads were activated by adding a reagent to increase surface activity, which included water and 2 mg / ml Tween 80, 0.05 mg / ml CHAPS, 0.8 M sodium fatty acid methyl ester sulfonate, 1 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 0.5 M N-hydroxybutanediamine sulfonic acid sodium salt;

[0042] In the third step, the magnetic beads obtained in the second step were separated and a surfactant-enhancing agent was added to prepare a magnetic bead suspension, wherein the surfactant agent was a phosphate buffer containing 2 mg / ml Tween 80, 0.05 mg / ml CHAPS, and 0.8 M sodium fatty acid methyl ester sulfonate. The magnetic bead suspension was incubated with 1 volume of Netrin-1 antibody (final concentration 5 mg / ml) at 23°C with shaking at 750 rpm for 60 min.

[0043] The fourth step is to separate the magnetic beads obtained in the third step and block the activity of the carboxyl groups on the magnetic beads with ethanolamine;

[0044] The fifth step is to separate the magnetic beads obtained in the fourth step, suspend them in a buffer solution to a density of 1 mg / mL, and store them. The buffer solution comprises 1‰ aminopyrine, 2 mol / L sucrose, 1% BSA, and 2‰ P300.

[0045] Preparation of Reagent 2:

[0046] Step 1: Label horseradish peroxidase on streptavidin;

[0047] Step 2: At room temperature, add the following components to 0.1M PB buffer to a final concentration of 3wt% BSA, 0.3wt% ADP protein protectant, 0.5M NaCl, 0.2wt% P300, and 0.06wt% Bro. Stir for at least 10 minutes until completely dissolved. Then, add Triton-X 100 to a final concentration of 1wt‰ and stir for at least 10 minutes until completely dissolved. Once all components are completely dissolved, measure the pH at 7.4±0.1 using a pH meter. Draw up to 1L with purified water to prepare the buffer. Add the enzyme conjugate to the buffer at a mass ratio of 1 / 500 to 1 / 5000.

[0048] Preparation of Reagent 3:

[0049] Step 1: The Netrin-1 antibody is covalently coupled to biotin to obtain a biotin-labeled Netrin-1 antibody.

[0050] Step 2: At room temperature, add the following components to 0.1M PB buffer to a final concentration of 3wt% BSA, 0.3wt% ADP protein protectant, 0.5M NaCl, 0.2wt% P300, and 0.06wt% Bro. Stir for at least 10 minutes until completely dissolved. Then, add Triton-X 100 to a final concentration of 1wt‰ and stir for at least 10 minutes until completely dissolved. Once all components are completely dissolved, measure the pH at 7.4±0.1 using a pH meter. Draw up to 1L with purified water to prepare the buffer. Add the enzyme conjugate to the buffer at a ratio of 1 / 500 to 1 / 5000.

[0051] 3. Preparation of Calibrators

[0052] Standard 1: Prepare the nerve axon guidance factor with 1% to 5% bovine serum albumin solution, calibrate it and freeze-dry it, assign a value, and prepare it at a concentration of 5 ng / mL.

[0053] Calibrator 2: Prepare the axon guidance factor with 1% to 5% bovine serum albumin solution, freeze-dry the calibrator, and assign the value to a concentration of 50 ng / mL.

[0054] Calibrator 3: Prepare the nerve axon guidance factor with 1% to 5% bovine serum albumin solution, freeze-dry the calibrator, and assign a value. The prepared concentration is 500 ng / mL.

[0055] Calibrator 4: Prepare the nerve axon guidance factor with 1% to 5% bovine serum albumin solution, freeze-dry the calibrator, and assign a value. The prepared concentration is 5000 ng / mL.

[0056] Calibrator 5: Prepare the nerve axon guidance factor with 1% to 5% bovine serum albumin solution, freeze-dry the calibrator, and assign a value. The prepared concentration is 50,000 ng / mL.

[0057] Comparative Example 1

[0058] Except for the surfactant added in the second step of the preparation of reagent 1, the preparation of other reagents is the same as that of Example 1.

[0059] The second step in the preparation of reagent 1 is to activate the separated magnetic beads by adding a reagent that increases surface activity. The reagent includes water and 5 mg / ml sodium dodecyl sulfate, 0.09 mg / ml polyethylene glycol-polypropylene glycol-polyethylene glycol, 0.8 M sodium fatty acid methyl ester sulfonate, 1 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 0.5 M N-hydroxybutanediamine sulfonic acid sodium salt.

[0060] Comparative Example 2

[0061] Except for the surfactant added in the second step of the preparation of reagent 1, the preparation of other reagents is the same as that of Example 1.

[0062] The second step in the preparation of reagent 1 is to activate the separated magnetic beads by adding a reagent that increases surface activity. The reagent includes water and 0.05 mg / ml CHAPS, 0.8 M sodium fatty acid methyl ester sulfonate, 1 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.5 M N-hydroxybutanediamine sulfonic acid sodium salt solution.

[0063] Comparative Example 3

[0064] During the coating process of the magnetic beads, no surfactant was used, and the other steps were the same as those in Example 1:

[0065] Step 2 is specifically as follows: the separated magnetic beads are activated by adding the following solution: the solution includes water and 0.8M sodium fatty acid methyl ester sulfonate, 1M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.5M sodium N-hydroxybutanediamine sulfonate.

[0066] Comparative Example 4

[0067] The preparation of reagent 1 is consistent with that of comparative example 3;

[0068] In reagents 2 and 3, the enzyme conjugate is the same as in Example 1, but the enzyme conjugate buffer is different, specifically comprising: adding the following components to 5M HEPES at room temperature to a final concentration of: 0.5M sodium chloride and 3wol‰ HCl, stirring for at least 5 minutes until completely dissolved, then adding Triton-X 100 to a concentration of 3mg / ml, adding sodium azide to a final concentration of 0.1%, and finally slowly adding bovine serum albumin to a concentration of 10-100g / L, stirring for at least 10 minutes until completely dissolved. After all components are completely dissolved, the pH value is measured with a pH meter and is qualified at 7.4±0.1. The enzyme conjugate is added to the buffer at a ratio of 1 / 500-1 / 5000.

[0069] Effect verification

[0070] 1. The standard product was tested using the reagents of Example 1 and Comparative Examples 1 to 3:

[0071] The steps include: taking 75 μL of the standard sample, adding 20 μL of reagent 1, 50 μL of reagent 2, and 50 μL of reagent 3. The standard sample is first mixed with reagent 1 and incubated, followed by the addition of reagent 2 and incubation, and finally reagent 3. After incubation, the sample is tested on the A2000PLUS platform. The back-calculation mechanism uses four parameters: LIN / LOG to determine the concentration value.

[0072] The results are shown in Table 1, where S0 is the test result for the negative control that does not contain axon guidance factors, and S1 to S5 are the test results for standard products 1 to 5, respectively. The results are summarized as follows:

[0073] Table 1

[0074]

[0075] As shown in Table 1, for the same analyte, the signal value detected by the reagent obtained in Example 1 is the highest, indicating that it has the best sensitivity. Figures 1 to 3 ), the linearity of the reagent detection in Example 1 is optimal.

[0076] 2. The reagents of Example 1 and Comparative Examples 4-5 were used to test samples of known concentrations to verify the accuracy of the reagents.

[0077] Table 2: Accuracy test table of Example 1

[0078]

[0079] Table 3: Accuracy test table of Example 4

[0080]

[0081] Table 4: Accuracy test table of Example 5

[0082]

[0083] The results showed that the data obtained from the detection of the detection reagent in Example 1 was the most reliable.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing magnetic particles coated with Netrin-1 antibody, characterized in that: After activating the carboxyl magnetic beads, the beads were mixed with the Netrin-1 antibody solution, and then incubated and blocked to prepare the Netrin-1 antibody-coated magnetic particles. The reagent for activating the carboxyl magnetic beads includes: Tween 80, CHAPS, sodium fatty acid methyl ester sulfonate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and sodium N-hydroxybutanediamine sulfonate.

2. The preparation method according to claim 1, characterized in that The activation reagent comprises Tween 80 at a concentration of 2 mg / ml, CHAPS at a concentration of 0.05 mg / ml, sodium fatty acid methyl ester sulfonate at a concentration of 0.3-1.0 M, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride at a concentration of 0.5-1.5 M, and sodium N-hydroxybutanediamine sulfonate at a concentration of 0.2-0.8 M.

3. The preparation method according to claim 1, characterized in that The Netrin-1 antibody solution includes 2 mg / ml Tween 80, 0.05 mg / ml CHAPS, 0.8 M sodium fatty acid methyl ester sulfonate, phosphate buffer and 5 mg / ml Netrin-1 antibody.

4. The preparation method according to claim 1, characterized in that The incubation conditions include: shaking at 750 rpm and incubating at 23° C. for 60 min.

5. The preparation method according to claim 1, characterized in that The blocking method uses ethanolamine.

6. Netrin-1 antibody-coated magnetic microparticles prepared by the method according to any one of claims 1 to 5.

7. A reagent combination for detecting Netrin-1, characterized in that: It includes reagent 1, reagent 2 and reagent 3, wherein, Reagent 1 consists of buffer A and magnetic particles coated with Netrin-1 antibodies prepared by the method of any one of claims 1 to 5; Reagent 2 consists of buffer B and horseradish peroxidase-labeled streptavidin; Reagent 3 consists of buffer C and biotinylated Netrin-1 antibody.

8. The reagent combination according to claim 7, characterized in that In reagent 1, the concentration of the Netrin-1 antibody-coated magnetic particles is 1 mg / mL; buffer A includes water, 1 wt‰ aminopyrine, 2 mol / L sucrose, 1 wt% BSA, and 2 wt‰ P300; In reagent 2, the concentration of the horseradish peroxidase-labeled streptavidin is 0.02 wt% to 0.2 wt%; the buffer B is a 0.1 mol / L PB buffer containing 3 wt% BSA, 0.3 wt% ADP protein protectant, 0.5 M NaCl, 0.2 wt% P300, 0.06 wt% Bro, and 1 wt‰ Triton-X 100; In reagent 3, the concentration of the biotin-labeled Netrin-1 antibody is 0.02wt%~0.2wt%; the buffer C includes 0.1mol / L PB buffer containing 3wt% BSA, 0.3wt% ADP protein protectant, 0.5M NaCl, 0.2wt% P300, 0.06wt% Bro, and 1wt‰ Triton-X 100.

9. The reagent combination according to claim 7 or 8, characterized in that The method further comprises a calibration solution comprising water, 5 ng / mL to 50,000 ng / mL Netrin-1 protein and 1 wt% to 5 wt% animal serum albumin.

10. Use of the reagent combination according to any one of claims 7 to 9 in the preparation of a Netrin-1 detection reagent.

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