ELISA kit and method of use

Abstract

The present invention relates to the field of immunoassays, and more particularly to an ELISA kit and its use. The kit comprises: an antibody for capturing an antigen to be detected, an enzyme-linked plate, an enzyme-linked complex, a coating solution, a washing solution, a blocking solution, a developer, and a stop solution. Compared to traditional ELISA methods, the kit utilizes an enzyme-linked complex comprising a polystyrene / polydopamine covalently coupled detection antibody and horseradish peroxidase to replace the horseradish peroxidase-labeled detection antibody, thereby amplifying the response signal and exhibiting high sensitivity and low background interference. Furthermore, the synthesis method of the enzyme-linked complex is simple, low-cost, and employs mild conditions. The enzyme-linked complex is highly stable, enabling detection under varying pH conditions.

Description

Technical Field

[0001] The present invention relates to the field of immunoassay, and in particular to an ELISA kit and a method of use. Background Art

[0002] The enzyme-linked immunosorbent assay (ELISA) is a technique designed to detect and quantify (bio)molecules in liquid samples. It is a powerful tool for clinical diagnostics, food safety, and environmental monitoring. However, the main limitation of traditional ELISA is its low sensitivity, which cannot meet the needs of analyzing and detecting targets in complex (biological) samples. Currently, increasing the number of enzyme molecules in the enzyme-labeled complex is an effective way to improve its sensitivity.

[0003] Nanoparticles, such as metal and metal oxide nanoparticles, can be used as carriers for antibodies and enzymes due to their large specific surface area, high loading capacity, ease of synthesis, good chemical stability, and ease of biobinding. Metals and their metal oxides, due to their high density, increase background interference caused by natural sedimentation, and their primary binding to antibodies and enzymes is through physical adsorption, which imposes certain limitations on synthesis and detection conditions. The present invention utilizes nanoparticles with a polystyrene / polydopamine core-shell structure as an ELISA signal amplification substrate, improving ELISA sensitivity while reducing background interference caused by the nanoparticles.

[0004] Dopamine self-polymerizes in an alkaline environment and can be modified by adhesion on different substrates. The generated polydopamine is evenly coated on the surface of the material. Polydopamine has good biocompatibility, many surface active groups, and can stably exist in acidic and weakly alkaline environments. It is a good surface modification material. Literature reports that dopamine can be modified on the surface of templates such as polystyrene and silica. After removing the template, polydopamine balls with a hollow structure are obtained. After carbonization, they are used as conductive materials in energy materials. However, polydopamine nanospheres with a hollow structure are rarely used in ELISA signal amplification. Since the hollow structure is easy to collapse and easily aggregate and overlap, the dispersibility is poor. Therefore, in the present invention, the template is retained to ensure the shape of the polydopamine nanospheres, and the presence of a low-density template is used to reduce the background interference of nanoparticles.

[0005] Polystyrene / polydopamine nanoparticles, as signal amplification substrates, can be covalently linked to antibodies and enzymes via Michael addition reactions. The unique biochemical properties of polydopamine ensure the activity of these antibodies and enzymes to a certain extent. Polystyrene / polydopamine nanoparticles are stable in both acidic and weakly alkaline environments, enabling detection in diverse acidic and alkaline environments.

[0006] Psoriasis is a chronic inflammatory skin disease characterized by abnormal activation and infiltration of T lymphocytes. Its etiology remains unclear, with multiple factors implicated, including genetics, immunomodulatory, environmental, and psychiatric factors. Numerous studies have implicated IL-17 in the pathogenesis of diseases such as psoriasis. IL-17A, a member of the IL-17 family, exhibits proinflammatory activity, inducing the expression of proinflammatory cytokines, colony-stimulating factors, and chemokines in dendritic cells, neutrophils, T cells, monocytes / macrophages, and epithelial cells. However, ELISA kits for IL-17A are mostly imported and present challenges such as low sensitivity and high background interference. Summary of the Invention

[0007] The present invention aims to at least solve the above-mentioned technical problems existing in the prior art. To this end, the primary purpose of the present invention is to provide an ELISA detection kit. Compared with conventional ELISA detection products, this kit uses a polystyrene / polydopamine covalently coupled detection antibody and horseradish peroxidase enzyme-labeled complex instead of the horseradish peroxidase-labeled detection antibody, thereby amplifying the detection signal, improving sensitivity, and reducing background interference.

[0008] An ELISA kit comprises: an antibody for capturing an antigen to be detected, an enzyme labeling plate, and an enzyme labeling complex; the enzyme labeling complex is obtained by coupling the antibody for detecting the antigen to be detected, horseradish peroxidase, and polystyrene / polydopamine nanoparticles.

[0009] The preparation method of the enzyme-labeled complex comprises the following steps: adding polystyrene nanospheres to a Tris-HCl solution containing dopamine or dopamine hydrochloride, stirring for reaction; washing the product after the reaction to obtain polystyrene / polydopamine nanoparticles; dispersing the polystyrene / polydopamine nanoparticles in a PBS solution, adding an antigen detection antibody to be detected and horseradish peroxidase to the solution for coupling reaction with the polystyrene / polydopamine nanoparticles; and washing the product to obtain the enzyme-labeled complex.

[0010] Nanoparticles with low density, size less than 100 nm and high stability can also replace polystyrene nanospheres.

[0011] Substances with a dopa-like structure can replace dopamine.

[0012] Furthermore, the Tris·HCl solution has a concentration of 10-50 mM, a pH of 7.5-8.5, and a mass ratio of polystyrene to dopamine or dopamine hydrochloride of 2-5:1.

[0013] Furthermore, dopamine or dopamine hydrochloride is reacted with polystyrene at 20-30° C. for at least 12 hours; the detection antibody for the antigen to be detected and horseradish peroxidase in the reaction system are evenly mixed and then added to the polystyrene / polydopamine nanoparticle solution, wherein the mass ratio of the detection antibody for the antigen to be detected to the horseradish peroxidase to the polystyrene / polydopamine particles is 1:4-10:200-400, and the reaction is allowed to stand at 2-6° C. for at least 12 hours (preferably 12-16 hours).

[0014] The test kit further comprises: a standard antigen to be tested, a washing solution, a color developing solution and a stop solution.

[0015] In the kit, the antigen standard to be tested is diluted to a target concentration by a sample diluent, and the sample diluent is a PBS solution containing 0.1-0.5% BSA.

[0016] The kit, the color developing solution is a colored conjugate of HRP, and further, the color developing solution includes substrate solution A and substrate solution B, substrate solution A is prepared as follows: 0.136g sodium acetate, 0.016g citric acid, 3μL 30% H2O2, and H2O is added to 5mL; substrate solution B is prepared as follows: 0.0021g disodium ethylenediaminetetraacetic acid, 0.0095g citric acid, 0.5mL glycerol, 0.0015g tetramethylbenzenediamine, and H2O is added to 5mL; when used, the volume ratio of substrate solution A to substrate solution B is 1:1.

[0017] The function of the color developing solution is to combine with the marker of the enzyme-labeled complex to develop color, and it can be selected according to actual needs.

[0018] In the kit, the stop solution is sulfuric acid with a concentration of 1.5-2.5 mol / L; and the washing solution is a PBS solution containing 0.1-0.5% Tween-20 with a pH of 7.4.

[0019] The purpose of the stop solution is to terminate the enzymatic reaction (or immunological reaction). An appropriate stop solution can be selected based on the actual enzyme marker selected.

[0020] A second object of the present invention is to provide a method for using the kit, comprising the following steps:

[0021] 1) Add the antigen-capturing antibody to the ELISA plate for antibody coating;

[0022] 2) Add blocking solution for blocking;

[0023] 3) Add standard solution or actual sample to be tested;

[0024] 4) Add enzyme-labeled complex to react;

[0025] 5) Add color developing solution;

[0026] 6) Add stop solution;

[0027] 7) Measure the absorbance and calculate the content of the substance to be tested in the sample based on the standard curve drawn based on the relationship between the concentration of the standard sample and the absorbance.

[0028] The method for using the kit comprises the following steps:

[0029] Step 1) and step 2) specifically include the following steps: first, coating the ELISA plate with the antigen-capturing antibody to be detected at 37° C. for 2 hours, then adding a blocking solution and blocking at 37° C. for 1-2 hours; wherein the concentration of the antigen-capturing antibody to be detected is 1-5 μg / mL, and the antigen-capturing antibody to be detected is diluted with the coating solution; the coating solution is a 10 mM PBS buffer solution with a pH of 7.4, and the blocking solution is a PBS solution containing 1-2% BSA.

[0030] The blocking solution is mainly used to block the active sites not occupied by monoclonal antibodies and reduce nonspecific adsorption in the experiment.

[0031] Compared with the prior art, the technical advantages of the present invention are:

[0032] 1) The present invention uses a standard sandwich method to detect the analyte, making the kit highly specific. PS / PDA is used as a signal amplification substrate, giving the kit a low detection limit and high sensitivity.

[0033] 2) PS / PDA can be covalently linked to capture antibodies and horseradish peroxidase, making the enzyme-labeled complex more stable and capable of detection under different pH environments.

[0034] 3) The detection process of the present invention is simple, the background signal is small, and the signal reproducibility is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the principle of detecting IL-17A by ELISA in the present invention.

[0036] Figure 2 The stability of the enzyme-labeled complex HRP-PS / PDA-Ab2 under different pH conditions.

[0037] Figure 3 The stability of the enzyme-labeled complex HRP-AuNPs-Ab2 with gold nanoparticles as the signal amplification substrate under different pH conditions.

[0038] Figure 4 It is the background signal generated by different enzyme-labeled complexes during ELISA detection.

[0039] Figure 5The response signal when different enzyme-labeled complexes detect the same antigen concentration.

[0040] Figure 6 The EIS method was used to demonstrate the successful construction of the ELISA model.

[0041] Figure 7 A standard curve of IL-17A concentration and optical density signal was established for the detection of IL-17A standard by ELISA method.

[0042] Figure 8 For the selectivity of ELISA kit. DETAILED DESCRIPTION

[0043] The following examples are intended to further illustrate the present invention, but are not intended to limit the scope of protection of the claims of the present invention.

[0044] Example 1:

[0045] 1. Synthesis of PS / PDA Nanoparticles

[0046] 5 mg of dopamine was added to 50 mL of a Tris buffer solution containing 0.2 mg / mL polystyrene nanoparticles. After ultrasonic dispersion, the solution was placed on a stirrer and allowed to react for 12 hours. After completion, the solid product was washed several times with water to obtain polystyrene / polydopamine (PS / PDA) nanoparticles. The PS nanoparticles had a particle size of 60-70 nm. Dopamine self-polymerized onto the PS surface under alkaline conditions, resulting in PS / PDA nanoparticles of 90-100 nm.

[0047] 2. Synthesis of enzyme-labeled complex HRP-PS / PDA-Ab2

[0048] Take 1.7mg of PS / PDA nanoparticles and dissolve them in 1mL of PBS buffer solution. After ultrasonic mixing, add 20μL of 1mg / mL HRP solution and 2.5μL of 1mg / mL Ab2 (antigen detection antibody to be tested, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.) solution, mix gently and evenly, and place it at 4°C for reaction for 12h. After the reaction is completed, the product is washed several times with PBS buffer solution to remove unbound HRP and Ab2. After washing, the product is dispersed in a PBS buffer solution containing 2% BSA and stored at 4°C in the dark. The polydopamine on the surface of the PS / PDA nanoparticles has many active groups, which can be covalently linked to HRP and Ab2 through Michael reaction and Schiff base reaction. The product is placed in a 2% BSA PBS solution, mainly using BSA to block unreacted active sites and avoid nonspecific binding.

[0049] 3.ELISA Experimental Procedure

[0050] 1) Dilute Ab1 (antigen capture antibody to be tested, purchased from Beijing Sino Biological Technology Co., Ltd.) to 1 μg / mL in 10 mM PBS (pH 7.4) buffer; add 100 μL of 1 μg / mL Ab1 to an ELISA plate and coat in an oven at 37°C for 2 h. After incubation, remove the solution from the wells and wash the plate three times with washing solution.

[0051] 2) Add 200 μL of blocking solution to block the ELISA plate and incubate at 37°C for 1-2 hours. After blocking, remove the solution from the wells and wash the plate three times with washing solution.

[0052] 3) Add 50 μL of IL-17A standard solution (purchased from Beijing Sino Biological Technology Co., Ltd.) or actual sample and incubate in an oven at 37°C for 1.0 h. The standard solution concentrations were 400, 200, 100, 80, 40, 20, 10, 5, 2.5, and 1.25 pg / mL. The sample diluent was 0.1-0.5% BSA in PBS. The actual sample was normal human serum. After incubation, remove the solution from the wells and wash the plate three times with washing solution.

[0053] 4) Add 50 μL of HRP-PS / PDA-Ab2 and incubate in a dark oven at 37°C for 1 h. After incubation, remove the solution from the wells and wash the plate six times with washing solution.

[0054] 5) Add 100 μL of colorimetric solution, with a 1:1 volume ratio of substrate solution A to substrate solution B, dilute 10-fold, and protect from light. Allow to develop for 15-30 minutes.

[0055] 6) Add 50 μL 2M H2SO4 to terminate the reaction.

[0056] 7) Measure the absorbance at OD450nm, plot a standard curve, and calculate the IL-17A content in the sample. The colorimetric solution includes Substrate Solution A and Substrate Solution B. Substrate Solution A is prepared as follows: 0.136g sodium acetate, 0.016g citric acid, 3μL 30% H2O2, and HO is added to 5mL. Substrate Solution B is prepared as follows: 0.0021g disodium EDTA, 0.0095g citric acid, 0.5mL glycerol, 0.0015g tetramethylbenzyl diamine, and HO is added to 5mL. The volume ratio of Substrate Solution A to Substrate Solution B is 1:1.

[0057] The washing solution is a PBS solution containing 0.1-0.5% Tween-20, pH=7.4.

[0058] from Figure 2It can be seen that different pH environments have little effect on the binding of PS / PDA and HRP. PS / PDA nanoparticles primarily bind to HRP and Ab2 through covalent bonding. Comparing the changes in HRP before and after PS / PDA adsorption shows that changes in pH have no significant impact on the stability of the enzyme-labeled complex.

[0059] from Figure 3 It can be seen that the enzyme-labeled complex using gold nanoparticles as the substrate for signal amplification is significantly affected by pH. Since gold nanoparticles bind to HRP and Ab2 primarily through physical adsorption, they are significantly affected by the environment, and the complex has poor stability.

[0060] from Figure 4 It can be seen that the background signals caused by different enzyme-labeled complexes are similar to the background signals of HRP-PS / PDA-Ab2 and HRP-Ab2, but the background signal of HRP-AuNPs-Ab2 is larger.

[0061] from Figure 5 It can be seen that the response signals of different enzyme-labeled complexes at the same antigen concentration are quite different. The response signal of HRP-AuNPs-Ab2 in the present invention is much higher than that of HRP-AuNPs-Ab2 and HRP-Ab2.

[0062] from Figure 6 EIS can be used to detect successful binding at each step of the ELISA, confirming the successful construction of the ELISA model. 11-Mercaptoundecanoic acid (MUA), Ab1, BSA, IL-17, and HRP-PS / PDA-Ab2 were sequentially incubated on the gold electrode surface. The impedance values gradually increased, indicating successful binding at each step of the ELISA.

[0063] from Figure 7 It can be seen that as the concentration of IL-17A increases, the response signal gradually increases and becomes linear in the range of 0-80 pg / mL.

[0064] from Figure 8 It can be seen that the ELISA of the present invention has excellent selectivity. The concentrations of IL-17A, IgG, MYOG, NFL, and BDNF are 1 ng / mL, 10 pg / mL, 1 ng / mL, and 1 ng / mL, respectively.

[0065] Table 1 IL-17A recovery determination (normal human serum)

[0066] sample Add (pg / mL) Determination (pg / mL) RSD (%) Recovery rate (%) 1 40 39.63±1.79 4.5 99.08±0.04 2 20 19.49±0.85 4.4 97.48±0.04 3 10 9.239±0.61 6.6 92.39±0.06

Claims

1. An ELISA kit, characterized in that include: The invention relates to a detection antigen capture antibody, an enzyme labeling plate, and an enzyme labeling complex; the enzyme labeling complex is obtained by coupling the detection antibody for the detection antigen, horseradish peroxidase and polystyrene / polydopamine nanoparticles; the detection antigen is IL-17A.

2. The ELISA kit according to claim 1, wherein The preparation method of the enzyme-labeled complex comprises the following steps: adding polystyrene nanospheres to a Tris-HCl solution containing dopamine or dopamine hydrochloride, stirring for reaction; washing the product after the reaction to obtain polystyrene / polydopamine nanoparticles; dispersing the polystyrene / polydopamine nanoparticles in a PBS buffer solution, adding an antigen detection antibody to be detected and horseradish peroxidase to the solution for coupling reaction with the polystyrene / polydopamine nanoparticles; and washing the product to obtain the enzyme-labeled complex.

3. The ELISA kit according to claim 2, wherein The mass ratio of polystyrene to dopamine or dopamine hydrochloride is 2-5:1 in a 10-50 mM Tris-HCl buffer solution at pH 7.5-8.

5.

4. The ELISA kit according to claim 2, wherein Dopamine or dopamine hydrochloride is reacted with polystyrene at 20-30°C for at least 12 hours. In the reaction system, a detection antibody for the antigen to be detected and horseradish peroxidase are evenly mixed and then added to a polystyrene / polydopamine nanoparticle solution, wherein the mass ratio of the detection antibody for the antigen to be detected to the horseradish peroxidase to the polystyrene / polydopamine particles is 1:4-10:200-400, and the reaction is allowed to stand at 2-6°C for at least 12 hours.

5. The kit according to claim 1, wherein The kit also includes: antigen standards to be tested, washing solution, color development solution and stop solution.

6. The kit according to claim 1, wherein The antigen standard to be tested is diluted to the target concentration with a sample diluent, which is a 0.1-0.5% BSA solution in PBS.

7. The kit according to claim 5, characterized in that The color developing solution is a colored conjugate of HRP.

8. The kit according to claim 7, characterized in that The color developing solution includes substrate solution A and substrate solution B. Substrate solution A is prepared as follows: 0.136 g sodium acetate, 0.016 g citric acid, 3 μL 30% H2O2, and H2O is added to 5 mL; substrate solution B is prepared as follows: 0.0021 g disodium ethylenediaminetetraacetic acid, 0.0095 g citric acid, 0.5 mL glycerol, 0.0015 g tetramethylbenzenediamine, and H2O is added to 5 mL; when used, the volume ratio of substrate solution A to substrate solution B is 1:

1.

9. The kit according to claim 5, characterized in that The stop solution is sulfuric acid; the concentration of sulfuric acid is 1.5-2.5 mol / L; the washing solution is a PBS solution containing 0.1-0.5% Tween-20, pH=7.

4.

10. The method for using the kit according to any one of claims 1 to 9, wherein the method is a method for non-disease diagnosis and treatment, characterized in that: The following steps are involved: 1) adding a capture antibody to the target antigen to be detected to coat the plate, wherein the target antigen is IL-17A; 2) Add blocking solution for blocking; 3) Add standard solution or actual sample to be tested; 4) Add enzyme-labeled complex to react; 5) Add color developing solution; 6) Add stop solution; 7) Measure the absorbance and calculate the content of the substance to be tested in the sample based on the standard curve drawn based on the relationship between the concentration of the standard sample and the absorbance.

11. The method for using the kit according to claim 10, characterized in that: The following steps are involved: Step 1) and step 2) specifically include the following steps: first, the ELISA plate is coated with the antigen-capturing antibody to be tested at 37°C for 2 hours, and then a blocking solution is added and blocked at 37°C for 1-2 hours; wherein the concentration of the antigen-capturing antibody to be tested is 1-5 μg / mL, and the antigen-capturing antibody to be tested is diluted with the coating solution; the coating solution is 10mM PBS buffer with a pH of 7.4, and the blocking solution is a PBS solution containing 1-2% BSA.

Images