Enzyme linked immunosorbent assay kit for detecting EGFR (epidermal growth factor receptor) completely based on gold antibody and application of enzyme linked immunosorbent assay kit

Abstract

The invention discloses an enzyme linked immunosorbent assay kit for detecting EGFR (epidermal growth factor receptor) completely based on a gold antibody and application of the enzyme linked immunosorbent assay kit. The enzyme linked immunosorbent assay kit comprises a capture antibody, an EGFR standard substance, a detection antibody, a blank elisa plate, a cleaning solution, a diluent, a developing solution and a stop solution. According to the present invention, the capture antibody can be fixed on the ELISA plate, and the detection antibody can efficiently react with the enzyme substrate so as to be used in the EGFR detection enzyme-linked immunosorbent assay kit. The invention also discloses a method for detecting a sample by using the kit. According to the present invention, the sandwich method principle in the enzyme-linked immunosorbent assay is adopted, and the EGFR can be quantitatively detected in the complex environment. A group of gold antibodies are used for replacing natural antibodies to participate in the kit for detection, so that the detection capability is better, the thermal stability is better, and the kit also has other advantages of the enzyme linked immunosorbent assay kit.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, in particular to an enzyme-linked immunosorbent assay kit for detecting EGFR completely based on gold antibodies and an application thereof, and is applied to the technical field of EGFR quantitative detection. Background Art

[0002] Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor involved in various cellular activities. EGFR (also known as ErbB-1 / HER1) is a 170KDs transmembrane glycoprotein that belongs to the ErbB family of receptor tyrosine kinases (RTKs). It is involved in signaling pathways related to cancer occurrence and progression and is associated with a variety of gene mutations. EGFR is highly expressed on the surface of most tumor cells, causing adverse clinical reactions. It has a certain relationship with tumor cell proliferation, angiogenesis, tumor invasion, metastasis and cell apoptosis, and is therefore a valuable diagnostic marker for early malignant tumors.

[0003] At present, a variety of methods for detecting EGFR have been developed, including high performance liquid chromatography, electrochemical biosensors, etc. These methods usually require long sample processing time, expensive instruments, and complex testing processes. Therefore, the development of fast and low-cost EGFR detection methods has a wide range of needs and broad prospects. Compared with these methods, the enzyme-linked immunosorbent assay has fast detection speed, low detection cost, and also has low detection limit and high sensitivity. However, the natural antibodies at the core of the enzyme-linked immunosorbent assay are usually susceptible to inactivation due to changes in ambient temperature, which will also limit the application of the enzyme-linked immunosorbent assay to a certain extent. For example, Chinese patent CN115792230A discloses an enzyme-linked immunosorbent assay sandwich kit based on biotinylated anti-lysozyme gold antibody and its application, wherein the detection antibody is replaced by a natural antibody with a biotinylated anti-lysozyme gold antibody to form an anti-lysozyme antibody-lysozyme-biotinylated anti-lysozyme gold antibody structure, which can perform enzyme-linked immunosorbent assay, has good thermal stability, and can quantitatively detect lysozyme in egg white. However, this patent still uses natural antibodies as capture antibodies, and does not realize the use of a pair of gold antibodies to simultaneously bind to the target antigen to form a gold antibody-antigen-gold antibody structure, and the enzyme-linked immunosorbent assay kit in which the constituent antibodies all use gold antibodies.

[0004] Therefore, using a pair of gold antibodies to simultaneously bind to the target antigen, constructing a gold antibody-antigen-gold antibody structure, and preparing an EGFR kit with excellent stability and sensitivity is a problem that urgently needs to be solved in the prior art. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the purpose of the present invention is to overcome the shortcomings of the existing technology and provide an enzyme-linked immunosorbent assay kit for detecting EGFR based entirely on gold antibodies and its application, using anti-EGFR gold antibodies as capture antibodies fixed on a well plate, using enzyme-labeled anti-EGFR gold antibodies that can simultaneously bind to EGFR and peroxidase substrates as detection antibodies, and applying them to an enzyme-linked immunosorbent assay kit to detect the content of EGFR in a sample to be tested, which has the advantages of high sensitivity, strong specificity, high accuracy, etc.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] One of the technical solutions of the present application provides an ELISA kit for detecting EGFR based entirely on gold antibodies, including: capture antibodies, EGFR standards, detection antibodies, blank ELISA plates, and detection reagents;

[0008] The detection reagent comprises: a washing solution, a diluent, a color developing solution and a stop solution.

[0009] Furthermore, the capture antibody is an anti-EGFR gold antibody EGFR-GB1, which is used to connect to a blank ELISA plate, and the blank ELISA plate serves as a solid phase carrier; the anti-EGFR gold antibody EGFR-GB1 is: a gold nanoparticle with a polypeptide sequence as shown in SEQ.ID.No.1 connected to the surface, and the polypeptide serves as an antibody determinant cluster.

[0010] Furthermore, the detection antibody is an enzyme-labeled anti-EGFR gold antibody EGFR-GB2-HRP, which is used to undergo a colorimetric reaction with a color developing solution; the enzyme-labeled anti-EGFR gold antibody EGFR-GB2-HRP is: a gold nanoparticle having a polypeptide and an enzyme connected to the surface as shown in SEQ.ID.No.2, and the polypeptide serves as an antibody determining cluster; the enzyme connected to the surface of the enzyme-labeled anti-EGFR gold antibody is horseradish peroxidase.

[0011] Furthermore, the anti-EGFR gold antibody EGFR-GB1 and the enzyme-labeled anti-EGFR gold antibody EGFR-GB2-HRP specifically bind to the antigenic determinant cluster on EGFR through their respective antibody determinants at the same time, forming a double antibody sandwich structure.

[0012] Furthermore, the anti-EGFR gold antibody and enzyme-labeled anti-EGFR gold antibody are both new artificial antibodies synthesized through conformational engineering, which not only inherit the natural thermal stability of gold nanoparticles, but also can specifically bind to EGFR, and have the potential to be used as antibody substitutes in enzyme-linked immunosorbent assay.

[0013] Preferably, the washing solution is PBS-T at pH 7.2-7.5, 0.1-0.2M, wherein the washing solution contains Tween-20 at a mass concentration of 0.04%-0.06%. More preferably, the washing solution is PBS-T at pH 7.4, 0.15M, wherein the washing solution contains Tween-20 at a mass concentration of 0.05%.

[0014] Preferably, the diluent is a buffer solution with a pH of 7-11; more preferably, it is a phosphate buffer solution or a carbonate buffer solution with a pH of 7-11.

[0015] Preferably, the color developing solution is tetramethylbenzidine (TMB) or o-phenylenediamine (OPD).

[0016] Preferably, the stop solution is sulfuric acid or hydrochloric acid, and the acid concentration in the stop solution is 0-2M, and is not zero.

[0017] Furthermore, anti-EGFR gold antibody and enzyme-labeled anti-EGFR gold antibody were used to detect EGFR by enzyme-linked immunosorbent assay sandwich method. The specific method steps are as follows:

[0018] The capture antibody was coated on a blank ELISA plate, and EGFR standard and detection antibody were added. After incubation for 10-60 minutes, a capture antibody-EGFR-detection antibody structure was formed on the ELISA plate. The substrate of horseradish peroxidase was added to produce a color reaction. After 5-30 minutes of reaction, the stop solution was added to terminate the reaction. The ELISA plate was placed in an ELISA reader for detection to obtain the OD values ​​corresponding to different concentrations of EGFR standard. 450 nm The standard curve was drawn to determine the concentration of EGFR in the sample to be tested.

[0019] The second technical solution of the present application provides an application of an ELISA kit for detecting EGFR based on a group of gold antibodies as described in the above technical solution, wherein the ELISA kit is used to detect the concentration of EGFR in a sample to be tested, comprising the following steps:

[0020] A. Add 20-200 μL of capture antibody to each well of the ELISA plate, incubate at 37°C for 30-120 min, wash the plate and pat dry;

[0021] B. Add EGFR standard or test sample: Add 20-200 μL EGFR standard or test sample to each well of the ELISA plate, incubate at room temperature for 10-90 min, wash the plate and pat dry;

[0022] C. Add 20-200 μL of detection antibody, incubate at room temperature for 10-60 min, wash the plate and pat dry;

[0023] D. Color development: Add 20-200 μL of color development solution to each well, incubate at room temperature in the dark for 10-30 minutes, and add stop solution to terminate the reaction;

[0024] E. Detection and analysis: Use a multifunctional microplate reader to measure the signal value OD of each well 450 nm , analyze the test results, with EGFR concentration as the x-axis and signal value OD 450 nm As the y-axis, a standard curve for detecting EGFR is drawn; the absorbance value of the sample to be tested is substituted into the standard curve of EGFR to calculate the EGFR concentration in the sample to be tested.

[0025] Furthermore, the concentration of the EGFR standard is 5-400 ng / mL, more preferably 400, 200, 100, 50, 25, 12.5, 6.25 ng / mL.

[0026] Furthermore, the EGFR test sample is a simulated sample in actual application, in which a standard concentration of EGFR is mixed in a high concentration of BSA, and does not involve the diagnosis and treatment of the disease.

[0027] Compared with the prior art, the present invention has the following obvious outstanding substantive features and significant advantages:

[0028] (1) The enzyme-linked immunosorbent assay kit for detecting EGFR of the present invention uses anti-EGFR gold antibodies as a substitute for natural antibodies, has a higher detection range, more flexible antibody selection, and avoids the disadvantage that natural antibodies are easily inactivated by temperature changes.

[0029] (2) The present invention adopts the sandwich method principle in the enzyme-linked immunosorbent assay, which can accurately quantify the content of EGFR in the sample to be tested; the kit of the present invention uses anti-EGFR gold antibodies instead of natural antibodies for detection, which has better detection ability and excellent thermal stability, and also has other advantages of the enzyme-linked immunosorbent assay kit.

[0030] (3) The enzyme-linked immunosorbent assay kit of the present invention, which is completely based on gold antibodies, provides a new direction for high-accuracy detection of other substances at low concentrations, and has important significance and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the principle of detecting EGFR in a preferred embodiment of the present invention;

[0032] Figure 2 A standard curve for detecting EGFR in a preferred embodiment of the present invention;

[0033] Figure 3This is a standard curve in which BSA is added when detecting EGFR in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, several variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0035] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0036] The anti-EGFR gold antibody in the preferred embodiment of the present invention uses the gold nanoparticle artificial antibody with EGFR targeting performance disclosed in the patent document with publication number CN106699890B. According to the polypeptide sequence SAWYGTLYEYD of 102-112 in the antibody determinant cluster CDR3 in the natural antibody 7D12 of EGFR, the polypeptide Pep2: CSAWYGTLYEYDGC (SEQ.ID.No.1) is designed and synthesized, and the preparation method and steps thereof refer to the patent CN106699890B. The thiol groups in the cysteine ​​(Cys) at both ends of the Pep2 sequence can form Au-S bonds with the nanogold surface, thereby fixing the two ends of the polypeptide Pep2 on the surface of the gold nanoparticles, and the anti-EGFR gold antibody can specifically bind to EGFR.

[0037] The enzyme-labeled anti-EGFR gold antibody used in the preferred embodiment of the present invention is designed and synthesized based on the peptide fragment CPPLMLYNPTTYQMDVNPEG of the EGFR extracellular domain II domain, and the polypeptide DA is designed and synthesized: CPPLMLYNPTTYQMDVNPEGC (SEQ.ID.No.2). There are cysteine ​​(Cys) residues at both ends of the DA sequence, which allows the polypeptide fragment to be fixed on the surface of the gold nanoparticles through the Au-S bonds at both ends, and through conformational regulation, its natural conformation is restored to form an anti-EGFR gold antibody that can specifically bind to EGFR. The diameter of the gold nanoparticles is about 14nm, and its surface is not only modified with the polypeptide DA, but also modified with horseradish peroxidase (HRP). The horseradish peroxidase (HRP) is modified by adding thiol polyethylene glycol horseradish peroxidase (SH-PEG-HRP), and the thiol polyethylene glycol horseradish peroxidase forms an Au-S bond with the gold nanoparticles through the thiol (-SH), and then is fixed on the surface of the gold nanoparticles. In summary, the enzyme-labeled anti-EGFR gold antibody has the antibody determinant cluster CPPLMLYNPTTYQMDVNPEGC and horseradish peroxidase corresponding to EGFR, so it can bind to EGFR and the substrate of horseradish peroxidase at the same time, and serve as the detection antibody of this method to participate in the detection of EGFR.

[0038] The preferred embodiment of the present invention uses the above-mentioned anti-EGFR gold antibody to detect EGFR according to the sandwich method principle of enzyme-linked immunosorbent assay. Figure 1 As shown, the detection principle is: Figure 1 As shown in a, first, anti-EGFR gold antibody is added to the well plate, and after incubation for a period of time, EGFR standard is added, such as Figure 1 As shown in b, an anti-EGFR gold antibody-EGFR structure is formed on the well plate. After adding enzyme-labeled anti-EGFR gold antibody and incubating for a period of time, a sandwich structure of anti-EGFR gold antibody-EGFR-enzyme-labeled anti-EGFR gold antibody is formed on the well plate, as shown in Figure 1 The cd part of the well is then added with horseradish peroxidase substrate. The gold antibody labeled with horseradish peroxidase will efficiently bind to the substrate. The enzymatic reaction between horseradish peroxidase and its substrate is used to generate the absorbance value OD on the well plate. 450 nm It is positively correlated with the concentration of EGFR, so a standard curve can be drawn.

[0039] The kit of the present invention comprises an anti-EGFR gold antibody, an ELISA plate, an enzyme-labeled anti-EGFR gold antibody, an EGFR standard, a diluent, a color developing solution, a washing solution and a stop solution. The steps of detecting EGFR of the present invention are as follows:

[0040] A. Add 20-200 μL of anti-EGFR gold antibody to each well of the ELISA plate, use carbonate buffer as diluent, incubate at 37°C for 30-120 min, wash the plate and pat dry;

[0041] B. Add EGFR standard and detection antibody: Add 20-200 μL of EGFR standard of different concentrations to each well of the ELISA plate, use phosphate buffer solution as diluent, incubate at room temperature for 10-60 minutes, wash the plate and pat dry;

[0042] C. Add 20-200 μL of enzyme-labeled anti-EGFR gold antibody, incubate at room temperature for 10-60 min, wash the plate and pat dry;

[0043] D. Color development: Add 20-200 μL of color development solution to each well, incubate at room temperature in the dark for 10-40 minutes, and add stop solution to terminate the reaction;

[0044] E. Detection and analysis: Use a multifunctional microplate reader to measure the signal value OD of each well 450 nm , analyze the test results, and draw a standard curve for detecting EGFR with the x-axis as the EGFR concentration and the y-axis as the OD value at 450nm.

[0045] In a preferred embodiment of the present invention, the diluent is a buffer solution with a pH of 7-11, the color developing solution is tetramethylbenzidine (TMB) or o-phenylenediamine (OPD), and the stop solution is sulfuric acid or hydrochloric acid.

[0046] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0047] Example 1

[0048] In this embodiment, the EGFR standard curve is drawn as follows:

[0049] Anti-EGFR gold antibody (capture antibody) was diluted with diluent (20 mM carbonate buffer, pH = 10.7) and coated into the ELISA plate, with a final concentration of 70 nM, and the amount added to each well was 100 μL; after incubation at 37°C for 2 hours, the plate was washed with a washing solution (the washing solution was: pH 7.4, 0.15 M PBS-T (containing 0.05% Tween-20), the same below) and patted dry, and repeated 4 times (each washing was left for 30 seconds);

[0050] Dilute the EGFR standard with diluent (20 mM pH 7.4 phosphate buffer solution) from 400 ng / mL to 6.25 ng / mL in two-fold gradient dilutions for 6 times, and start the test according to the following steps:

[0051] Add EGFR standard and detection antibody (the detection antibody is: enzyme-labeled anti-EGFR gold antibody): EGFR standard concentration is 400, 200, 100, 50, 25, 12.5, 6.25 ng / mL; add 100 μL of standard to the well plate, incubate at room temperature for 1 hour, wash the plate with washing solution and pat dry, repeat 4 times (each time the plate is washed for 30 seconds); add 100 μL of enzyme-labeled anti-EGFR gold antibody, incubate at room temperature for 1 hour, wash the plate with washing solution and pat dry, repeat 4 times (each time the plate is washed for 30 seconds);

[0052] Color development: Add 100 μL of the color developing agent TMB (tetramethylbenzidine) to each well, incubate at room temperature in the dark for 10 min, then add 100 μL of the stop solution;

[0053] Detection and analysis: Use an enzyme-labeled instrument to test the absorbance of each well at a wavelength of 450 nm (OD 450 nm ). The EGFR concentration in the standard is used as the X-axis, OD 450 nm As the Y axis, a standard curve for EGFR detection was constructed. The OD corresponding to the detection limit 450 nm is the OD of the blank sample 450 nm Add 3 times the standard deviation.

[0054] Figure 2 To obtain the standard curve for detecting EGFR, the standard curve equation is: The detection limit was 15.84 ng / mL.

[0055] Example 2

[0056] This embodiment is basically the same as Embodiment 1, except that:

[0057] In this embodiment, EGFR is detected in a complex environment by the following method:

[0058] In order to verify the effectiveness of the ELISA kit in detecting EGFR in a complex solution environment, the present application selected bovine serum albumin (BSA) as a non-specific protein. BSA was added to the EGFR standard of each concentration, and its concentration was set to 100 times the molar concentration of EGFR, and the standard curve was constructed according to the steps of Example 1. Figure 3 The obtained standard curve is not much different from the original standard curve, which also means that the detection range and detection limit have not changed. It can be preliminarily demonstrated that the enzyme-linked immunosorbent assay kit has the ability to detect EGFR in a complex solution environment.

[0059] Example 3

[0060] This embodiment is basically the same as the above-mentioned embodiment 1 or 2, with the special features that:

[0061] In this example, the gold antibody ELISA was spiked and tested. The spike recovery rate refers to the ratio of the result obtained by adding a known concentration of a standard substance to the sample and then analyzing it according to the normal sample processing steps to the theoretical value in the ELISA experiment. Specifically, the spike recovery rate is to add a known amount of analyte to the sample matrix, then detect it, and compare the test result with the theoretical value to evaluate the accuracy and reliability of the ELISA method. The method is as follows:

[0062] The concentration of EGFR in the spiked samples was set at 40 ng / mL, 60 ng / mL and 150 ng / mL, and the gold antibody ELISA was used for quantitative testing and the recovery rate was calculated. The recovery rate formula here is: recovery rate = (detection concentration / spiked concentration) × 100%.

[0063] Table 1. Results of spike-in test for EGFR by gold antibody ELISA

[0064]

[0065] The above results indicate that the ELISA kit provided in the present application has good quantitative accuracy, and the spike recovery rate is between 87.30% and 110.87%, which meets the recovery rate standard (80%-120%).

[0066] Example 4

[0067] This embodiment is basically the same as the above-mentioned embodiments 1 to 3, with the special features that:

[0068] In this example, the gold antibody pretreated at different temperatures was used for spike testing, and the method was as follows:

[0069] The gold antibody was pretreated for 1 h at room temperature and in a water bath at 60°C and 100°C, and then cooled to room temperature. The concentration of EGFR in the spiked sample was set to 60 ng / mL, and the pretreated gold antibody was used for quantitative testing and the recovery rate was calculated to study the thermal stability of the gold antibody. The test results are shown in Table 2 (the calculation formula is the same as in Example 3). Even after the gold antibody was treated at 100°C, the recovery rate of the quantitative spiked sample can still reach 93.44%, indicating that the gold antibody has excellent thermal stability.

[0070] Table 2. Results of gold antibody spike test after treatment at different temperatures

[0071]

[0072] The results in Table 2 show that the quantitative accuracy of the ELISA kit provided in the present application can be maintained at a good state at different temperatures, and the spiked recovery rate is between 93.44% and 107.22%, which meets the recovery rate standard (80%-120%).

[0073] The above embodiments are anti-EGFR gold antibodies and ELISA kits for detecting EGFR and their applications. The kit is a detection kit completely based on gold antibodies. The ELISA kit includes anti-EGFR gold antibodies, enzyme-labeled anti-EGFR gold antibodies, EGFR standards, diluents, developer solutions, washing solutions and stop solutions. The present invention also discloses a method for sample detection using the kit. The kits in the above embodiments completely use gold antibodies instead of natural antibodies for detection, which has better detection capabilities, excellent thermal stability, and other advantages of ELISA kits.

[0074] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

[0075] The sequences involved in this application are as follows:

[0076] SEQ.ID.No.1 (polypeptide sequence on anti-EGFR gold antibody):

[0077] CSAWYGTLYEYDGC;

[0078] SEQ.ID.No.2 (polypeptide sequence on enzyme-labeled anti-EGFR gold antibody): CPPLMLYNPTTYQMDVNPEGC.

Claims

1. An enzyme-linked immunosorbent assay kit for detecting EGFR based entirely on gold antibodies, characterized in that: include: Capture antibody, EGFR standard, detection antibody, blank ELISA plate, detection reagent; The capture antibody is an anti-EGFR gold antibody EGFR-GB1, which is a gold nanoparticle with a polypeptide with a sequence as shown in SEQ.ID.No.1 connected to the surface, and the polypeptide with a sequence as shown in SEQ.ID.No.1 serves as an antibody determinant cluster; The detection antibody is an enzyme-labeled anti-EGFR gold antibody EGFR-GB2-HRP, and the enzyme-labeled anti-EGFR gold antibody EGFR-GB2-HRP is: a gold nanoparticle with a polypeptide and an enzyme connected to the surface as shown in SEQ.ID.No.2, and the polypeptide as shown in SEQ.ID.No.2 is used as an antibody determinant cluster; The anti-EGFR gold antibody EGFR-GB1 and the enzyme-labeled anti-EGFR gold antibody EGFR-GB2-HRP respectively bind to the antigenic determinant on EGFR through their respective antibody determinants at the same time to form a double antibody sandwich structure; The detection reagent comprises: a washing solution, a diluent, a color developing solution and a stop solution.

2. The ELISA kit for detecting EGFR based entirely on gold antibodies according to claim 1, characterized in that: The capture antibody is used to connect to a blank ELISA plate, and the blank ELISA plate serves as a solid phase carrier.

3. The ELISA kit for detecting EGFR based entirely on gold antibodies according to claim 1, characterized in that: The detection antibody is used to react with the color developing solution.

4. The ELISA kit for detecting EGFR based entirely on gold antibodies according to claim 1, characterized in that: The enzyme connected to the surface of the enzyme-labeled anti-EGFR gold antibody is horseradish peroxidase.

5. The ELISA kit for detecting EGFR based entirely on gold antibodies according to claim 1, characterized in that: The washing solution is PBS-T with a pH of 7.2 to 7.5 and a 0.1 to 0.2 M content, wherein the washing solution contains Tween-20 with a mass concentration of 0.04% to 0.06%.

6. The ELISA kit for detecting EGFR based entirely on gold antibodies according to claim 1, characterized in that: The diluent is a buffer solution with a pH of 7-11, and the buffer solution is a phosphate buffer solution or a carbonate buffer solution.

7. The ELISA kit for detecting EGFR based entirely on gold antibodies according to claim 1, characterized in that: The color developing solution is tetramethylbenzidine or o-phenylenediamine.

8. The ELISA kit for detecting EGFR based entirely on gold antibodies according to claim 1, characterized in that: The stop solution is sulfuric acid or hydrochloric acid, and the acid concentration in the stop solution is 0-2M, and is not zero.

9. An application of an ELISA kit for detecting EGFR based entirely on gold antibodies as claimed in any one of claims 1 to 9, characterized in that: The enzyme-linked immunosorbent assay kit is used to detect the EGFR concentration in a sample to be tested, and comprises the following steps: A. Add 20-200 μL of capture antibody to each well of the ELISA plate, incubate at 37°C for 30-120 min, wash the plate and pat dry; B. Add EGFR standard or test sample: Add 20-200 μL EGFR standard or test sample to each well of the ELISA plate, incubate at room temperature for 10-90 min, wash the plate and pat dry; C. Add 20-200 μL of detection antibody, incubate at room temperature for 10-60 min, wash the plate and pat dry; D. Color development: Add 20-200 μL of color development solution to each well, incubate at room temperature in the dark for 10-30 minutes, and add stop solution to terminate the reaction; E. Detection and analysis: Use a multifunctional microplate reader to measure the signal value OD of each well 450nm , analyze the test results, with EGFR concentration as the x-axis and signal value OD 450nm The y-axis is used to draw a standard curve for detecting EGFR; the absorbance value of the sample to be tested is substituted into the standard curve of EGFR to calculate the EGFR concentration in the sample to be tested.

10. The use of an ELISA kit for detecting EGFR based entirely on gold antibodies according to claim 9, characterized in that: The concentration of the EGFR standard is 5-400 ng / mL.

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