An enzyme-linked immunosorbent assay kit for detecting EGFR based entirely on gold antibodies and its application

Abstract

The present application discloses an enzyme-linked immunosorbent assay kit for detecting EGFR that is completely based on gold antibodies and its application. The enzyme-linked immunosorbent assay kit includes: a capture antibody, an EGFR standard, a detection antibody, a blank enzyme-linked immunosorbent assay plate, a washing solution, a diluent, a developer and a stop solution. In the present application, the capture antibody can be fixed on the enzyme-linked immunosorbent assay plate, and the detection antibody can efficiently react with the enzyme substrate, thereby being applied to an enzyme-linked immunosorbent assay kit for detecting EGFR. The present application also discloses a method for using the kit for sample detection. The present application adopts the sandwich method principle in the enzyme-linked immunosorbent assay, which can quantitatively detect EGFR in a complex environment. The present application uses a group of gold antibodies instead of natural antibodies to participate in the kit for detection, which has better detection ability and better thermal stability, and also has the other advantages of the enzyme-linked immunosorbent assay kit.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, in particular to an enzyme-linked immunosorbent assay kit for detecting EGFR based entirely on gold antibodies and an application thereof, and is applied to the field of EGFR quantitative detection technology. 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 170 kDa transmembrane glycoprotein that belongs to the ErbB family of receptor tyrosine kinases (RTKs). It participates in signaling pathways associated with cancer development 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 and being associated with tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and apoptosis. Therefore, it is a valuable diagnostic marker for early-stage malignancies.

[0003] Currently, a variety of methods for detecting EGFR have been developed, including high-performance liquid chromatography and electrochemical biosensors. These methods typically require long sample processing times, expensive equipment, and complex testing procedures. Therefore, there is a widespread demand and promising prospects for developing rapid, low-cost EGFR detection methods. Compared to these methods, enzyme-linked immunosorbent assay (ELISA) offers faster detection speeds, lower costs, and similarly low detection limits and high sensitivity. However, the natural antibodies at the core of ELISA are often susceptible to inactivation by changes in ambient temperature, which to some extent limits the application of ELISA. For example, Chinese patent CN115792230A discloses an ELISA sandwich assay kit based on a biotinylated anti-lysozyme gold antibody and its applications. By replacing the detection antibody with a biotinylated anti-lysozyme gold antibody instead of a natural antibody, the resulting structure is an anti-lysozyme antibody-lysozyme-biotinylated anti-lysozyme gold antibody. This allows for ELISA testing, exhibits excellent thermal stability, and is capable of quantitatively detecting 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 an urgent problem to be solved in the existing technology. 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 that is completely based on gold antibodies and its application. Anti-EGFR gold antibodies are used as capture antibodies fixed on a well plate, and enzyme-labeled anti-EGFR gold antibodies that can simultaneously bind to EGFR and peroxidase substrates are used as detection antibodies. The enzyme-linked immunosorbent assay kit is used to detect the content of EGFR in a sample to be tested, which has the advantages of high sensitivity, strong specificity, and high accuracy.

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

[0007] One of the technical solutions of the present application provides an enzyme-linked immunosorbent assay kit for detecting EGFR that is completely based on gold antibodies, comprising: a capture antibody, an EGFR standard, a detection antibody, a blank ELISA plate, and a detection reagent;

[0008] The detection reagents include: washing solution, diluent, color developing solution and 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 the 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 color reaction with a color developing solution; 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 serves as an antibody determinant 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 determinants on EGFR through their respective antibody determinants, thereby 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. They not only inherit the natural thermal stability of gold nanoparticles, but can also 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 a pH of 7.2 to 7.5, 0.1 to 0.2 M, containing 0.04% to 0.06% Tween-20 by mass. More preferably, the washing solution is PBS-T at a pH of 7.4, 0.15 M, containing 0.05% Tween-20 by mass.

[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 steps are as follows:

[0018] The capture antibody is coated on a blank ELISA plate, and the EGFR standard and detection antibody are added. After incubation for 10-60 minutes, a capture antibody-EGFR-detection antibody structure will be formed on the ELISA plate. The substrate of horseradish peroxidase is added to produce a color reaction. After 5-30 minutes of reaction, the stop solution is added to terminate the reaction. The ELISA plate is 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 minutes, wash the plate, and pat dry.

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

[0022] C. Add 20-200 μL of detection antibody, incubate at room temperature for 10-60 minutes, 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 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.

[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 substantial 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 of 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 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 the high-accuracy detection of other substances at low concentrations, and has important significance and value. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0032] Figure 2 The standard curve for detecting EGFR in the 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 with reference to specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but are not intended to 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 be made without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0035] All raw materials of the present invention are not particularly limited in their sources and can be purchased from the market or prepared according to conventional methods well known to those skilled in the art.

[0036] The anti-EGFR gold antibody in a preferred embodiment of the present invention utilizes the gold nanoparticle-based artificial antibody with EGFR-targeting properties disclosed in patent publication number CN106699890B. Based on the polypeptide sequence SAWYGTLYEYD at positions 102-112 in the CDR3 of the natural EGFR antibody 7D12, the peptide Pep2: CSAWYGTLYEYDGC (SEQ. ID. No. 1) was designed and synthesized. The preparation method and steps are described in patent CN106699890B. The thiol groups in the cysteine ​​(Cys) residues at both ends of the Pep2 sequence can form Au-S bonds with the gold nanoparticle surface, thereby anchoring the ends of the Pep2 peptide to the surface of the gold nanoparticle, enabling the anti-EGFR gold antibody to specifically bind to EGFR.

[0037] The enzyme-labeled anti-EGFR gold antibody used in a preferred embodiment of the present invention is a peptide DA: CPPLMLYNPTTYQMDVNPEGC (SEQ.ID.No.2), designed and synthesized based on the peptide fragment CPPLMLYNPTTYQMDVNPEG of the EGFR extracellular domain II. The DA sequence contains cysteine ​​(Cys) residues at both ends, allowing the peptide fragment to be fixed to the surface of gold nanoparticles via Au-S bonds. Through conformational manipulation, its native conformation is restored, forming an anti-EGFR gold antibody that can specifically bind to EGFR. The gold nanoparticles are approximately 14 nm in diameter, and their surfaces are modified not only with the peptide DA but also with horseradish peroxidase (HRP). The HRP is modified by adding thiol-polyethylene glycol horseradish peroxidase (SH-PEG-HRP). The thiol-polyethylene glycol horseradish peroxidase forms an Au-S bond with the gold nanoparticles through the thiol (-SH) group, thereby fixing it to the surface of the gold nanoparticles. In summary, the enzyme-labeled anti-EGFR gold antibody has the antibody determinant cluster CPPLMLYNPTTYQMDVNPEGC corresponding to EGFR and horseradish peroxidase, so it can simultaneously bind to EGFR and the substrate of horseradish peroxidase, 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 add anti-EGFR gold antibody to the well plate, incubate for a period of time, and then add EGFR standard, as shown in Figure 1 As shown in b, an anti-EGFR gold antibody-EGFR structure is formed on the well plate. Add enzyme-labeled anti-EGFR gold antibody and incubate for a period of time. At this time, a sandwich structure of anti-EGFR gold antibody-EGFR-enzyme-labeled anti-EGFR gold antibody will be formed on the well plate, as shown in Figure 1 The cd part in 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 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 includes an anti-EGFR gold antibody, an ELISA plate, an enzyme-labeled anti-EGFR gold antibody, an EGFR standard, a diluent, a developer, a washing solution, and a stop solution. The steps for detecting EGFR in the present invention are as follows:

[0040] A. Add 20-200 μL of anti-EGFR gold antibody to each well of the ELISA plate, using carbonate buffer as the diluent. Incubate at 37°C for 30-120 minutes, 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, using phosphate buffered saline as the 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 minutes, 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 EGFR detection 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 was drawn as follows:

[0049] Anti-EGFR gold antibody (capture antibody) was diluted in diluent (20 mM carbonate buffer, pH 10.7) and coated onto an ELISA plate to a final concentration of 70 nM. 100 μL was added to each well. After incubation at 37°C for 2 hours, the plate was washed with a wash buffer (0.15 M PBS-T, pH 7.4 (containing 0.05% Tween-20), the same below) and patted dry. This was repeated four times (each wash was for 30 seconds).

[0050] Dilute the EGFR standard with 20 mM pH 7.4 phosphate buffer solution (400 ng / mL) six times in a two-fold serial dilution from 400 ng / mL to 6.25 ng / mL. Start the assay as follows:

[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 detergent and pat dry, repeat 4 times (each wash plate stay 30 seconds); add 100 μL of enzyme-labeled anti-EGFR gold antibody, incubate at room temperature for 1 hour, wash the plate with detergent and pat dry, repeat 4 times (each wash plate stay 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, and then add 100 μL of the stop solution;

[0053] Detection and analysis: Use a microplate reader 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, the 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 EGFR detection standard curve, the standard curve equation is: The detection limit was 15.84 ng / mL.

[0055] Example 2

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

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

[0058] To verify the effectiveness of the enzyme-linked immunosorbent assay (ELISA) for detecting EGFR in complex solution environments, bovine serum albumin (BSA) was selected as a nonspecific protein. BSA was added to EGFR standards at various concentrations, and its concentration was set to 100 times the molar concentration of EGFR. Standard curves were constructed according to the procedures in 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 following special features:

[0061] In this example, a gold antibody ELISA was spiked. Spike recovery 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 normal sample processing steps to the theoretical value in an ELISA experiment. Specifically, spike recovery is achieved by adding a known amount of analyte to the sample matrix and then comparing the test results with the theoretical value to evaluate the accuracy and reliability of the ELISA method. The method is as follows:

[0062] The concentrations of EGFR in the spiked samples were set at 40 ng / mL, 60 ng / mL, and 150 ng / mL. 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. Gold antibody ELISA test results for EGFR spike

[0064]

[0065] The above results indicate that the ELISA kit provided in this application has good quantitative accuracy, with spiked recovery rates ranging from 87.30% to 110.87%, meeting 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 following special features:

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

[0069] The gold antibody was pretreated for 1 hour at room temperature, 60°C, and 100°C in a water bath, then cooled to room temperature. The EGFR concentration in the spiked sample was set at 60 ng / mL. The pretreated gold antibody was used for quantitative analysis and recovery calculations to investigate the thermal stability of the gold antibody. The test results are shown in Table 2 (using the same calculation formula as in Example 3). Even after treatment at 100°C, the gold antibody achieved a quantitative recovery of 93.44% in the spiked sample, demonstrating its excellent thermal stability.

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

[0071]

[0072] The results in Table 2 show that the quantitative accuracy of the enzyme-linked immunosorbent assay kit provided in this application can maintain 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 EGFR enzyme-linked immunosorbent assay kits and their applications. This kit is a detection kit based entirely on gold antibodies. The enzyme-linked immunosorbent assay kit includes an anti-EGFR gold antibody, an enzyme-labeled anti-EGFR gold antibody, an EGFR standard, a diluent, a developer, a wash solution, and a stop solution. The present invention also discloses a method for sample detection using the kit. The above embodiment kits completely use gold antibodies instead of natural antibodies for detection, which not only has better detection capabilities but also has excellent thermal stability and combines the other advantages of enzyme-linked immunosorbent assay kits.

[0074] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. 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 having a sequence as shown in SEQ.ID.No.1 connected to its surface, and the polypeptide having a sequence as shown in SEQ.ID.No.1 serves as an antibody determinant; The detection antibody is an enzyme-labeled anti-EGFR gold antibody EGFR-GB2-HRP, which is a gold nanoparticle with a polypeptide and an enzyme linked to its surface as shown in SEQ.ID.No.2, wherein the polypeptide is used as an antibody determinant. The anti-EGFR gold antibody EGFR-GB1 and the enzyme-labeled anti-EGFR gold antibody EGFR-GB2-HRP specifically bind to the antigenic determinants on EGFR through their respective antibody determinants, forming a double antibody sandwich structure; The detection reagents include: washing solution, diluent, color developing solution and stop solution.

2. The enzyme-linked immunosorbent assay 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 enzyme-linked immunosorbent assay kit for detecting EGFR based entirely on gold antibodies according to claim 1, characterized in that: The detection antibody is used to generate a color reaction with the color developing solution.

4. The enzyme-linked immunosorbent assay 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 enzyme-linked immunosorbent assay 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 7.2-7.5 and 0.1-0.2 M, wherein the washing solution contains Tween-20 with a mass concentration of 0.04%-0.06%.

6. The enzyme-linked immunosorbent assay 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 enzyme-linked immunosorbent assay 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 enzyme-linked immunosorbent assay 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 0-2 M, and is not 0.

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