CN119936402B

CN119936402B - An enzyme-linked immunosorbent assay kit for detecting EGFR based entirely on gold antibodies and its application

Info

Publication number: CN119936402B
Application number: CN202510051868.2A
Authority: CN
Inventors: 曹傲能; 戴静静; 王海芳
Original assignee: University of Shanghai for Science and Technology
Current assignee: University of Shanghai for Science and Technology
Priority date: 2025-01-14
Filing date: 2025-01-14
Publication date: 2025-09-23
Anticipated expiration: 2045-01-14
Also published as: CN119936402A

Abstract

The application discloses an enzyme-linked immunosorbent assay kit for detecting EGFR (epidermal growth factor receptor) based on gold antibodies and application thereof. The ELISA kit comprises a capture antibody, an EGFR standard, a detection antibody, a blank ELISA plate, a washing solution, a diluent, a color development solution and a stop solution. The capturing antibody can be fixed on the ELISA plate, and the detection antibody can react with an enzyme substrate efficiently, so that the capturing antibody can be applied to an ELISA kit for detecting EGFR. The application also discloses a method for detecting the sample by using the kit. The application adopts the sandwich method principle in the enzyme-linked immunosorbent assay, and can quantitatively detect EGFR in a complex environment. The application uses a group of gold antibodies to replace natural antibodies to participate in the detection of the kit, has better detection capability and better thermal stability, and also has other advantages of the enzyme-linked immunosorbent assay kit.

Description

ELISA kit for detecting EGFR (epidermal growth factor receptor) based on gold antibody completely and application thereof

Technical Field

The invention relates to the technical field of biological detection, in particular to an enzyme-linked immunosorbent assay kit for detecting EGFR based on gold antibodies and application thereof, which are applied to the technical field of EGFR quantitative detection.

Background

The Epidermal Growth Factor Receptor (EGFR) is a tyrosine kinase receptor involved in various cellular activities. EGFR (also known as ErbB-1/HER 1) is a 170KDS transmembrane glycoprotein, belongs to the ErbB family of Receptor Tyrosine Kinases (RTKs), is involved in signaling pathways associated with the development and progression of cancer, and is associated with a variety of genetic mutations. EGFR is highly expressed on the surfaces of most tumor cells, causes adverse clinical reactions, has certain relation with proliferation, angiogenesis, tumor invasion, metastasis and apoptosis of tumor cells, and is a valuable diagnostic marker of early malignant tumors.

Various methods for detecting EGFR have been developed, including high performance liquid chromatography, electrochemical biosensors, etc., which generally require long sample processing times, expensive instruments, and complex test procedures. Therefore, there is a broad need and broad prospect to develop a rapid, low cost EGFR detection method. Compared with the methods, the ELISA method has the advantages of high detection speed, low detection cost, low detection limit and high sensitivity. However, natural antibodies, which are the core of the enzyme-linked immunosorbent method, are usually susceptible to inactivation by changes in ambient temperature, which also limits the use of the enzyme-linked immunosorbent method to some extent. For example, chinese patent CN115792230a discloses an enzyme-linked immunosorbent assay kit based on a biotinylated anti-lysozyme gold antibody and application thereof, wherein the detection antibody is replaced by a natural antibody to form an anti-lysozyme antibody-lysozyme-biotinylated anti-lysozyme gold antibody structure, so that an enzyme-linked immunosorbent assay can be performed, the thermal stability is good, and the quantitative detection of lysozyme in egg white can be performed. However, the patent still uses a natural antibody as a capture antibody, and does not realize that a pair of gold antibodies are simultaneously combined with a target antigen to form a gold antibody-antigen-gold antibody structure, so that an enzyme-linked immunosorbent kit is formed by using gold antibodies for all antibodies.

Therefore, the EGFR kit with excellent stability and sensitivity is prepared by using a pair of gold antibodies to combine with target antigen simultaneously to construct a gold antibody-antigen-gold antibody structure, and the EGFR kit is a problem to be solved in the prior art.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to overcome the defects in the prior art, provides an enzyme-linked immunosorbent assay kit for detecting EGFR based on gold antibodies and application thereof, uses anti-EGFR gold antibodies as capture antibodies to be fixed on a pore plate, the enzyme-labeled anti-EGFR gold antibody capable of simultaneously combining EGFR and peroxidase substrates is used as a detection antibody, and is applied to an enzyme-linked immunosorbent assay kit to detect the content of EGFR in a sample to be detected, and the method has the advantages of high sensitivity, strong specificity, high accuracy and the like.

In order to achieve the above purpose, the invention adopts the following technical scheme:

according to one of the technical schemes, the application provides an ELISA kit for detecting EGFR based on gold antibodies, which comprises a capture antibody, an EGFR standard, a detection antibody, a blank ELISA plate and a detection reagent;

the detection reagent comprises a washing solution, a diluent, a chromogenic solution and a stop solution.

Further, the capture antibody is an anti-EGFR gold antibody EGFR-GB1, and is used for being connected with a blank ELISA plate, wherein the blank ELISA plate is used as a solid phase carrier, the anti-EGFR gold antibody EGFR-GB1 is gold nanoparticles, the surfaces of which are connected with polypeptides with the sequence shown as SEQ.ID.No.1, and the polypeptides are used as antibody determinants.

The detection antibody is an enzyme-labeled anti-EGFR gold antibody EGFR-GB2-HRP and is used for carrying out a color reaction with a color development liquid, wherein the enzyme-labeled anti-EGFR gold antibody EGFR-GB2-HRP is gold nanoparticles with the surfaces connected with polypeptides and enzymes with sequences shown as SEQ.ID.No.2, the polypeptides are used as antibody determinants, and the enzyme connected with the surfaces of the enzyme-labeled anti-EGFR gold antibodies is horseradish peroxidase.

Further, the anti-EGFR gold antibody EGFR-GB1 and the enzyme-labeled anti-EGFR gold antibody EGFR-GB2-HRP respectively pass through respective antibody determinants and simultaneously specifically bind with an antigenic determinant on EGFR to form a double-antibody sandwich structure.

Furthermore, the anti-EGFR gold antibody and the enzyme-labeled anti-EGFR gold antibody are all brand-new artificial antibodies synthesized through conformational engineering, not only inherit the natural thermal stability of gold nanoparticles, but also can specifically bind EGFR, and have the potential of being applied to an enzyme-linked immunosorbent assay as an antibody substitute.

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

Preferably, the diluent is a buffer solution with pH of 7-11, and more preferably a phosphate buffer solution or a carbonate buffer solution with pH of 7-11.

Preferably, the color developing solution is Tetramethylbenzidine (TMB) or o-phenylenediamine (OPD).

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

Further, the EGFR is detected by an enzyme-linked immunosorbent assay sandwich method by using an anti-EGFR gold antibody and an enzyme-labeled anti-EGFR gold antibody, and the specific method comprises the following steps:

Coating a capture antibody on a blank ELISA plate, adding an EGFR standard substance and a detection antibody, incubating for 10-60min, forming a capture antibody-EGFR-detection antibody structure on the ELISA plate, adding a substrate of horseradish peroxidase to generate a chromogenic reaction, adding a stop solution to stop the reaction after reacting for 5-30min, putting the ELISA plate into an ELISA instrument to detect, obtaining OD ₄₅₀ _nm values corresponding to EGFR standard substances with different concentrations, drawing a standard curve, and determining the concentration of EGFR in a sample to be detected.

According to a second technical scheme, the application provides application of the EGFR enzyme-linked immunosorbent assay kit based on a group of gold antibodies, wherein the enzyme-linked immunosorbent assay kit is used for detecting EGFR concentration in a sample to be detected, and comprises the following steps:

A. adding 20-200 mu L of capture antibody into each hole of the ELISA plate, incubating for 30-120min at 37 ℃, washing the plate and beating to dryness;

B. Adding EGFR standard substance or sample to be tested, namely adding 20-200 mu L EGFR standard substance or sample to be tested into each hole of an ELISA plate, incubating for 10-90min at room temperature, washing the plate and beating to dry;

C. adding 20-200 mu L of detection antibody, incubating for 10-60min at room temperature, washing the plate and beating to dry;

D. color development, namely adding 20-200 mu L of color development liquid into each hole, incubating for 10-30min at room temperature in a dark place, and adding stop solution to stop the reaction;

E. The detection analysis comprises the steps of measuring a signal value OD ₄₅₀ _nm of each hole by using a multifunctional enzyme-labeled instrument, analyzing a detection result, drawing a standard curve for detecting EGFR by taking the EGFR concentration as an x axis and the signal value OD ₄₅₀ _nm as a y axis, taking the light absorption value of a sample to be detected into the standard curve of the EGFR, and calculating to obtain the EGFR concentration in the sample to be detected.

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

Furthermore, the EGFR sample to be tested is a simulated sample in practical application, and EGFR with standard concentration is mixed in BSA with high concentration, so that diagnosis and treatment of diseases are not involved.

Compared with the prior art, the invention has the following obvious prominent substantive features and obvious advantages:

(1) The ELISA kit for detecting EGFR uses the anti-EGFR gold antibody as a substitute of a natural antibody, has a higher detection range and more flexible antibody selection, and avoids the defect that the natural antibody is easy to be inactivated due to temperature change.

(2) The kit disclosed by the invention adopts a sandwich method principle in an ELISA method, can accurately quantify the content of EGFR in a sample to be detected, uses an anti-EGFR gold antibody to replace a natural antibody for detection, has better detection capability and excellent thermal stability, and has other advantages of the ELISA kit.

(3) The ELISA kit completely based on the gold antibody provides a new direction for high-accuracy detection of other substances at low concentration, and has important significance and value.

Drawings

FIG. 1 is a schematic diagram of EGFR detection in accordance with a preferred embodiment of the present invention;

FIG. 2 is a standard curve of EGFR detection in accordance with a preferred embodiment of the present invention;

FIG. 3 is a standard curve of BSA addition when EGFR is detected in accordance with a preferred embodiment of the present invention.

Detailed Description

In order to enable those skilled in the art to better understand the technical scheme of the present invention, the present invention will be described in detail with reference to specific embodiments. It should be noted that the following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the present invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.

All the raw materials of the present invention are not particularly limited in their sources, and may be commercially available or prepared according to conventional methods well known to those skilled in the art.

The anti-EGFR gold antibody in the preferred embodiment of the invention uses the gold nano-artificial antibody with EGFR targeting property disclosed in the patent document publication No. CN106699890B, and the polypeptide Pep2: CSAWYGTLYEYDGC (SEQ. ID. No. 1) is designed and synthesized according to the 102-112 polypeptide sequence SAWYGTLYEYD in the antibody determinant CDR3 in the natural antibody 7D12 of EGFR, and the content of the preparation method steps and the like are referred to in the patent CN106699890B. The sulfhydryl groups in the cysteine (Cys) at the two ends of the Pep2 sequence can form Au-S bond with the surface of the nano gold, so that the two ends of the polypeptide Pep2 are fixed on the surface of the gold nano particle, and the anti-EGFR gold antibody of EGFR can be specifically combined.

The enzyme-labeled anti-EGFR gold antibody used in the preferred embodiment of the present invention was designed to synthesize the polypeptide DA CPPLMLYNPTTYQMDVNPEGC (SEQ. ID. No. 2) based on the peptide fragment CPPLMLYNPTTYQMDVNPEG of EGFR extracellular domain II domain. The DA sequence has cysteine (Cys) residues at both ends, which allows the polypeptide fragment to be immobilized on the gold nanoparticle surface via Au-S bond at both ends, and to resume its natural conformation via conformational regulation, forming an anti-EGFR gold antibody capable of specifically binding EGFR. The diameter of the gold nanoparticle is about 14nm, and the surface of the gold nanoparticle is modified with polypeptide DA and horseradish peroxidase (HRP). The horseradish peroxidase (HRP) is modified by adding sulfhydryl polyethylene glycol horseradish peroxidase (SH-PEG-HRP), and the sulfhydryl polyethylene glycol horseradish peroxidase forms an Au-S bond with the gold nanoparticles through sulfhydryl (-SH), so that the horseradish peroxidase is fixed on the surfaces of the gold nanoparticles. In summary, the enzyme-labeled anti-EGFR gold antibody has an antibody determinant CPPLMLYNPTTYQMDVNPEGC and horseradish peroxidase corresponding to EGFR, so that the enzyme-labeled anti-EGFR gold antibody can be combined with substrates of EGFR and horseradish peroxidase simultaneously, and can be used as a detection antibody for the method to participate in EGFR detection.

The preferred embodiment of the invention uses the anti-EGFR gold antibody to detect EGFR according to the sandwich method principle in an enzyme-linked immunoassay method. The detection principle is that an anti-EGFR gold antibody is firstly added into an orifice plate as shown in figure 1a, and after a period of incubation, an EGFR standard substance is added, and the structure of the anti-EGFR gold antibody-EGFR is formed on the orifice plate as shown in figure 1 b. And adding an enzyme-labeled anti-EGFR gold antibody and incubating for a period of time, wherein a sandwich structure of the anti-EGFR gold antibody-EGFR-enzyme-labeled anti-EGFR gold antibody is formed on the pore plate, such as a c-d part in figure 1, and then adding a horseradish peroxidase substrate, wherein the gold antibody labeled with horseradish peroxidase is combined with the substrate efficiently, and the light absorption value OD ₄₅₀ _nm on the pore plate is positively correlated with the concentration of EGFR by utilizing the enzymatic reaction of horseradish peroxidase and the substrate, so that a standard curve can be drawn.

The kit comprises an anti-EGFR gold antibody, an ELISA plate, an ELISA anti-EGFR gold antibody, an EGFR standard substance, a diluent, a chromogenic solution, a washing solution and a stop solution. The invention performs the following steps of:

A. Adding 20-200 mu L of anti-EGFR gold antibody into each hole of an ELISA plate, using carbonate buffer solution as diluent, incubating for 30-120min at 37 ℃, washing the plate and beating to dry;

B. adding EGFR standard and detection antibody, namely adding 20-200 mu L EGFR standard with different concentrations into each hole of an ELISA plate, adopting phosphate buffer solution as diluent, incubating for 10-60min at room temperature, washing the plate and beating to dry;

C. Adding 20-200 mu L of enzyme-labeled anti-EGFR gold antibody, incubating for 10-60min at room temperature, washing the plate and beating to dryness;

D. Color development, namely adding 20-200 mu L of color development liquid into each hole, incubating for 10-40min at room temperature in a dark place, and adding stop solution to stop reaction;

E. And (3) detecting and analyzing by using a multifunctional enzyme-labeled instrument, measuring a signal value OD ₄₅₀ _nm of each hole, analyzing a detection result, taking an x axis as EGFR concentration, taking a y axis as an OD value at 450nm, and drawing a standard curve for detecting EGFR.

In the preferred embodiment of the invention, the diluent is a buffer solution with pH of 7-11, the chromogenic solution is tetramethyl benzidine (TMB) or o-phenylenediamine (OPD), and the stop solution is sulfuric acid or hydrochloric acid.

In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention will be rendered by reference to the appended drawings and appended detailed description.

Example 1

In this example, an EGFR standard curve was drawn as follows:

anti-EGFR gold antibody (capture antibody) was diluted with a diluent (20 mM carbonate buffer pH=10.7) and coated on an ELISA plate with a final concentration of 70nM, each well was added at 100. Mu.L, incubated at 37℃for 2 hours, washed with a wash solution (the wash solution: PBS-T at pH 7.4,0.15M (containing 0.05% Tween-20), the same as the following) and dried by pipetting, and repeated 4 times (30 s per wash plate);

EGFR standards were diluted with a dilution (20 mM pH 7.4 phosphate buffer) from a 400ng/mL two-fold gradient 6 times to 6.25ng/mL, starting the assay as follows:

Adding EGFR standard substance and detection antibody (the detection antibody is enzyme-labeled anti-EGFR gold antibody), wherein the concentration of the EGFR standard substance is 400,200,100,50,25,12.5 and 6.25ng/mL, adding 100 mu L of standard substance into an orifice plate, incubating for 1 hour at room temperature, washing the plate with washing liquid and beating, repeating for 4 times (30 s for each washing plate), adding 100 mu L of enzyme-labeled anti-EGFR gold antibody, incubating for 1 hour at room temperature, washing the plate with washing liquid and beating, repeating for 4 times (30 s for each washing plate);

color development, namely adding 100 mu L of a color developing agent TMB (tetramethyl benzidine) into each hole, incubating for 10min at room temperature in a dark place, and adding 100 mu L of a stop solution;

Detection analysis Each well was tested for absorbance (OD ₄₅₀ _nm) at a wavelength of 450nm using a microplate reader. EGFR standard curves were constructed with EGFR concentration in the standard as the X-axis and OD ₄₅₀ _nm as the Y-axis. The OD ₄₅₀ _nm corresponding to the limit of detection was 3 standard deviations added to the OD ₄₅₀ _nm of the blank sample.

Fig. 2 is a standard curve of the detected EGFR obtained, the standard curve equation being: the limit of detection was 15.84ng/mL.

Example 2

This embodiment is substantially the same as embodiment 1, except that:

In this example, EGFR was detected in a complex environment as follows:

In order to verify the effect of an enzyme-linked immunosorbent assay kit on detecting EGFR in a complex solution environment, bovine Serum Albumin (BSA) is selected as a non-specific protein. BSA was added to EGFR standards at each concentration and the concentration was set to 100 times the EGFR molar concentration, and standard curves were constructed following the procedure of example 1. FIG. 3 shows the standard curve obtained, and the standard curve is not greatly different from the original standard curve, which also means that the detection range and the detection limit are not changed, and can initially show that the ELISA kit has the capability of detecting EGFR in a complex solution environment.

Example 3

This embodiment is substantially the same as the foregoing embodiment 1 or 2, except that:

In this example, the gold antibody ELISA was subjected to a labeling test, and the labeling recovery rate was the ratio of the result to the theoretical value obtained by adding a standard substance of a known concentration to the sample in ELISA experiments, and then analyzing the sample according to a normal sample treatment procedure. Specifically, the labeled recovery is achieved by adding a known amount of analyte to the sample matrix, then performing a test, and comparing the test result to a theoretical value to evaluate the accuracy and reliability of the ELISA method. The method comprises the following steps:

The concentration of EGFR in the labeled samples was set at 40ng/mL, 60ng/mL and 150ng/mL, and quantitative tests were performed using gold antibody ELISA and recovery was calculated, where the recovery was expressed as = (detection concentration/labeled concentration) ×100%.

TABLE 1 labeling test results of gold antibody ELISA for EGFR detection

The results show that the ELISA kit provided by the application has good quantitative accuracy, the labeling recovery rate is 87.30% -110.87%, and the recovery rate meets the recovery rate standard (80% -120%).

Example 4

This embodiment is substantially the same as the previous embodiments 1 to 3, and is characterized in that:

in this example, the gold antibodies pretreated at different temperatures were used for the labeling test as follows:

The gold antibodies were pretreated in a water bath at room temperature and 60, 100 ℃ for 1h, after which they were cooled to room temperature. The EGFR concentration in the labeled sample was set at 60ng/mL, and the heat stability of the gold antibody was studied by quantitatively testing and calculating the recovery rate using the pretreated gold antibody. The test results are shown in Table 2 (the calculation formula is the same as that of example 3), and the recovery rate of the quantitative labeled sample can still reach 93.44% even after the treatment at 100 ℃, which shows that the gold antibody has excellent thermal stability.

TABLE 2 results of gold antibody labelling test after treatment with different temperatures

The results in Table 2 show that the quantitative accuracy of the ELISA kit provided by the application can be kept in a good state at different temperatures, the standard recovery rate is 93.44% -107.22%, and the standard recovery rate (80% -120%) is met.

The embodiment is an anti-EGFR gold antibody, an EGFR detection enzyme-linked immunosorbent assay kit and application thereof. The kit is a detection kit based on gold antibodies completely. The ELISA kit comprises an anti-EGFR gold antibody, an ELISA anti-EGFR gold antibody, an EGFR standard substance, a diluent, a chromogenic solution, a washing solution and a stop solution. The invention also discloses a method for detecting the sample by using the kit. The kit of the embodiment completely uses gold antibodies to replace natural antibodies for detection, has better detection capability, also has excellent thermal stability, and has other advantages of the enzyme-linked immunosorbent assay kit.

The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.

The sequences involved in the present application are as follows:

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

CSAWYGTLYEYDGC;

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

Claims

1. An ELISA kit for detecting EGFR based on gold antibody is characterized by comprising a capture antibody, an EGFR standard, a detection antibody, a blank ELISA plate and a detection reagent;

The capture antibody is an anti-EGFR gold antibody EGFR-GB1, wherein the anti-EGFR gold antibody EGFR-GB1 is gold nano-particles with the surface connected with polypeptide with a sequence shown as SEQ.ID.No.1, and the polypeptide with the sequence shown as SEQ.ID.No.1 is used as an antibody determinant;

The detection antibody is an enzyme-labeled anti-EGFR gold antibody EGFR-GB2-HRP, wherein the enzyme-labeled anti-EGFR gold antibody EGFR-GB2-HRP is gold nanoparticles with the surfaces connected with polypeptides and enzymes with the sequences shown as SEQ.ID.No.2, and the polypeptides with the sequences shown as SEQ.ID.No.2 are used as antibody determinants;

the anti-EGFR gold antibody EGFR-GB1 and the enzyme-labeled anti-EGFR gold antibody EGFR-GB2-HRP respectively pass through respective antibody determinants and are simultaneously combined with an antigenic determinant on EGFR in a specific way to form a double-antibody sandwich structure;

2. The enzyme-linked immunosorbent assay kit for detecting EGFR based entirely on gold antibodies according to claim 1, wherein the capture antibodies are used for being connected with a blank ELISA plate as a solid phase carrier.

3. The enzyme-linked immunosorbent assay kit for detecting EGFR based entirely on gold antibodies according to claim 1, wherein the detection antibodies are used for chromogenic reaction with a chromogenic solution.

4. The enzyme-linked immunosorbent assay kit for detecting EGFR based entirely on gold antibodies according to claim 1, wherein the enzyme linked 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 on gold antibodies completely according to claim 1, wherein the washing solution is PBS-T with pH 7.2~7.5,0.1~0.2M, and wherein the washing solution contains tween-20 with 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, wherein the diluent is a buffer solution having a pH of 711, and wherein 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, wherein the chromogenic solution is tetramethylbenzidine or o-phenylenediamine.

8. The enzyme-linked immunosorbent assay kit for detecting EGFR based on gold antibodies completely according to claim 1, wherein the stop solution is sulfuric acid or hydrochloric acid, and the acid concentration in the stop solution is 0-2M and is not 0.