Organic Mass Spectrometry Methods for Detecting Tumor Biomarkers

Abstract

The present invention relates to the technical field of mass spectrometry detection and analysis, and specifically discloses an organic mass spectrometry analysis method for detecting tumor biomarkers, comprising the following steps: S1, synthesizing gold nanoparticles; S2, assembling the CEA detection antibody and the mass spectrometry label molecule modified by the connecting arm on the surface of the gold nanoparticles through Au-S bonds in sequence to obtain a mass spectrometry probe; S3, preparing modified magnetic beads; S4, using the modified magnetic beads as capture units, adding the sample to be tested and the mass spectrometry probe for immune reaction; then releasing the gold nanoparticles from the immune complex, collecting the supernatant; S5, detecting the supernatant in a high-resolution mass spectrometer to obtain the concentration of the antigen in the sample to be tested. The present invention uses the mass spectrometry label molecule to perform organic mass spectrometry analysis to detect tumor biomarkers, and can simultaneously label multiple antibodies to achieve simultaneous detection of multiple targets; and has good sensitivity and detection stability.

Description

Technical Field

[0001] The present invention relates to the technical field of mass spectrometry detection and analysis, and in particular to an organic mass spectrometry analysis method for detecting tumor biomarkers. Background Art

[0002] Protein tumor biomarkers can be used to assist in early diagnosis, treatment monitoring, and prognosis assessment of tumors, but their content in the body is extremely low, making detection difficult. In addition, biomarkers and diseases do not have a one-to-one correspondence. Generally speaking, multiple directional biomarkers will appear during the development of a disease, and a single biomarker usually does not have sufficient predictive value. Therefore, testing multiple tumor markers at the same time can improve the accuracy of diagnosis.

[0003] At present, the mainstream detection technology for sensitive quantification of tumor markers is still the labeling strategy combined with immunoassay based on the specific interaction between antigen and antibody, and the gold standard for detection is still enzyme-linked immunosorbent assay. However, the signal of this type of immunological method is based on optical or electrical readout system, which is usually significantly limited in dynamic range and repeatability, and the accuracy of the test results needs to be improved; and in most cases, only one or a few targets can be detected simultaneously, because the spectral signal overlaps, and it is impossible to achieve simultaneous quantitative detection of multiple biomarkers.

[0004] Mass spectrometry immunoassay technology combines immune response and mass spectrometry detection, which can effectively make up for the above shortcomings and significantly improve the detection sensitivity and specificity of protein biomarkers. It has great application potential in the field of clinical disease screening and detection. Compared with direct mass spectrometry detection analysis, mass spectrometry immunoassay based on mass spectrometry labels overcomes the limitation of low ionization efficiency of biomacromolecules by converting the detection signal from biomacromolecules into the signal of mass labels marked on mass spectrometry probes. At the same time, it uses mass labels to achieve signal amplification, providing the possibility of high-sensitivity detection.

[0005] The main disadvantage of the existing organic mass spectrometry immunoassay technology is that it utilizes an open mass spectrometer, the detection results are greatly affected by the environment, and the detection device is complex and cannot be promoted and applied clinically. Summary of the invention

[0006] The purpose of the present invention is to provide a mass spectrometry analysis method for detecting tumor biomarkers, which can label multiple antibodies at the same time to achieve simultaneous detection of multiple targets; and has good sensitivity and detection stability.

[0007] The present invention is achieved through the following technical solutions:

[0008] The organic mass spectrometry analysis method for detecting tumor biomarkers comprises the following steps:

[0009] S1, synthesizing gold nanoparticles by reducing chloroauric acid with sodium citrate;

[0010] S2, assembling the CEA detection antibody modified with the linker arm and the mass spectrometry tag molecule on the surface of the gold nanoparticles prepared in step S1 through Au-S bonds in sequence to obtain a mass spectrometry probe;

[0011] The general structure of the mass spectrometry tag molecule is as follows:

[0012]

[0013] In the formula, R is a group having a mass spectrometry signal; n is a positive integer greater than or equal to 1; wherein R includes but is not limited to a quaternary ammonium group or a pyridine group;

[0014] S3, using streptavidin magnetic beads to capture biotinylated CEA capture antibodies to obtain modified magnetic beads;

[0015] S4, using the modified magnetic beads prepared in step S3 as capture units, adding the sample to be tested and the mass spectrometry probe prepared in step S2 to perform immune reaction to form an immune complex with a sandwich structure; then releasing the gold nanoparticles from the immune complex and collecting the supernatant;

[0016] S5. Detect the supernatant obtained in step S4 using a high-resolution mass spectrometer to obtain the concentration of the antigen in the sample to be tested.

[0017] The thiol group (-SH) can enable small molecule compounds to be modified on the surface of gold nanoparticles through the Au-S bond; the mass spectrometry label molecule of the present invention contains a thiol group (-SH), which can be modified on the surface of gold nanoparticles through the Au-S bond; and the present invention can synthesize mass spectrometry labels with different mass numbers by designing different n in the molecular structure formula, and can simultaneously label multiple antibodies to achieve simultaneous detection of multiple targets.

[0018] The mass spectrometry label molecule of the present invention is applied to the organic mass spectrometry analysis method for detecting tumor biomarkers, with a detection limit as low as 0.5 ng / mL, good sensitivity, and can be directly detected using a commercial mass spectrometer. The stability of the test results is improved to a certain extent, and it has the advantage of good detection stability.

[0019] Gold nanoparticles modified with CEA capture antibodies and mass spectrometry label molecules on the surface are used as mass spectrometry probes. After specific binding with the target detection antigen, they are separated by magnetic beads and the excess mass spectrometry probes are washed away. A small amount of organic solvent can be used to dissociate the mass spectrometry probe from the immune complex, and the supernatant can be directly analyzed by mass spectrometry. The detection time for a single sample is less than 1 min. Since commercial mass spectrometry instruments are used for detection, the stability of the test results is improved to a certain extent. Combined with magnetic beads for separation and enrichment, it is easy to realize the automation of pre-processing, which proposes a more convenient and fast high-throughput detection method for multiple detection of tumor biomarkers in clinical practice.

[0020] Specifically, the specific process of step S1 is:

[0021] The chloroauric acid aqueous solution is heated to boiling, and then trisodium citrate solution is added, and heating and stirring are continued for 20-30 minutes. The heat source is removed and cooled to room temperature to obtain gold nanoparticles.

[0022] Specifically, in step S2, the linker used for the linker-modified detection antibody includes NHS-PEG7-SS-PEG7-NHS.

[0023] Specifically, in step S2, the preparation process of the detection antibody modified with the connecting arm is as follows:

[0024] The HEPES solution, the detection antibody and the linker are mixed and reacted at room temperature.

[0025] Specifically, the specific process of step S2 is:

[0026] The detection antibody modified with the linker arm is added to the gold nanoparticles, and K2CO3 solution is added to react for a period of time so that the antibody is assembled on the surface of the gold nanoparticles through the Au-S bond; then the mass spectrometry label molecule is added and the reaction is continued for a period of time so that the mass spectrometry label molecule is also assembled on the surface of the gold nanoparticles through the Au-S bond.

[0027] Specifically, the specific process of step S3 is:

[0028] Add biotinylated CEA capture antibody to the washed streptavidin magnetic beads, react at room temperature, and remove excess antibody by magnetic separation; then add buffer and perform blocking treatment on the surface of the magnetic beads at room temperature to obtain modified magnetic beads.

[0029] Specifically, the buffer is PBS buffer containing bovine serum albumin.

[0030] Specifically, in step S4, the sample to be tested includes a CEA antigen solution or a serum sample.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0032] 1. The mass spectrometry label molecule of the present invention has a structure of a thiol mass spectrometry label molecule with mass spectrometry signal enhancement effect. The thiol group can modify the mass spectrometry label molecule on the surface of gold nanoparticles through the Au-S bond; by designing different n in the molecular structure of the mass spectrometry label molecule, mass spectrometry label molecules with different mass numbers can be synthesized, and multiple antibodies can be labeled at the same time to achieve simultaneous detection of multiple targets.

[0033] 2. The present invention uses gold nanoparticles as carriers of small molecule mass spectrometry labels to synthesize organic mass spectrometry probes, and uses magnetic beads for separation to achieve accurate quantitative analysis of CEA; this method has simple pre-treatment and is easy to automate, providing a new idea for the detection of protein biomarkers. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0035] Figure 1 This is the electrospray dissociation mass spectrum of the mass spectrometry probe in Example 1 of the present invention;

[0036] Figure 2 This is the EIC diagram of TIC and label molecules detected by mass spectrometry of a single sample in Example 1 of the present invention;

[0037] Figure 3 The antigen concentration and mass spectrometry report signal of the sample subjected to mass spectrometry analysis in Example 1 of the present invention m / z Linear correlation graph of the peak area of ​​245.2172. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. The schematic implementation mode of the present invention and its description are only used to explain the present invention and are not intended to limit the present invention. The embodiments described below are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0039] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present invention. However, it is apparent to those of ordinary skill in the art that these specific details do not have to be employed to practice the present invention. In other embodiments, in order to avoid confusing the present invention, known structures, materials or methods are not specifically described. The materials, instruments and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. The technical means used in the examples, unless otherwise specified, are conventional means well known to those skilled in the art.

[0040] Example:

[0041] Enzyme-linked immunosorbent assay (ELISA) is the mainstream method for protein detection, but it has inevitable spectral overlap problems and certain limitations in multiple labeling, and cannot simultaneously detect multiple tumor biomarkers in a single analysis.

[0042] In order to achieve the simultaneous detection of multiple tumor biomarkers in a single analysis, this embodiment provides a mass spectrometry tag molecule for detecting tumor biomarkers. The general structural formula of the mass spectrometry tag molecule is as follows:

[0043]

[0044] In the formula, R is a group having a strong mass spectrometry signal, including but not limited to a quaternary ammonium group or a pyridine group; and n is a positive integer greater than or equal to 1.

[0045] The mass spectrometry label molecule of this embodiment has a structure of a thiol mass spectrometry label molecule with a mass spectrometry signal enhancement effect. The thiol group can enable the mass spectrometry label molecule to be modified on the surface of gold nanoparticles through the Au-S bond; by designing different n in the molecular structure formula of the mass spectrometry label molecule, mass spectrometry label molecules with different mass numbers can be synthesized, and multiple antibodies can be labeled at the same time to achieve simultaneous detection of multiple targets.

[0046] In order to achieve accurate quantitative detection of extremely low concentration protein tumor markers, this example uses carcinoembryonic antigen (CEA) as a research model and develops an organic mass spectrometry analysis method for detecting tumor biomarkers based on the above mass spectrometry label molecules. The analysis method has the advantages of high sensitivity and good detection stability. The designed small molecule label structure (mass spectrometry label molecule) has high scalability and can be used for multiple labeling.

[0047] Specifically, the organic mass spectrometry analysis method for detecting tumor biomarkers comprises the following steps:

[0048] S1. Using sodium citrate to reduce chloroauric acid to synthesize gold nanoparticles; specifically: heating the chloroauric acid aqueous solution to boiling, then adding trisodium citrate solution, continuing heating and stirring for 20-30 min, removing the heat source and cooling to room temperature to obtain gold nanoparticles.

[0049] S2, assembling the CEA detection antibody modified by the linker and the mass spectrometry label molecule on the surface of the gold nanoparticles prepared in step S1 through Au-S bonds in sequence to obtain a mass spectrometry probe; specifically: adding the detection antibody modified by the linker to the gold nanoparticles, and adding K2CO3 solution, reacting for a period of time, so that the antibody is assembled on the surface of the gold nanoparticles through Au-S bonds; then adding the mass spectrometry label molecule, continuing to react for a period of time, so that the mass spectrometry label molecule is also assembled on the surface of the gold nanoparticles through Au-S bonds, wherein the preparation process of the detection antibody modified by the linker is as follows: mixing the HEPES solution, the detection antibody and the linker and reacting at room temperature to obtain; the linker used for the detection antibody modified by the linker includes NHS-PEG7-SS-PEG7-NHS. The surface of the modified gold nanoparticles has capture antibodies and label molecules, which can be used as mass spectrometry probes to identify target detection antigens, and the label molecules on the surface can reflect the concentration level of the target detection object as mass spectrometry groups.

[0050] S3. Using streptavidin magnetic beads to capture biotinylated CEA capture antibodies to obtain modified magnetic beads; specifically: adding biotinylated CEA capture antibodies to the washed streptavidin magnetic beads, reacting at room temperature, and removing excess antibodies by magnetic separation; then adding buffer, blocking the surface of the magnetic beads at room temperature to obtain modified magnetic beads; wherein the buffer is PBS buffer containing bovine serum albumin.

[0051] S4, using the modified magnetic beads prepared in step S3 as capture units, adding the sample to be tested and the mass spectrometry probe prepared in step S2 to carry out immune reaction, forming an immune complex with a sandwich structure; then washing and organic solvent release are performed in sequence to release the gold nanoparticles from the immune complex, and collecting the supernatant, wherein the sample to be tested includes a CEA antigen solution or a serum sample; the organic solvent includes acetonitrile, and washing can be performed by a combination of PBS washing and H2O washing.

[0052] S5. Detect the supernatant obtained in step S4 in a high-resolution mass spectrometer to obtain the concentration of the antigen in the sample to be tested: Specifically, the collected supernatant is transferred to a 1.5 mL liquid phase vial and detected in a high-resolution mass spectrometer. No chromatographic column separation is required. Acetonitrile is used as a solvent to transport 10 μL of the supernatant through the pipeline of the liquid phase system to the mass spectrometer for detection. The Au-S bond of the gold nanoparticles will break in the electrospray ion source, so that the mass spectrometry label molecule dissociates from the surface of the nanoparticle and generates a dimer with two charges, producing m / z The mass spectrometer reported a signal of 245.21717.

[0053] This embodiment develops a new type of quaternary ammonium salt organic mass spectrometry small molecule compound (mass spectrometry label molecule), using the mass spectrometry label molecule as the group of the mass spectrometry probe, assembled on the surface of gold nanoparticles through Au-S bonds, and the surface of the gold nanoparticles is also modified with CEA detection antibodies, and a mass spectrometry probe based on gold nanoparticles is synthesized. The magnetic beads are modified with CEA capture antibodies through a streptavidin-biotin system. The capture antibody, CEA antigen, and the detection antibody on the mass spectrometry probe form an immune complex with a sandwich structure. The nanoparticles with label molecules on the magnetic beads can indirectly reflect the concentration information of the target detection object, and the nanoparticles are eluted from the magnetic beads for mass spectrometry detection. The gold nanoparticles with mass spectrometry label molecules break the Au-S bond under the action of electrospray, generating a dimer with two charges, so that the mass spectrometer detects the quaternary ammonium salt small molecule compound. Different concentrations of target detection objects will produce mass spectrometry reporting signals of different intensities. m / z 245.21717, from which a linear equation can be established to calculate the concentration of the target analyte in the sample.

[0054] The organic mass spectrometry analysis method of this embodiment can directly use a commercial mass spectrometer to perform quantitative analysis, which greatly simplifies the detection process and can provide more stable detection results.

[0055] Embodiment 1:

[0056] This example uses the mass spectrometry tag molecule with the structure shown below as an example for illustration:

[0057] (1).

[0058] The organic mass spectrometry analysis method for detecting tumor biomarkers comprises the following steps:

[0059] S1. Synthesis of gold nanoparticles:

[0060] Gold nanoparticles with a diameter of about 25 nm were synthesized by reducing chloroauric acid with trisodium citrate. 50 mL of 0.01% (w / v) chloroauric acid aqueous solution was heated to boiling, and 0.75 mL of 1% (w / v) trisodium citrate solution was added. The mixture was heated and stirred for 20 min, and the heat source was removed and cooled to room temperature to obtain gold nanoparticles.

[0061] S2. Modification and synthesis of mass spectrometry probes:

[0062] In 1 mL of 10 mM HEPES solution, add 160 μg of detection antibody, and add the linker NHS-PEG7-SS-PEG7-NHS at a molar ratio of detection antibody: linker = 1:2, and react at room temperature for 12 h. Take 30 μL of the linker-modified detection antibody and add it to 1 mL of gold nanoparticles, and add K2CO3 (final concentration of 1.8 mM), and continue to react for 12 h, so that the antibody is assembled on the surface of the gold nanoparticles through Au-S bonds. Then add 10 μL of mass spectrometry label molecules with a concentration of 4 μM and continue to react for 12 h. The mass spectrometry label molecules are also assembled on the surface of the gold nanoparticles through Au-S bonds. Subsequently, centrifugation is performed to remove excess unbound small molecule labels, and the gold nanoprobe is obtained after washing twice with PBS, which is the mass spectrometry probe.

[0063] The structure of the mass spectrometry tag molecule used in step S2 is shown in formula (1).

[0064] Electrospray dissociation mass spectrometry of the mass spectrometry probe prepared in step S2 Figure 1 As shown, the structure of the mass spectrometry probe is shown in formula (2):

[0065] (2).

[0066] During mass spectrometry detection, the label molecules dissociate from the surface of the gold nanoparticles and exist mainly in the form of dimers, producing m / z The signal of 245.2172 (Z=2) is shown in the following formula:

[0067]

[0068] S3, magnetic bead-bound capture antibody:

[0069] Take 30 μL of 10 mg / mL streptavidin magnetic beads, wash twice with PBS (10 mM, pH = 7.4), add 50 μL of 190 μg / mL biotinylated CEA capture antibody, react at room temperature for 2 h, and remove excess antibodies by magnetic separation. Subsequently, add 100 μL of PBS buffer containing 1% (w / v) bovine serum albumin, and block the surface of the magnetic beads at room temperature for 1 h. After blocking, wash 3 times with PBS, resuspend in PBS buffer, and store at 4°C.

[0070] S4. Immunoassay:

[0071] The modified magnetic beads were used as capture units, and 10 μL of a series of gradient concentrations (1, 20, 40, 60, 100, 200 ng / mL) of CEA antigen solution was added, and then 30 μL of mass spectrometry probe was added, and the mixture was reacted at 37°C for 1 h to form a sandwich structure immune complex. The beads were then washed 3 times with PBS and 2 times with H2O, and finally 100 μL of acetonitrile solution containing 5% (w / v) formic acid was added, vortexed for 10 min, the gold nanoparticles were released from the immune complex, and the supernatant was collected.

[0072] S5. Mass spectrometry analysis:

[0073] The collected supernatant was transferred to a 1.5 mL liquid phase vial and detected in a high-resolution mass spectrometer. Without the need for chromatographic column separation, 10 μL of the supernatant was transported to the mass spectrometer through the pipeline of the liquid phase system using acetonitrile as a solvent for detection. The Au-S bond of the gold nanoparticles will break in the electrospray ion source, causing the mass spectrometry label molecule to dissociate from the surface of the nanoparticle and generate a dimer with two charges, producing m / z The mass spectrometer reported a signal of 245.21717.

[0074] Mass spectrometry of a single sample to detect TIC and EIC of the tag molecule Figure 2 shown.

[0075] The detection time for each sample is less than 1 min. The signal is reported by mass spectrometry. m / z The peak area of ​​245.2172 is taken as the ordinate and the antigen concentration is taken as the abscissa, and a linear equation can be obtained, such as Figure 3 As shown, Y = 609126.3486X (ng / mL) - 4938304.2908, R 2 =0.9911, and the detection limit was 0.5 ng / mL. This shows that this analytical method has good sensitivity and excellent analytical ability.

[0076] In order to verify that the organic mass spectrometry analysis method of the present invention can accurately and quantitatively detect extremely low concentration protein tumor markers, the following specific cases are used to illustrate:

[0077] Case 1:

[0078] A CEA antigen solution with a concentration of 2.00 ng / mL was prepared, and the CEA antigen solution was detected using the organic mass spectrometry analysis method described in Example 1. The antigen concentration of the CEA antigen solution was measured to be 2.23 ng / mL.

[0079] Case 2:

[0080] A CEA antigen solution with a concentration of 30.00 ng / mL was prepared, and the CEA antigen solution was detected using the organic mass spectrometry analysis method described in Example 1. The antigen concentration of the CEA antigen solution was measured to be 30.06 ng / mL.

[0081] Case 3:

[0082] A CEA antigen solution with a concentration of 100.00 ng / mL was prepared, and the CEA antigen solution was detected using the organic mass spectrometry analysis method described in Example 1. The antigen concentration of the CEA antigen solution was measured to be 104.73 ng / mL.

[0083] The above specific implementation methods further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific implementation methods of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An organic mass spectrometry method for detecting tumor biomarkers, characterized in that: The following steps are involved: S1, synthesizing gold nanoparticles by reducing chloroauric acid with sodium citrate; S2, assembling the CEA detection antibody modified with the connecting arm and the mass spectrometry tag molecule on the surface of the gold nanoparticle prepared in step S1 through Au-S bonds in sequence to obtain a mass spectrometry probe; the general structural formula of the mass spectrometry tag molecule is as follows: In the formula, R is a group having a mass spectrometry signal; n is a positive integer greater than or equal to 1; S3, using streptavidin magnetic beads to capture biotinylated CEA capture antibodies to obtain modified magnetic beads; S4, using the modified magnetic beads prepared in step S3 as capture units, adding the sample to be tested and the mass spectrometry probe prepared in step S2 to perform immune reaction to form an immune complex with a sandwich structure; then releasing the gold nanoparticles from the immune complex and collecting the supernatant; S5, the supernatant obtained in step S4 is tested in a high-resolution mass spectrometer to obtain the concentration of the antigen in the sample to be tested; during the detection, the mass spectrometer probe breaks the Au-S bond under the action of electrospray, and the label molecule generates a dimer with two charges, producing m / z 245.21717 mass spectrometry report signal; mass spectrometry report signal m / z The peak area of ​​245.21717 is taken as the ordinate and the antigen concentration is taken as the abscissa to obtain a linear equation based on the mass spectrometry report signal in the sample. m / z The peak area of ​​245.21717 was used to obtain the antigen concentration of the sample to be tested.

2. The organic mass spectrometry analysis method according to claim 1, characterized in that: The specific process of step S1 is: The chloroauric acid aqueous solution is heated to boiling, and then the trisodium citrate solution is added, and the heating and stirring are continued for 20-30 min. The heat source is removed and cooled to room temperature to obtain the gold nanoparticles.

3. The organic mass spectrometry analysis method according to claim 1, characterized in that: In step S2, the linker used for the linker-modified detection antibody includes NHS-PEG7-SS-PEG7-NHS.

4. The organic mass spectrometry analysis method according to claim 1, characterized in that: In step S2, the preparation process of the detection antibody modified with the linker arm is as follows: The HEPES solution, the detection antibody and the linker are mixed and reacted at room temperature.

5. The organic mass spectrometry analysis method according to claim 1, characterized in that: The specific process of step S2 is: The detection antibody modified with the connecting arm is added to the gold nanoparticles, and a K2CO3 solution is added to react for a period of time so that the antibody is assembled on the surface of the gold nanoparticles through the Au-S bond; then the mass spectrometry label molecule is added and the reaction is continued for a period of time so that the mass spectrometry label molecule is also assembled on the surface of the gold nanoparticles through the Au-S bond.

6. The organic mass spectrometry analysis method according to claim 1, characterized in that: In step S2, R includes a quaternary ammonium group or a pyridine group.

7. The organic mass spectrometry analysis method according to claim 1, characterized in that: The specific process of step S3 is: Add biotinylated CEA capture antibody to the washed streptavidin magnetic beads, react at room temperature, and remove excess antibody by magnetic separation; then add buffer and perform blocking treatment on the surface of the magnetic beads at room temperature to obtain modified magnetic beads.

8. The organic mass spectrometry analysis method according to claim 7, characterized in that: The buffer is PBS buffer containing bovine serum albumin.

9. The organic mass spectrometry analysis method according to any one of claims 1 to 8, characterized in that: In step S4, the sample to be tested includes a CEA antigen solution or a serum sample.

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