Chemiluminescence kit for detecting CEACAM1 as well as use method and application of chemiluminescence kit

Abstract

The invention belongs to the field of biology, and particularly relates to a chemiluminescence kit for detecting CEACAM1, a use method and application, the chemiluminescence kit comprises a streptavidin magnetic particle working solution, a biotinylated carcino-embryonic antigen related cell adhesion molecule 1 capture antibody solution and an acridinium ester labeled carcino-embryonic antigen related cell adhesion molecule 1 detection antibody solution. Coupling of acridinium ester and the antibody is efficient and simple, repeatability is good, large-scale production and application are easy, a biotin-avidin amplification system (BAS system) is used in a reaction system, quantitative detection of ultramicro markers is more sensitive and more efficient, and compared with a traditional enzyme-linked immunosorbent assay method, the method has the advantages that the detection sensitivity is high, and the sensitivity is high. The magnetic particle chemiluminescence reagent disclosed by the invention has the advantages of low cost, stable and reliable result, high test flux, convenience in production, small batch difference, easiness in large-scale application and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a chemiluminescence kit for detecting CEACAM1, a use method and an application thereof. Background Art

[0002] CEACAM1 (Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1) is a member of the immunoglobulin superfamily and is widely expressed in epithelial cells, endothelial cells, granulocytes, lymphocytes and tumor cells.

[0003] The role of CEACAM1 in tumors is complex, potentially acting as both a tumor suppressor and a tumor promoter. As a multifunctional cell adhesion molecule, CEACAM1 plays a crucial role in tumor, immune, and metabolic diseases. Its expression levels and functions vary across tissues and cell types, suggesting that its mechanisms of action may differ across different diseases.

[0004] Currently, the detection methods for CEACAM1 mainly include the following:

[0005] 1. ELISA (enzyme-linked immunosorbent assay): This is one of the most commonly used methods, particularly the double-antibody sandwich method. Commercially available ELISA kits are available for detecting CEACAM1. These kits typically utilize pre-coated antibodies that bind to CEACAM1 in the sample. Biotinylated antibodies and HRP-labeled streptavidin are then added, and quantitative analysis is performed using a TMB colorimetric reaction.

[0006] 2. Flow cytometry: Flow cytometry is used to detect CEACAM1 expression. For example, in the study "The Effect of Abnormal CEACAM1 Expression on the Proliferation of Leukemia BALL-1 Cells" by Zhao Wenjin et al. from China Medical University, FITC-labeled pan-CEACAM monoclonal antibody was used to detect CEACAM1 expression on the cell surface.

[0007] 3. Immunohistochemistry (IHC): Use immunohistochemistry to detect the expression of CEACAM1.

[0008] 4. Real-time quantitative PCR (QRT-PCR): In the study “Carcinoembryonic antigen-related adhesion molecule 1 and tumors” by Yang Changcheng et al. from the Sixth People’s Hospital affiliated to Shanghai Jiao Tong University, the use of QRT-PCR technology to detect the mRNA level of CEACAM1 was mentioned.

[0009] 5. Immunoprecipitation (IP): Use immunoprecipitation to detect CEACAM1 expression. For example, immunoprecipitation can be performed using Fc-8G5 antibody and biotin-labeled UM tumor cells.

[0010] 6. Recombinant proteins and antibodies: Wuhan Cloud-Clone Technology Co., Ltd. mentioned "carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) recombinant protein", which uses recombinant proteins and polyclonal antibodies to detect CEACAM1. These products can be used in ELISA, IHC, ICC and other detection methods.

[0011] Among the above methods, the most commonly used method for detecting CEACAM1 is the enzyme-linked immunosorbent assay (ELISA) scientific research kit.

[0012] Chemiluminescence has been widely used in clinical testing. For example, it can detect various protein molecules such as CA125, CA15-3, CA199, and ferritin. The detection method is mature, and its precision, accuracy, detection range, detection speed, and anti-interference ability are superior to other detection methods.

[0013] Problems with existing technologies:

[0014] 1. Detection of CEACAM1 is limited to enzyme-linked immunosorbent assay (ELISA) research kits, and there is no clinical diagnostic kit available.

[0015] 2. The precision, accuracy, detection range, detection speed and anti-interference ability of the ELISA detection method are inferior to those of the chemiluminescence method.

[0016] 3. Chemiluminescence has been widely used in clinical testing, but chemiluminescence detection of CEACAM1 has not yet been established.

[0017] The present invention establishes a chemiluminescence method to detect the expression level of CEACAM1 in serum or plasma, which can be used for the diagnosis of diseases such as tumors. Summary of the Invention

[0018] The object of the present invention is to provide a chemiluminescence kit for detecting CEACAM1, a method for use and an application thereof, so as to solve the above-mentioned problems.

[0019] First, a chemiluminescence kit for detecting CEACAM1 includes a streptavidin magnetic particle working solution, a biotinylated carcinoembryonic antigen-related cell adhesion molecule-1 capture antibody solution, and an acridinium ester-labeled carcinoembryonic antigen-related cell adhesion molecule-1 detection antibody solution.

[0020] As a further improvement, the streptavidin magnetic particle working solution is prepared by the following method:

[0021] Take the streptavidin magnetic particle solution, add phosphate buffer and mix thoroughly, then place it on a magnetic separator until the supernatant is clear, discard the supernatant, retain the magnetic particles, wash them, and dilute them with magnetic bead dilution buffer to obtain a streptavidin magnetic particle working solution.

[0022] In a further improvement, the biotinylated carcinoembryonic antigen-related cell adhesion molecule-1 capture antibody solution is prepared by the following method:

[0023] Step S11: 1 mg of mouse anti-human carcinoembryonic antigen-related cell adhesion molecule-1 monoclonal antibody was taken, and a PBS buffer solution with a pH of 7.2 to 7.4 was added to obtain a carcinoembryonic antigen-related cell adhesion molecule-1 capture antibody solution with a final concentration of 1 mg / mL, and the solution was added to a dialysis bag;

[0024] Step S12: adding PBS buffer with a pH of 7.2 to 7.4 into the dialysis bag for dialysis, and then transferring the dialyzed antibody solution into an Eppendorf tube;

[0025] Step S13, adding NHS-LC-Biotin to dimethyl sulfoxide to obtain an NHS-LC-Biotin solution;

[0026] Step S14, adding the NHS-LC-Biotin solution to the Eppendorf tube containing the carcinoembryonic antigen-related cell adhesion molecule 1 capture antibody in step S12, mixing and reacting to form a reaction mixture;

[0027] Step S15, transferring the reaction mixture in step S14 into a dialysis bag, and placing the dialysis bag into a PBS buffer solution with a pH of 7.2 to 7.4 for dialysis, and then collecting the biotin-labeled CEACAM1-mAb (Bio-CEACAM1-mAb) stock solution;

[0028] Step S16: Add bovine serum albumin (PBS) buffer to the collected biotin-labeled CEACAM1-mAb (Bio-CEACAM1-mAb) stock solution to obtain a Bio-mAb stock solution; before use, dilute the Bio-mAb stock solution with a biotin-labeled antibody diluent to obtain a biotinylated carcinoembryonic antigen-related cell adhesion molecule 1 capture antibody solution.

[0029] As a further improvement, the acridinium ester-labeled carcinoembryonic antigen-related cell adhesion molecule 1 detection antibody solution is prepared by the following method:

[0030] Step S21, accurately pipetting the carcinoembryonic antigen-related cell adhesion molecule 1 detection antibody solution into the purification column;

[0031] Step S22: centrifugation to collect the purified detection antibody;

[0032] Step S23: measuring the absorbance at 280 nm and 260 nm using an ultra-microphotometer to accurately calculate the concentration of the detection antibody;

[0033] Step S24, adding the acridinium ester solution, mixing thoroughly, and shaking the mixture at room temperature in the dark; after the reaction is completed, adding glycine to the reaction mixture to terminate the cross-linking reaction, to obtain a mixture X;

[0034] Step S25, placing the mixture X into a dialysis bag for dialysis, with the dialysate being a phosphate buffer solution;

[0035] Step S26 , adding a storage buffer and mixing thoroughly to obtain an acridinium ester-labeled carcinoembryonic antigen-related cell adhesion molecule 1 detection antibody solution.

[0036] In a second aspect, the method for using the chemiluminescence kit for detecting CEACAM1 comprises the following steps:

[0037] The test sample, streptavidin magnetic particle working solution, biotinylated carcinoembryonic antigen-related cell adhesion molecule 1 capture antibody solution and acridinium ester-labeled carcinoembryonic antigen-related cell adhesion molecule 1 detection antibody solution are added in sequence, and a pair of carcinoembryonic antigen-related cell adhesion molecule 1 monoclonal antibodies bind to carcinoembryonic antigen-related cell adhesion molecule 1 antigen molecules in the test sample to form an immune complex; after washing to remove unbound antibodies and impurities, pre-excitation solution and excitation solution (NaOH) are added in sequence, and the relative luminescence intensity is measured; the carcinoembryonic antigen-related cell adhesion molecule 1 content in the test sample is calculated from the standard curve based on the relative luminescence intensity.

[0038] In a further improvement, the pre-excitation liquid is nitric acid or H2O2.

[0039] In a further improvement, the exciting liquid is NaOH.

[0040] In a third aspect, the chemiluminescence kit for detecting CEACAM1 is used to detect CEACAM1 protein in human serum or plasma.

[0041] In a fourth aspect, the chemiluminescence kit for detecting CEACAM1 is used in the preparation or screening of agents for diagnosing and / or preventing and / or treating benign thyroid diseases / thyroid cancer, wherein the kit is used for in vitro diagnosis and / or risk stratification of benign thyroid diseases / thyroid cancer.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The coupling of acridinium esters and antibodies in the present invention is efficient and simple, with good reproducibility, and is easy to produce and apply on a large scale. In addition, the biotin-avidin amplification system (BAS system) is used in the reaction system, which is more sensitive and efficient for the quantitative detection of ultra-trace markers. Compared with the traditional enzyme-linked immunosorbent assay method, the magnetic microparticle chemiluminescence reagent disclosed in the present invention has the advantages of low cost, stable and reliable results, high test throughput, convenient production, small batch-to-batch variability, and easy large-scale application.

[0044] 2. Early screening of solid tumors such as thyroid tumors and breast tumors has important clinical value in tumor treatment. Early detection of tumors can significantly improve the cure rate. For example, the 5-year survival rate of early breast cancer exceeds 90%, while it drops sharply in the late stage. Ultrasound, fine needle aspiration and other means are used to distinguish between benign and malignant nodules, avoid missed diagnosis of malignant nodules (such as thyroid cancer), and reduce unnecessary treatment (such as surgery for benign nodules). Early detection of breast cancer (such as through mammography, ultrasound or MRI) can reduce mortality, and the clear diagnosis of benign lesions (such as fibroadenomas) can avoid excessive intervention. The differentiation of benign or malignant tumors helps to optimize treatment strategies and reduce over-medicalization. At the same time, the cost of early treatment is much lower than the comprehensive treatment cost of advanced tumors, reducing the waste of resources caused by misdiagnosis and mistreatment (such as lifelong medication after the mistaken removal of benign thyroid nodules).

[0045] With the continuous development of diagnostic technology, more and more innovative technical means are being applied to clinical diagnosis and treatment. The magnetic particle chemiluminescence technology platform can detect the concentration of tumor-related markers with a sensitivity of up to 10 -19 -10 -22 mol / L, significantly improving the detection rate during the onset window of malignant tumors. Traditional immunological and histological diagnostic methods are unable to detect elevated levels of related tumor marker proteins early on, leading to treatment delays. This invention combines single-photon detection technology, magnetic particle technology, and other immune response systems with a chemiluminescence analysis system to mitigate these delays in diagnosis and treatment to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the result of detecting the CEACAM1 expression levels of different populations using the present invention. DETAILED DESCRIPTION

[0047] The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0048] Example 1

[0049] 1. Covalent coupling of biotin derivative NHS-LC-Biotin and mouse anti-human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) monoclonal antibody:

[0050] Step S11: 1 mg of mouse anti-human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) monoclonal antibody was taken, and an appropriate amount of 0.01 mol / L PBS buffer (pH 7.2-7.4) was added to obtain a carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) capture antibody solution with a final concentration of 1 mg / mL, and the solution was added to the dialysis bag;

[0051] Step S12: Add not less than 500 ml of 0.01 mol / L PBS buffer (pH 7.2-7.4) to the dialysis bag for dialysis. Each dialysis time is not less than 4 hours. Repeat the dialysis solution 3-4 times, and then transfer the dialyzed antibody solution to an Eppendorf tube.

[0052] Step S13: Weigh 1 mg of NHS-LC-Biotin (N-hydroxysuccinimide 6-(biotinamido) hexanoate, an active ester biotinylation reagent used for biomolecule labeling, with a CAS number of 72040-63-2), and add an appropriate amount of DMSO (dimethyl sulfoxide) to obtain a final concentration of 2 mg / mL NHS-LC-Biotin solution;

[0053] Step S14: labeling the cells with an appropriate volume of NHS-LC-Biotin solution at a molar ratio of 20 to carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) capture antibody, adding the NHS-LC-Biotin solution to the Eppendorf tube containing the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) capture antibody in step S12, mixing the solution, and placing the tube on a rotating reactor for reaction at room temperature for 120 minutes to form a reaction mixture;

[0054] Step S15, transferring the reaction mixture from step S14 into a dialysis bag, and placing the dialysis bag into not less than 500 ml of 0.01 mol / L PBS buffer (pH 7.2-7.4) for dialysis. Repeat the dialysis solution replacement 3-4 times, each dialysis time being not less than 4 hours, and then collect the biotin-labeled CEACAM1-mAb (Bio-CEACAM1-mAb) stock solution, the volume of which is V1;

[0055] Step S16: Add (0.1×V1) volume of 0.01 mol / L PBS buffer (pH 7.2-7.4) containing 5% BSA (bovine serum albumin) to the collected biotin-labeled CEACAM1-mAb (Bio-CEACAM1-mAb) stock solution to obtain a Bio-mAb stock solution, which is then stored at 2-8°C. Prior to use, dilute the Bio-mAb stock solution with a biotin-labeled antibody diluent (using 0.01 mol / L PBS buffer at pH 7.4 as an example) to obtain a Bio-CEACAM1-mAb working solution, i.e., a biotinylated carcinoembryonic antigen-related cell adhesion molecule 1 capture antibody solution.

[0056] 2. Covalent conjugation of acridinium ester and mouse anti-human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) monoclonal antibody:

[0057] Step S21, accurately pipetting the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) detection antibody solution into the purification column;

[0058] Step S22, centrifuging at 1000 rpm for 2 minutes to collect the purified detection antibody;

[0059] Step S23: measuring the absorbance at 280 nm and 260 nm using an ultra-microphotometer to accurately calculate the concentration of the detection antibody;

[0060] Step S24: Add the acridinium ester solution, mix thoroughly, and react in the dark at room temperature with shaking for 120 minutes. The molar ratio of acridinium ester to antibody is 1:50. After the reaction is completed, 10% by volume of 5% glycine is added to the reaction solution to terminate the cross-linking reaction, to obtain a mixture X.

[0061] Step S25: Place the mixture X into a dialysis bag and dialyze at 2-8° C. in the dark. Replace the dialysate once every 2 hours, 5 hours, and 12 hours after dialysis. The dialysate is a 0.02 mol / L phosphate buffer solution.

[0062] Step S26, add an appropriate amount of storage buffer and mix thoroughly, measure the absorbance at 280nm and 260nm with an ultramicrophotometer, accurately calculate the concentration of the marker, and store in a sealed container at 2-8°C to obtain the acridinium ester-labeled carcinoembryonic antigen-related cell adhesion molecule 1 detection antibody solution.

[0063] 3. Preparation method of streptavidin magnetic particle working solution

[0064] In this example, commercial streptavidin magnetic particles were prepared as a streptavidin magnetic particle working solution: a streptavidin magnetic particle solution was added to a phosphate buffer solution and thoroughly mixed, and then placed on a magnetic separator until the supernatant was clear. The supernatant was discarded, and the magnetic particles were retained. After repeated washing three times, the solution was diluted with a magnetic bead dilution buffer to obtain a streptavidin magnetic particle working solution.

[0065] Example 2

[0066] 1. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) main analytical performance evaluation

[0067] 1.1、Blank limit

[0068] The zero-concentration calibrator or sample dilution was used as the sample for detection, and the measurement was repeated 20 times. The mean (M) and standard deviation (SD) of the signal value were calculated. A two-point regression fit was performed based on the concentration-signal value results between the zero-concentration calibrator and the adjacent calibrators to obtain a linear equation. The signal value (M+2SD) was substituted into the above equation to obtain the corresponding concentration value. The results are shown in Table 1:

[0069] Table 1

[0070]

[0071]

[0072] 1.2 Linear range

[0073] Dilute high-value samples near the upper limit of the linear range to at least five concentrations at a certain ratio, with the lowest diluted sample concentration close to the lower limit of the linear range. Repeat the measurement at least twice for each concentration, calculate the average value, and use the least squares method to perform a linear fit between the average measured concentration and the theoretical concentration or dilution ratio. Calculate the linear correlation coefficient r. The results are shown in Table 2:

[0074] Table 2

[0075]

[0076] 1.3 Precision

[0077] Under repeated conditions, the samples with concentrations of (80.0-120.0) ng / mL and (800.0-1200.0) ng / mL were tested at least 10 times (n≥10) using the same batch of reagents, and the average values ​​of the measured values ​​were calculated. And standard deviation (SD), calculate the coefficient of variation (CV), the result should be ≤8.0%, see Table 3:

[0078]

[0079] Where: SD is the standard deviation, and the calculation formula is

[0080] d n is the deviation of the measured values ​​at the same level, and the calculation formula is

[0081] x n These are the values ​​measured at the same level at each time;

[0082] is the average value, and the calculation formula is

[0083] n is the number of repetitions.

[0084] Table 3

[0085] Number of repetitions Concentration value 1 982.85 2 1028.86 3 932.41 4 1010.77 5 972.58 6 958.63 7 990.06 8 951.22 9 1013.28 10 1068.83 mean 990.949 Standard deviation 40.61714155 CV value 4.10％

[0086] 1.4. Common Interference Anti-interference Test

[0087] Bilirubin (50 mg / dL), hemoglobin (2.0 g / dL), triglycerides (3000 mg / dL), and biotin (60 ng / mL) were added and mixed according to the ratios shown in Table 4 to prepare samples with different dilution ratios. The CEACAM1 concentration was measured twice for each sample. When the interfering substances, bilirubin ≤25 mg / dL, hemoglobin ≤1.0 g / dL, triglycerides ≤1500 mg / dL, and biotin ≤30 ng / mL, the deviation between group A and control group B should be ≤10%.

[0088] Table 5

[0089]

[0090] 1.5. Differences in reagent production batches

[0091] Test the same sample with three different batches of kits, repeating 10 times each. Calculate the mean and standard deviation of each concentration measurement result. Calculate the coefficient of variation (CV) according to the formula. The result should be ≤15.0%. See Table 5:

[0092] Table 5

[0093]

[0094] 2. Clinical evaluation of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)

[0095] 2.1 Detection Method

[0096] The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) produced by this method was tested on fresh clinical sera of normal subjects, lung cancer, breast cancer, thyroid cancer, and cervical cancer. The statistical differences between different groups were calculated, and data analysis and comparison were performed to verify the specificity and sensitivity of this marker for the incidence of lung cancer, breast cancer, thyroid cancer, and cervical cancer.

[0097] This kit utilizes a double-antibody one-step sandwich assay, combining chemiluminescent immunoassay with magnetic microparticle separation technology. The assay principle involves sequentially adding the test sample, streptavidin magnetic microparticle working solution, biotinylated CEACAM1 capture antibody solution, and acridinium ester-labeled CEACAM1 detection antibody solution. A pair of CEACAM1 monoclonal antibodies bind to CEACAM1 antigen molecules in the test sample, forming an immune complex. After washing to remove unbound antibody and impurities, a pre-stimulation solution (nitric acid, H2O2) and a stimulation solution (NaOH) are sequentially added, and the relative luminescence intensity (RLU) is measured. Within a certain range, the RLU is positively correlated with the CEACAM1 antigen concentration. The CEACAM1 content in the test sample can be calculated from the standard curve using the RLU.

[0098] 2.2. How to use the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) quantitative detection kit:

[0099] 1. Add 20 μL of calibrator, quality control material or sample to be tested into the test tube;

[0100] 2. Add 50 μL of biotinylated carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) capture antibody solution to the test tube;

[0101] 3. Add 50 μL of acridinium ester-labeled carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) detection antibody solution to the test tube;

[0102] 4. After mixing, incubate at 37±0.5℃ for 10 minutes;

[0103] 5. Add 30 μL of streptavidin magnetic particle working solution to the test tube;

[0104] 6. After mixing, incubate at 37±0.5℃ for 5 minutes;

[0105] 7. Add 300 μL of cleaning solution to the test tube and mix well;

[0106] 8. Magnetic separation to remove supernatant;

[0107] 9. Repeat steps 7 and 8 twice;

[0108] 10. Add 100 μL of pre-stimulation solution and 100 μL of stimulation solution to the test tube;

[0109] 11. Detect the luminescence intensity RLU after 1 second;

[0110] 12. Based on the working curve established with the calibrator, convert the RLU of the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in the test sample into the corresponding concentration (ng / mL) using the four-parameter fitting method.

[0111] 2.3 Analysis of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) concentration characteristics in different populations

[0112] The present invention was used to detect the CEACAM1 expression levels in different populations, including: 201 healthy individuals, 34 patients with benign lung diseases, 35 patients with lung cancer, 98 patients with benign breast diseases, 72 patients with breast cancer, 33 patients with benign thyroid diseases, 48 ​​patients with thyroid cancer, 13 patients with benign cervical diseases, and 15 patients with cervical cancer. The CEACAM1 expression level of all cancer patients (including lung cancer, breast cancer, thyroid cancer, cervical cancer, etc.) (13.00 (5.55, 13.00) ng / mL) was significantly higher than that of the healthy people (1.89 (1.35, 2.80) ng / mL) (P<0.0001). Among the cancer patients, there was no statistical difference between lung cancer patients (6.25 (3.30, 8.00) ng / mL) and patients with benign lung diseases (6.01 (3.34, 7.64) ng / mL). There was no statistical difference between cervical cancer patients (6.70 (6.33, 10.62) ng / mL) and patients with benign cervical diseases (6.25 (5.77, 8.86) ng / mL). The serum levels of thyroid cancer patients (6.73 (5.98, 15.35) ng / mL) were significantly higher than those of patients with benign thyroid diseases (6.61 (5.42, 7.42) ng / mL) and healthy people (1.89 (1.35, 2.80) ng / mL), and the results were statistically different (P = 0.0367 and P < 0.0001). The serum levels of breast cancer patients (8.33 (3.27, 16.86) ng / mL) were significantly higher than those of patients with benign breast diseases (1.89 (0.89, 6.15) ng / mL) and healthy people (1.89 (1.35, 2.80) ng / mL), and the results were statistically different (P < 0.0001). The results are shown in Tables 6 and Figure 1Therefore, chemiluminescence detection of CEACAM1 has important clinical application value in solid tumor screening, differentiation of benign and malignant thyroid diseases, and differentiation of benign and malignant breast diseases.

[0113] A 2002 study by researchers including Singer BB, Scheffrahn I, and Heymann R demonstrated that CEACAM1 expression levels vary in various tumors and are closely associated with tumor growth, invasion, and metastasis. Based on this research, drug development targeting CEACAM1 has primarily focused on tumor treatment, often targeting colorectal, breast, and lung cancers, with little attention paid to the treatment of thyroid cancer. The results of this study demonstrate significant differences in CEACAM1 expression levels among thyroid-related diseases (thyroid cancer and benign diseases), suggesting the potential of CEACAM1 as a target for drug development in thyroid cancer.

[0114] Table 6 CEACAM1 concentrations (Median (P 25 ,P 75 ))

[0115] Testing population Content level (ng / mL) Healthy people (n=201) 1.89(1.35，2.80) All cancer patients (n=170) 13.00(5.55，13.00) Benign lung disease (n=34) 6.01(3.34,7.64) Lung cancer (n=35) 6.25(3.30，8.00) Benign cervical diseases (n=13) 6.25(5.77，8.86) Cervical cancer (n=15) 6.70(6.33，10.62) Benign thyroid disease (n=33) 6.61(5.42，7.42) Thyroid cancer (n=48) 6.73(5.98，15.35) Benign breast diseases (n=98) 1.89(0.89，6.15) Breast cancer (n=72) 8.33(3.27，16.86)

[0116] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A chemiluminescent kit for detecting CEACAM1, characterized in that: It includes streptavidin magnetic particle working solution, biotinylated carcinoembryonic antigen-related cell adhesion molecule-1 capture antibody solution and acridinium ester-labeled carcinoembryonic antigen-related cell adhesion molecule-1 detection antibody solution.

2. The chemiluminescence kit for detecting CEACAM1 according to claim 1, wherein: The streptavidin magnetic particle working solution is prepared by the following method: Take the streptavidin magnetic particle solution, add phosphate buffer and mix thoroughly, then place it on a magnetic separator until the supernatant is clear, discard the supernatant, retain the magnetic particles, wash them, and dilute them with magnetic bead dilution buffer to obtain a streptavidin magnetic particle working solution.

3. The chemiluminescence kit for detecting CEACAM1 according to claim 1, wherein: The biotinylated carcinoembryonic antigen-related cell adhesion molecule-1 capture antibody solution was prepared by the following method: Step S11: 1 mg of mouse anti-human carcinoembryonic antigen-related cell adhesion molecule-1 monoclonal antibody was taken, and a PBS buffer solution with a pH of 7.2 to 7.4 was added to obtain a carcinoembryonic antigen-related cell adhesion molecule-1 capture antibody solution with a final concentration of 1 mg / mL, and the solution was added to a dialysis bag; Step S12: adding PBS buffer with a pH of 7.2 to 7.4 into the dialysis bag for dialysis, and then transferring the dialyzed antibody solution into an Eppendorf tube; Step S13, adding NHS-LC-Biotin to dimethyl sulfoxide to obtain an NHS-LC-Biotin solution; Step S14, adding the NHS-LC-Biotin solution to the Eppendorf tube containing the carcinoembryonic antigen-related cell adhesion molecule 1 capture antibody in step S12, mixing and reacting to form a reaction mixture; Step S15, transferring the reaction mixture in step S14 into a dialysis bag, and placing the dialysis bag into a PBS buffer solution with a pH of 7.2 to 7.4 for dialysis, and then collecting the biotin-labeled CEACAM1-mAb (Bio-CEACAM1-mAb) stock solution; Step S16: Add bovine serum albumin in PBS buffer to the collected biotin-labeled CEACAM1-mAb (Bio-CEACAM1-mAb) stock solution to obtain a Bio-mAb stock solution; before use, dilute the Bio-mAb stock solution with a biotin-labeled antibody diluent to obtain a biotinylated carcinoembryonic antigen-related cell adhesion molecule 1 capture antibody solution.

4. The chemiluminescence kit for detecting CEACAM1 according to claim 1, characterized in that: The acridinium ester-labeled carcinoembryonic antigen-related cell adhesion molecule 1 detection antibody solution is prepared by the following method: Step S21, accurately pipetting the carcinoembryonic antigen-related cell adhesion molecule 1 detection antibody solution into the purification column; Step S22: centrifugation to collect the purified detection antibody; Step S23: measuring the absorbance at 280 nm and 260 nm using an ultra-microphotometer to accurately calculate the concentration of the detection antibody; Step S24, adding the acridinium ester solution, mixing thoroughly, and shaking the mixture at room temperature in the dark; after the reaction is completed, adding glycine to the reaction mixture to terminate the cross-linking reaction, to obtain a mixture X; Step S25, placing the mixture X into a dialysis bag for dialysis, with the dialysate being a phosphate buffer solution; Step S26 , adding a storage buffer and mixing thoroughly to obtain an acridinium ester-labeled carcinoembryonic antigen-related cell adhesion molecule 1 detection antibody solution.

5. The method for using the chemiluminescence kit for detecting CEACAM1 according to any one of claims 1 to 4, wherein: The following steps are involved: The test sample, streptavidin magnetic particle working solution, biotinylated carcinoembryonic antigen-related cell adhesion molecule 1 capture antibody solution and acridinium ester-labeled carcinoembryonic antigen-related cell adhesion molecule 1 detection antibody solution are added in sequence, and a pair of carcinoembryonic antigen-related cell adhesion molecule 1 monoclonal antibodies bind to carcinoembryonic antigen-related cell adhesion molecule 1 antigen molecules in the test sample to form an immune complex; after washing to remove unbound antibodies and impurities, pre-excitation solution and excitation solution (NaOH) are added in sequence, and the relative luminescence intensity is measured; the carcinoembryonic antigen-related cell adhesion molecule 1 content in the test sample is calculated from the standard curve based on the relative luminescence intensity. 6 . The method for using the chemiluminescence kit for detecting CEACAM1 according to claim 5 , wherein the pre-excitation solution is nitric acid or H 2 O 2 . 7 . The method for using the chemiluminescence kit for detecting CEACAM1 according to claim 5 , wherein the excitation solution is NaOH.

8. Use of the chemiluminescence kit for detecting CEACAM1 according to any one of claims 1 to 4 in detecting CEACAM1 protein in human serum or plasma.

9. Use of the chemiluminescent kit for detecting CEACAM1 according to any one of claims 1 to 4 in the preparation or screening of agents for diagnosing and / or preventing and / or treating benign thyroid diseases / thyroid cancer, wherein the kit is used for in vitro diagnosis and / or risk stratification of benign thyroid diseases / thyroid cancer.

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