A high specificity carcinoembryonic antigen (CEA) quantitative detection kit and a preparation method thereof

Abstract

This invention belongs to the field of detection technology. Specifically, it discloses a highly specific carcinoembryonic antigen (CEA) quantitative detection kit and its preparation method. The highly specific CEA quantitative detection kit includes reagent R1 and reagent R2. Reagent R1 contains sodium azide, bovine serum albumin, Tween 20, and glycine buffer. Reagent R2 contains diethylenetriaminepentaacetic acid, reduced glutathione, bovine serum albumin, hydroxyethyl starch, glycerol, a coagulant, labeled particle solution, and phosphate buffer. This application uses a specific combination of reagent R2 and reagent R1 to amplify the detection signal of low-concentration samples, improve detection repeatability, eliminate false negatives in samples with extremely high concentrations, and ensure the stability of the R2 and R1 reagent system. Using the detection kit described in this application can effectively lower the detection limit, effectively improve the reliability of detection results, and has good detection repeatability and stability.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to a highly specific carcinoembryonic antigen (CEA) quantitative detection kit and its preparation method. Background Technology

[0002] Carcinoembryonic antigen (CEA) is a polysaccharide-protein complex synthesized by embryonic digestive tract tissues. Its concentration in the serum of healthy adults is extremely low, but it is significantly elevated in patients with various malignant tumors such as colon cancer, lung cancer, gastric cancer, breast cancer, and pancreatic cancer. Mild elevations may also be observed in individuals with cirrhosis, chronic enteritis, and long-term smokers. It is a broad-spectrum tumor marker widely used in clinical practice for auxiliary tumor screening, postoperative recurrence monitoring, and treatment efficacy evaluation. Accurate quantification of serum CEA has important guiding value for clinical diagnosis and treatment.

[0003] Currently, the mainstream technologies used in clinical practice for quantitative detection of serum CEA include three types: enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CIA), and immunoturbidimetric assay. Among them, ELISA has a cumbersome procedure and takes more than 2 hours, making it unsuitable for rapid testing of large batches of samples in emergency settings, and its batch repeatability is poor. Chemiluminescence immunoassay has excellent sensitivity and linearity, but the cost of specialized testing instruments and reagents is high, making it difficult to widely adopt in primary healthcare institutions. Immunoturbidimetric assay can be directly adapted to existing fully automated biochemical analyzers in hospitals, offering fast testing speed and low cost per sample, making it the preferred testing solution for hospitals at all levels.

[0004] Therefore, this application is submitted. Summary of the Invention

[0005] This invention provides a highly specific carcinoembryonic antigen (CEA) quantitative detection kit and its preparation method. The CEA quantitative detection kit described in this application can effectively reduce the detection limit, effectively improve the reliability of detection results, and has good detection repeatability and stability.

[0006] The present invention solves its technical problem by adopting the following technical solution: A highly specific carcinoembryonic antigen (CEA) quantitative detection kit, comprising reagent R1 and reagent R2; The R1 reagent contains sodium azide, bovine serum albumin, Tween 20 and glycine buffer; The R2 reagent contains diethylenetriaminepentaacetic acid, reduced glutathione, bovine serum albumin, hydroxyethyl starch, glycerol, a coagulant, labeled particle solution, and phosphate buffer.

[0007] As an embodiment of this application, the R1 reagent comprises the following components: 0.05~0.2wt% sodium azide, 0.4~1wt% bovine serum albumin, 0.05~0.2wt% Tween 20, and the balance being 0.1~0.2M glycine buffer.

[0008] As an embodiment of this application, the R2 reagent comprises the following components: 0.05~0.2wt% diethylenetriaminepentaacetic acid, 0.4~0.8wt% reduced glutathione, 0.5~1.2wt% bovine serum albumin, 0.8~1.5wt% hydroxyethyl starch, 3~6wt% glycerol, 0.1~0.4wt% coagulant, 0.6~0.9wt% labeled particle solution, and the balance being 0.02~0.08M phosphate buffer.

[0009] As an embodiment of this application, the coagulant is polyethylene glycol.

[0010] As an embodiment of this application, the method for preparing the labeled particle liquid is as follows: (1) The first polystyrene microspheres and the second polystyrene microspheres were dispersed in MES buffer to obtain a mixture; (2) Add dopamine hydrochloride to the mixture, then adjust the pH to 8.2-8.6 with tris(hydroxymethyl)aminomethane hydrochloride buffer, react, and obtain the modified solution; (3) Add N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and casein to the modified solution, react, filter, and obtain the precursor solution; (4) Add the CEA monoclonal antibody to the precursor solution and react to obtain the labeled particle solution.

[0011] As an embodiment of this application, the diameter of the first polystyrene microsphere is 30~50nm.

[0012] As an embodiment of this application, the diameter of the second polystyrene microsphere is 200~300nm.

[0013] As an embodiment of this application, the mass ratio of the first polystyrene microspheres, the second polystyrene microspheres, and the MES buffer is (0.8~1.2):(0.4~0.8):100.

[0014] As an embodiment of this application, the concentration of the MES buffer is 0.02~0.1M.

[0015] As an embodiment of this application, the mass ratio of dopamine hydrochloride to the mixture is (0.5~0.8):100.

[0016] As an embodiment of this application, the mass ratio of N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, casein, and the modifying liquid is (0.02~0.04):(0.02~0.04):(0.08~0.15):100.

[0017] As an embodiment of this application, the final concentration of the CEA monoclonal antibody in the precursor solution is 0.5–2 mg / mL.

[0018] As an implementation scheme of this application, the reaction temperature in step (2) is 45~60℃ and the time is 1~5h.

[0019] As an implementation scheme of this application, the reaction temperature in step (3) is 20~30℃ and the time is 0.5~4h.

[0020] As an implementation scheme of this application, the reaction temperature in step (4) is 20~30℃ and the time is 0.4~4h.

[0021] The beneficial effects of this invention are as follows: This application uses a specific R2 reagent in combination with an R1 reagent, which can amplify the detection signal of low-concentration samples, improve detection repeatability, eliminate false negatives in samples with extremely high values, and ensure that the R2 and R1 reagent systems are stable. Using the detection kit described in this application can effectively reduce the detection limit, effectively improve the reliability of detection results, and has good detection repeatability and stability. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0024] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0025] In this invention, there are no particular limitations on the specific dispersion and stirring methods.

[0026] Unless otherwise specified, all reagents or instruments used in this invention are commercially available conventional products. Unless otherwise specified, the raw materials used in each comparative example and the parallel experiments of each embodiment are the same commercially available products.

[0027] This application provides a highly specific carcinoembryonic antigen (CEA) quantitative detection kit, including reagent R1 and reagent R2; The R1 reagent contains sodium azide, bovine serum albumin, Tween 20 and glycine buffer; The R2 reagent contains diethylenetriaminepentaacetic acid, reduced glutathione, bovine serum albumin, hydroxyethyl starch, glycerol, a coagulant, labeled particle solution, and phosphate buffer.

[0028] This application uses a specific R2 reagent in combination with an R1 reagent to amplify the detection signal of low-concentration samples, improve detection repeatability, eliminate false negatives in samples with extremely high concentrations, and ensure that the R2 and R1 reagent systems are stable. Using the detection kit described in this application can effectively reduce the detection limit, effectively improve the reliability of detection results, and has good detection repeatability and stability.

[0029] In some embodiments, the R1 reagent comprises the following components: 0.05-0.2 wt% sodium azide, 0.4-1 wt% bovine serum albumin, 0.05-0.2 wt% Tween 20, and the balance being 0.1-0.2 M glycine buffer.

[0030] In some embodiments, the R2 reagent comprises the following components: 0.05-0.2 wt% diethylenetriaminepentaacetic acid, 0.4-0.8 wt% reduced glutathione, 0.5-1.2 wt% bovine serum albumin, 0.8-1.5 wt% hydroxyethyl starch, 3-6 wt% glycerol, 0.1-0.4 wt% coagulant, 0.6-0.9 wt% labeled particle solution, and the balance being 0.02-0.08 M phosphate buffer.

[0031] In some embodiments, the coagulant is polyethylene glycol.

[0032] In some embodiments, the method for preparing the labeled particulate liquid is as follows: (1) The first polystyrene microspheres and the second polystyrene microspheres were dispersed in MES buffer to obtain a mixture; (2) Add dopamine hydrochloride to the mixture, then adjust the pH to 8.2-8.6 with tris(hydroxymethyl)aminomethane hydrochloride buffer, react, and obtain the modified solution; (3) Add N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and casein to the modified solution, react, filter, and obtain the precursor solution; (4) Add the CEA monoclonal antibody to the precursor solution and react to obtain the labeled particle solution.

[0033] This application uses two types of polystyrene microspheres with different diameters, which are then modified by reacting with dopamine hydrochloride and tris(hydroxymethyl)aminomethane hydrochloride buffer. Subsequently, they are treated with N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and casein, and finally conjugated with CEA monoclonal antibody to obtain the labeled particle solution. This solution can improve the repeatability of low-value detection, broaden the linear range of high concentrations, eliminate false negatives in ultra-high value samples, improve structural stability, avoid the aggregation of polystyrene microspheres, reduce interference from factors such as serum proteins, amplify the detection signal of low-concentration samples, effectively lower the detection limit, effectively improve the reliability of detection results, and have good detection repeatability and stability.

[0034] In some embodiments, the diameter of the first polystyrene microsphere is 30-50 nm.

[0035] In some embodiments, the diameter of the second polystyrene microsphere is 200-300 nm.

[0036] The polystyrene microspheres mentioned in this application are commercially available, and it is preferred to purchase polystyrene microspheres with the same diameter as those used in Xidian Experimental.

[0037] In some embodiments, the mass ratio of the first polystyrene microsphere, the second polystyrene microsphere, and the MES buffer is (0.8~1.2):(0.4~0.8):100.

[0038] In some embodiments, the concentration of the MES buffer is 0.02~0.1M.

[0039] In some embodiments, the mass ratio of dopamine hydrochloride to the mixture is (0.5~0.8):100.

[0040] In some embodiments, the mass ratio of N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, casein, and the modifying solution is (0.02~0.04):(0.02~0.04):(0.08~0.15):100.

[0041] In some embodiments, the final concentration of the CEA monoclonal antibody in the precursor solution is 0.5–2 mg / mL.

[0042] In some embodiments, the reaction temperature in step (2) is 45~60°C and the time is 1~5h.

[0043] In some embodiments, the reaction temperature in step (3) is 20~30°C and the time is 0.5~4h.

[0044] In some embodiments, the reaction temperature in step (4) is 20~30°C and the time is 0.4~4h.

[0045] In some embodiments, the highly specific carcinoembryonic antigen (CEA) quantitative detection kit further includes a calibrator comprising the following components: 0.4–1 wt% bovine serum albumin, 0.8–1.2 wt% sodium chloride, 0.2–0.6 wt% sodium azide, 5–1000 ng / mL carcinoembryonic antigen, and the balance being 0.02–0.1 M Hepes buffer.

[0046] The present application is further illustrated below with specific embodiments:

[0047] Example 1 A highly specific carcinoembryonic antigen (CEA) quantitative detection kit, comprising reagent R1, reagent R2, and calibrators.

[0048] The R1 reagent contains the following components: 0.12 wt% sodium azide, 0.5 wt% bovine serum albumin, 0.15 wt% Tween 20, and the balance being 0.15 M glycine buffer.

[0049] The R2 reagent contains the following components: 0.15 wt% diethylenetriaminepentaacetic acid, 0.5 wt% reduced glutathione, 0.8 wt% bovine serum albumin, 1 wt% hydroxyethyl starch, 5 wt% glycerol, 0.2 wt% polyethylene glycol 2000, 0.8 wt% labeled particle solution, and the balance being 0.05 M phosphate buffer.

[0050] The method for preparing the labeled particulate liquid is as follows: (1) The first polystyrene microspheres (50 nm in diameter) and the second polystyrene microspheres (200 nm in diameter) were dispersed in a 0.05 M MES buffer solution to obtain a mixture; wherein the mass ratio of the first polystyrene microspheres, the second polystyrene microspheres and the MES buffer solution was 1:0.6:100.

[0051] (2) Add dopamine hydrochloride to the mixture, then adjust the pH to 8.5 with tris(hydroxymethyl)aminomethane hydrochloride buffer, and react at 50°C for 2.5 h to obtain the modified solution; wherein, the mass ratio of dopamine hydrochloride to the mixture is 0.6:100.

[0052] (3) N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and casein were added to the modification solution and reacted at 25°C for 2 h. After filtration, the precursor solution was obtained. The mass ratio of N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, casein and modification solution was 0.03:0.03:0.12:100.

[0053] (4) Add the CEA monoclonal antibody to the precursor solution at a final concentration of 1 mg / mL and react at 25°C for 2 h to obtain the labeled particle solution.

[0054] The calibrator contains the following components: 0.5 wt% bovine serum albumin, 1 wt% sodium chloride, 0.5 wt% sodium azide, 5-80 ng / mL carcinoembryonic antigen, and the balance 0.05 M Hepes buffer.

[0055] Carcinoembryonic antigen was prepared into six concentration gradients: 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, and 80 ng / mL.

[0056] Example 2 The difference between Example 2 and Example 1 is that the diameter of the polystyrene microspheres is different, but everything else is the same.

[0057] In this embodiment, the diameter of the first polystyrene microsphere is 30 nm, and the diameter of the second polystyrene microsphere is 300 nm.

[0058] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the preparation method of the labeled particulate liquid is different, but everything else is the same.

[0059] The preparation method of labeled particulate liquid is as follows: (1) The first polystyrene microspheres (50 nm in diameter) and the second polystyrene microspheres (200 nm in diameter) were dispersed in a 0.05 M MES buffer solution to obtain a mixture; wherein the mass ratio of the first polystyrene microspheres, the second polystyrene microspheres and the MES buffer solution was 1:0.6:100.

[0060] (2) Add CEA monoclonal antibody to the mixture at a final concentration of 1 mg / mL and react at 25°C for 2 h to obtain labeled particle solution.

[0061] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the preparation method of the labeled particulate liquid is different, but everything else is the same.

[0062] The preparation method of labeled particulate liquid is as follows: (1) The first polystyrene microspheres (50 nm in diameter) and the second polystyrene microspheres (200 nm in diameter) were dispersed in a 0.05 M MES buffer solution to obtain a mixture; wherein the mass ratio of the first polystyrene microspheres, the second polystyrene microspheres and the MES buffer solution was 1:0.6:100.

[0063] (2) Add dopamine hydrochloride to the mixture, then adjust the pH to 8.5 with tris(hydroxymethyl)aminomethane hydrochloride buffer, and react at 50°C for 2.5 h to obtain the modified solution; wherein, the mass ratio of dopamine hydrochloride to the mixture is 0.6:100.

[0064] (3) Add CEA monoclonal antibody to the modification solution at a final concentration of 1 mg / mL and react at 25°C for 2 h to obtain labeled particle solution.

[0065] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the preparation method of the labeled particulate liquid is different, but everything else is the same.

[0066] The preparation method of labeled particulate liquid is as follows: (1) The first polystyrene microspheres (50 nm in diameter) and the second polystyrene microspheres (200 nm in diameter) were dispersed in a 0.05 M MES buffer solution to obtain a mixture; wherein the mass ratio of the first polystyrene microspheres, the second polystyrene microspheres and the MES buffer solution was 1:0.6:100.

[0067] (2) N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and casein were added to the mixture and reacted at 25°C for 2 hours. The mixture was then filtered to obtain the precursor solution. The mass ratio of N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, casein and the mixture was 0.03:0.03:0.12:100.

[0068] (3) Add the CEA monoclonal antibody to the precursor solution at a final concentration of 1 mg / mL and react at 25°C for 2 h to obtain the labeled particle solution.

[0069] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that the preparation method of the labeled particulate liquid is different, but everything else is the same.

[0070] The preparation method of labeled particulate liquid is as follows: (1) The first polystyrene microspheres (50 nm in diameter) and the second polystyrene microspheres (200 nm in diameter) were dispersed in a 0.05 M MES buffer solution to obtain a mixture; wherein the mass ratio of the first polystyrene microspheres, the second polystyrene microspheres and the MES buffer solution was 1:0.6:100.

[0071] (2) Add dopamine hydrochloride to the mixture, then adjust the pH to 8.5 with tris(hydroxymethyl)aminomethane hydrochloride buffer, and react at 50°C for 2.5 h to obtain the modified solution; wherein, the mass ratio of dopamine hydrochloride to the mixture is 0.6:100.

[0072] (3) N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added to the modification solution and reacted at 25°C for 2 h. The mixture was then filtered to obtain the precursor solution. The mass ratio of N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and the modification solution was 0.03:0.03:100.

[0073] (4) Add the CEA monoclonal antibody to the precursor solution at a final concentration of 1 mg / mL and react at 25°C for 2 h to obtain the labeled particle solution.

[0074] Test case 1. Minimum detection limit: 1.1 Zero blank sample: Pure negative mixed serum, without added antigens; 1.2 Concentration gradient samples: The standard antigen was diluted with blank matrix to prepare a series of low concentrations: 0.1 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, and 0.5 ng / mL; 1.3 Reagent kit parameters (fixed): 3 μL sample, 240 μL R1 reagent, incubate at 37℃ for 5 min, add 60 μL R2 reagent, use the two-point endpoint method, read the difference in absorbance at 540 nm before and after adding R2 to calculate the concentration.

[0075] 1.4.1 Determine the baseline of blank absorbance and calculate the blank limit (LOB). Take blank negative serum samples and perform repeated testing 20 times consecutively; record the equivalent concentration values ​​corresponding to the 20 tests; calculate the average value M0 and standard SD0 of the 20 test results; Blank limit calculation formula: LOB = M0 + 1.645 × SD0.

[0076] 1.4.2 Repeated testing of concentration samples Five low-concentration antigen samples (0.1, 0.2, 0.3, 0.4, and 0.5 ng / mL) were prepared and divided into 10 parallel tubes for each group. Each group was tested 10 times consecutively, and the concentration of each test was recorded. The mean Mx and standard deviation SDx of each group were calculated.

[0077] 1.4.3 Calculate the Limit of Detection (LOD) Substituting into the formula: LOD = LOB + 1.645 × SDmin, where SDmin is the standard deviation of the lowest gradient sample detection.

[0078] Table 1

[0079] As can be seen from Table 1, the detection kit described in this application can effectively reduce the limit of detection, and the prepared labeled particle liquid can significantly reduce the limit of detection.

[0080] 2. Precision: Three groups of serum samples (5, 50, 300 ng / mL) were measured 20 times consecutively, and intra-day and inter-day precision were calculated.

[0081] Table 2

[0082] As can be seen from Table 2, the detection kit described in this application has excellent precision, can effectively improve the reliability of detection results, and has good detection repeatability and stability.

[0083] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A highly specific carcinoembryonic antigen (CEA) quantitative detection kit, characterized in that, Including reagents R1 and R2; The R1 reagent contains sodium azide, bovine serum albumin, Tween 20, and glycine buffer. The R2 reagent contains diethylenetriaminepentaacetic acid, reduced glutathione, bovine serum albumin, hydroxyethyl starch, glycerol, a coagulant, labeled particle solution, and phosphate buffer.

2. The highly specific carcinoembryonic antigen (CEA) quantitative detection kit according to claim 1, characterized in that, The R1 reagent comprises the following components: 0.05-0.2 wt% sodium azide, 0.4-1 wt% bovine serum albumin, 0.05-0.2 wt% Tween 20, and the balance being 0.1-0.2 M glycine buffer.

3. The highly specific carcinoembryonic antigen (CEA) quantitative detection kit according to claim 1, characterized in that, The R2 reagent comprises the following components: 0.05-0.2 wt% diethylenetriaminepentaacetic acid, 0.4-0.8 wt% reduced glutathione, 0.5-1.2 wt% bovine serum albumin, 0.8-1.5 wt% hydroxyethyl starch, 3-6 wt% glycerol, 0.1-0.4 wt% coagulant, 0.6-0.9 wt% labeled particle solution, and the balance being 0.02-0.08 M phosphate buffer.

4. The highly specific carcinoembryonic antigen (CEA) quantitative detection kit according to claim 1, characterized in that, The coagulant is polyethylene glycol.

5. The highly specific carcinoembryonic antigen (CEA) quantitative detection kit according to claim 1, characterized in that, The method for preparing the labeled particle liquid is as follows: (1) The first polystyrene microspheres and the second polystyrene microspheres were dispersed in MES buffer to obtain a mixture; (2) Add dopamine hydrochloride to the mixture, then adjust the pH to 8.2-8.6 with tris(hydroxymethyl)aminomethane hydrochloride buffer, react, and obtain the modified solution; (3) Add N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and casein to the modified solution, react, filter, and obtain the precursor solution; (4) Add the CEA monoclonal antibody to the precursor solution and react to obtain the labeled particle solution.

6. The highly specific carcinoembryonic antigen (CEA) quantitative detection kit according to claim 5, characterized in that, The diameter of the first polystyrene microsphere is 30~50 nm; The diameter of the second polystyrene microsphere is 200~300 nm; The mass ratio of the first polystyrene microsphere, the second polystyrene microsphere, and the MES buffer is (0.8~1.2):(0.4~0.8):100; The concentration of the MES buffer solution is 0.02~0.1M.

7. The highly specific carcinoembryonic antigen (CEA) quantitative detection kit according to claim 5, characterized in that, The mass ratio of dopamine hydrochloride to the mixture is (0.5~0.8):

100.

8. The highly specific carcinoembryonic antigen (CEA) quantitative detection kit according to claim 5, characterized in that, The mass ratio of N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, casein, and the modifying solution is (0.02~0.04):(0.02~0.04):(0.08~0.15):

100.

9. The highly specific carcinoembryonic antigen (CEA) quantitative detection kit according to claim 5, characterized in that, The final concentration of the CEA monoclonal antibody in the precursor solution is 0.5–2 mg / mL.

10. The highly specific carcinoembryonic antigen (CEA) quantitative detection kit according to claim 5, characterized in that, The reaction temperature in step (2) is 45~60℃, and the reaction time is 1~5h; The reaction temperature in step (3) is 20~30℃, and the reaction time is 0.5~4h; The reaction temperature in step (4) is 20~30℃ and the reaction time is 0.4~4h.

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