Pancreatic cancer tumor marker kit and preparation method thereof

Abstract

The invention relates to the technical field of tumor marker kits, in particular to a pancreatic cancer tumor marker kit and a preparation method thereof. The kit comprises a CA19-9 antibody coupling magnetic bead, the CA242 antibody is coupled with magnetic beads; the CA50 antibody is coupled with magnetic beads; coupling the CEA antibody with magnetic beads; coupling the LIF antibody with magnetic beads; a fluorescent second antibody; and a sample processing reagent. According to the pancreatic cancer tumor marker kit and the preparation method thereof, a double antibody sandwich detection principle is utilized, a capture antibody is coupled to a magnetic microsphere, a corresponding antigen in serum is captured and then combined with a fluorescent secondary antibody, a flow cytometry is used for detection, and a novel pancreatic cancer tumor marker detection scheme including CA19-9, CA242, CA50, CEA and LIF is established; the kit can improve the sensitivity and specificity of pancreatic cancer detection and diagnosis, and improve the ability of evaluating prognosis and metastasis.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor marker kits, and in particular to a pancreatic cancer tumor marker kit and a preparation method thereof. Background Art

[0002] Pancreatic cancer is a common malignant tumor with a very high degree of malignancy and rapid progression. There is a lack of sensitive and specific diagnostic indicators in the early stages. Currently, the main tumor markers for pancreatic cancer detection are carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 242 (CA242), CA50 and carcinoembryonic antigen (CEA).

[0003] When the high value of CA19-9 is 37U / mL, the sensitivity is 79%-89% and the specificity is 72%-90%. If the high value is 1000U / mL, the specificity can reach 100%, but the sensitivity is reduced to 24%-41%. In addition, CA19-9 is also elevated in other gastrointestinal malignancies and benign diseases such as chronic pancreatitis. The sensitivity of CEA for the diagnosis of pancreatic cancer is 30% to 68%, but its specificity is poor as a broad-spectrum tumor marker. Elevated CA242 is mainly seen in pancreatic cancer. Its sensitivity is slightly lower than that of CA199, but its specificity is higher. CA50 is highly correlated with CA19-9, so the diagnostic value of CA50 testing for gastrointestinal tumors is similar to that of CA19-9. CA50 levels will be elevated in 66.6% of lung cancer, 88.2% of liver cancer, 68.9% of gastric cancer, 88.5% of ovarian or cervical cancer, 94.4% of pancreatic or bile duct cancer, and more than 70% of rectal cancer and bladder cancer. Elevated CA50 levels can also be seen in kidney cancer and breast cancer.

[0004] Currently, there are commercial reagents for single tumor markers and multiple combined tests, but these known tumor markers still have shortcomings in sensitivity and specificity. Summary of the invention

[0005] The purpose of the present invention is to provide a pancreatic cancer tumor marker kit and a preparation method thereof to solve the problems raised in the above background technology.

[0006] To achieve the above object, on the one hand, the present invention provides a pancreatic cancer tumor marker kit, comprising: CA19-9 antibody coupled to magnetic beads; CA242 antibody coupled to magnetic beads; CA50 antibody coupled to magnetic beads; CEA antibody coupled to magnetic beads; LIF antibody coupled to magnetic beads; Fluorescent secondary antibodies; Sample processing reagents.

[0007] As a further improvement of the technical solution, the synthesis of LIF adopts genetic engineering technology, and the specific steps are as follows: S1.1, extracting the DNA sequence of the LIF gene from the source; S1.2, inserting the LIF gene into the expression vector; S1.3, introducing the recombinant vector into the host cell; S1.4, culturing the transfected host cells to allow the host cells to express LIF protein; S1.5. Collect the host cell culture supernatant and obtain the purified LIF protein through separation and purification steps.

[0008] As a further improvement of the technical solution, the preparation method of the LIF antibody-coupled magnetic beads is as follows: S2.1, prepare magnetic microsphere material, select Fe3O4 microsphere; S2.1, modifying the surface of the magnetic microspheres, coupling the LIF antibody to the surface of the magnetic microspheres using a carboxylation reagent; S2.1. Coating and immobilization of magnetic beads are performed to enable them to have specific recognition capability for LIF protein.

[0009] As a further improvement of the technical solution, the sample processing reagent includes a buffer solution, a washing buffer solution, a blocking agent and a standard substance.

[0010] In another aspect, the present invention provides a method for preparing a pancreatic cancer tumor marker kit as described in any one of the above, comprising the following steps: S3.1. Select antibodies that specifically recognize pancreatic cancer markers, including antibodies for CA19-9, CA242, CA50, CEA, and LIF, and purify and concentrate the antibodies; S3.2, selecting magnetic microsphere materials, performing surface modification, and coupling antibodies to the surface of the magnetic microspheres so that the antibodies have specific recognition capabilities for the corresponding antigens; S3.3, select a fluorescent secondary antibody different from the antibody on the magnetic microspheres to detect the binding of the antigen; S3.4. Assemble the magnetic microspheres, fluorescent secondary antibodies and sample processing reagents together to form a complete kit.

[0011] Compared with the prior art, the present invention has the following beneficial effects: In the pancreatic cancer tumor marker kit and preparation method thereof, the detection principle of double antibody sandwich is utilized to couple the capture antibody to the magnetic microspheres, and the corresponding antigen in the serum is captured, and then combined with the fluorescent secondary antibody, and then detected by flow cytometry. By establishing a new pancreatic cancer tumor marker detection scheme, including CA19-9, CA242, CA50, CEA, and LIF, the kit can improve the sensitivity and specificity of pancreatic cancer detection and diagnosis, and improve the ability to evaluate prognosis and metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is one of the schematic diagrams for analyzing the sensitivity and specificity of the biomarkers of the test examples of the present invention; Figure 2 This is the second schematic diagram of sensitivity and specificity analysis of the biomarkers of the test example of the present invention. DETAILED DESCRIPTION

[0013] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0014] Example

[0015] An embodiment of the present invention provides a pancreatic cancer tumor marker kit, comprising: CA19-9 antibody coupled to magnetic beads; CA242 antibody coupled to magnetic beads; CA50 antibody coupled to magnetic beads; CEA antibody coupled to magnetic beads; LIF antibody coupled to magnetic beads; Fluorescent secondary antibodies; Sample processing reagents, including buffers, wash buffers, blocking agents, and standards.

[0016] The synthesis of LIF protein is achieved through genetic engineering technology, and the specific steps are as follows: (1) Obtaining the LIF gene: extracting the DNA sequence of the LIF gene from a source; (2) Cloning the LIF gene: inserting the LIF gene into an expression vector, such as a plasmid or viral vector; (3) Transfection of host cells: introducing the recombinant vector into host cells, such as bacteria, mammalian cells, etc. (4) Culture and expression: Cultivate the transfected host cells and provide appropriate culture conditions, such as temperature and culture medium, so that the host cells express LIF protein; (5) Extraction and purification: Collect the host cell culture supernatant and obtain the purified LIF protein through separation and purification steps such as column chromatography, filtration, centrifugation, etc.

[0017] Preparation of LIF antibody-coupled magnetic beads: (1) Prepare magnetic microsphere material, and select Fe3O4 microspheres; (2) Modifying the surface of the magnetic microspheres by coupling the LIF antibody to the surface of the magnetic microspheres using a carboxylation reagent; (3) Coating and immobilizing the magnetic beads to enable them to have specific recognition capabilities for LIF protein.

[0018] The present invention provides a method for preparing a pancreatic cancer tumor marker kit, and the specific steps are as follows: (1) Preparation of antibodies: Select antibodies that specifically recognize pancreatic cancer markers as needed, including antibodies for CA19-9, CA242, CA50, CEA, and LIF, and purify and concentrate the antibodies; (2) Preparing magnetic microspheres: Select appropriate magnetic microsphere materials, perform surface modification, and couple antibodies to the surface of magnetic microspheres to enable them to have specific recognition capabilities for the corresponding antigens; (3) Prepare fluorescent secondary antibodies: Select fluorescent secondary antibodies that are different from the antibodies on the magnetic microspheres to detect the binding of antigens; (4) Assembling the kit: Assemble the magnetic microspheres, fluorescent secondary antibodies and sample processing reagents together to form a complete kit; (5) Quality control testing: Perform quality control testing on the prepared test kit to ensure its performance and reliability; (6) Packaging and labeling: Package the test kit and mark the relevant information on the package, such as instructions for use, storage conditions, etc. (7) Verification and Validation: The kit is verified and validated to ensure its suitability and accuracy in the laboratory environment.

[0019] In the embodiment of the present invention, the detection principle of double antibody sandwich is utilized, the capture antibody is coupled to the magnetic microspheres, the corresponding antigen in the serum is captured, and then combined with the fluorescent secondary antibody, and then detected by flow cytometry, and a new pancreatic cancer tumor marker detection scheme is established, including CA19-9, CA242, CA50, CEA, and LIF; the kit can improve the sensitivity and specificity of pancreatic cancer detection and diagnosis, and improve the ability to evaluate prognosis and metastasis.

[0020] In order to verify that the LIF provided in the embodiment of the present invention can be used as a marker, the following experimental examples are used for illustration.

[0021] Test example 1. Support for serum LIF as a diagnostic indicator for pancreatic cancer Serum LIF levels in pancreatic cancer patients were significantly higher than those in healthy controls (193.15±87.71 vs 83.85±57.32 pg / mL) (P<.001).

[0022] It was higher in PDAC than in PNET (193.15±87.71 vs 67.03±35.18 pg / mL) (P<.001) or PCA (193.15±87.71 vs 126.61±80.13 pg / mL) (P<.001).

[0023] The area under the ROC curve of LIF was 0.848 (P < .001) when LIF was set to 132.52 pg / mL; the Youden index was the largest; the Youden index was the largest, and the sensitivity and specificity were 68.9% and 85.0%, respectively.

[0024] 2. Support for serum LIF as a prognostic marker for predicting lymph node metastasis in PDAC patients There were significant differences in serum levels between patients with and without lymph node metastasis.

[0025] There were significant differences in serum levels in patients with lymph node metastasis (training set: 123.14±71.80 vs 187.75±78.26 pg / mL, validation set: 142.83±64.83 vs 219.97±75.04 pg / mL).

[0026] CA199 and CEA are common pancreatic cancer biomarkers in clinical practice; therefore, we compared the efficacy of serum LIF levels with other markers in predicting lymph node metastasis, and the ROC curve of LIF expression was better than that of CA199 and CEA.

[0027] The ROC curves for predicting lymph node metastasis in both the training group and the validation group were better than those of CA199 and CEA.

[0028] according to Figure 1 As can be seen in Tables A and C, the sensitivity and specificity analysis of these biomarkers in the training group showed that the sensitivity of LIF (62.5%) was lower than that of CA199 (62.5%); lower than CA199 (87.5%), while the former has better specificity (78.5%); The former (78.5%) was better than the latter (35.7%); the sensitivity and specificity of LIF (62.5% and 78.5%, respectively) were higher than those of CEA (34.4% and 75.0%, respectively) for predicting lymph node metastasis; in the validation group, the sensitivity of LIF (67.6%) was lower than that of CA199 (79.0%); lower than that of CA199 (79.4%), while the specificity of LIF (80.6%) was higher than that of CA199; (80.6%) was higher than CA199 (48.3%); the sensitivity and specificity (67.6% and 80.6%) were similar to those of the training group and were higher than CEA (50.3%); the sensitivity and specificity (67.6% and 80.6%) were higher than CEA (50.0% and 70.9%), see Figure 1 Table B and Table D.

[0029] 3. Serum LIF as a prognostic biomarker Predicting distant metastasis in patients with PDAC When patients were divided into two groups according to TNM staging [no distant metastasis (stage I-III)]; stage (stage I-III) and metastasis (stage IV)], LIF levels differed between the groups [no distant metastasis (stage I-III) vs metastasis (stage IV)].

[0030] (stage I-III) vs metastasis (stage IV)] (training group: 163.46±70.98 vs 249.79±86.55 pg / mL; validation set: 183.88±70.62 vs 268.04±76.83 pg / mL); the analysis of LIF as a biomarker for predicting distant metastasis showed that the ROC curve of LIF expression was better than that of other biomarkers.

[0031] The ROC curves of the training set and CEA were better than those of the two biomarkers (CA199 and CEA), indicating that the expression of LIF in the training set and the validation set was better than that of the two biomarkers (CA199 and CEA). Figure 2 Table A and Table C.

[0032] In the training set, the sensitivity of LIF (61.4%) was lower than that of CA199 (65.9%), but higher than that of CEA (31.8%); while the specificity of LIF (91.0%) was higher than that of CA199 (55.3%) and CEA (78.5%); In the validation group, the specificity of LIF (69.4%) was lower than that of CA199 (79.6%) and higher than that of CEA (49.0%). However, the specificity of LIF (83.6%) was higher than that of CA199 (41.5%) and CEA (67.2%). Figure 2 Table B and Table D.

[0033] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A pancreatic cancer tumor marker kit, characterized in that: include: CA19-9 antibody coupled to magnetic beads; CA242 antibody coupled to magnetic beads; CA50 antibody coupled to magnetic beads; CEA antibody coupled to magnetic beads; LIF antibody coupled to magnetic beads; Fluorescent secondary antibodies; Sample processing reagents.

2. The pancreatic cancer tumor marker kit according to claim 1, characterized in that: The synthesis of LIF adopts genetic engineering technology, and the specific steps are as follows: S1.1, extracting the DNA sequence of the LIF gene from the source; S1.2, inserting the LIF gene into the expression vector; S1.3, introducing the recombinant vector into the host cell; S1.4, culturing the transfected host cells to allow the host cells to express LIF protein; S1.

5. Collect the host cell culture supernatant and obtain the purified LIF protein through separation and purification steps.

3. The pancreatic cancer tumor marker kit according to claim 1, characterized in that: The preparation method of the LIF antibody-coupled magnetic beads is as follows: S2.1, prepare magnetic microsphere material, select Fe3O4 microsphere; S2.1, modifying the surface of the magnetic microspheres, coupling the LIF antibody to the surface of the magnetic microspheres using a carboxylation reagent; S2.

1. Coating and immobilization of magnetic beads are performed to enable them to have specific recognition capability for LIF protein.

4. The pancreatic cancer tumor marker kit according to claim 1, characterized in that: The sample processing reagents include a buffer, a washing buffer, a blocking agent and a standard substance.

5. A method for preparing the pancreatic cancer tumor marker kit according to any one of claims 1 to 4, characterized in that: The following steps are involved: S3.

1. Select antibodies that specifically recognize pancreatic cancer markers, including antibodies for CA19-9, CA242, CA50, CEA, and LIF, and purify and concentrate the antibodies; S3.2, selecting magnetic microsphere materials, performing surface modification, and coupling antibodies to the surface of the magnetic microspheres so that the antibodies have specific recognition capabilities for the corresponding antigens; S3.3, select a fluorescent secondary antibody different from the antibody on the magnetic microspheres to detect the binding of the antigen; S3.

4. Assemble the magnetic microspheres, fluorescent secondary antibodies and sample processing reagents together to form a complete kit.

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