CN113759116A - An ELISA kit for the detection of gastric cancer tumor markers - Google Patents
An ELISA kit for the detection of gastric cancer tumor markers Download PDFInfo
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- 206010017758 gastric cancer Diseases 0.000 title claims abstract description 58
- 208000005718 Stomach Neoplasms Diseases 0.000 title claims abstract description 57
- 201000011549 stomach cancer Diseases 0.000 title claims abstract description 57
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 33
- 238000001514 detection method Methods 0.000 title claims abstract description 18
- 238000008157 ELISA kit Methods 0.000 title claims abstract description 16
- 210000002966 serum Anatomy 0.000 claims abstract description 39
- 102100033011 Integrin beta-6 Human genes 0.000 claims abstract description 38
- 101001015064 Homo sapiens Integrin beta-6 Proteins 0.000 claims abstract description 36
- 238000005406 washing Methods 0.000 claims abstract description 25
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- 201000011510 cancer Diseases 0.000 claims abstract description 11
- 238000004393 prognosis Methods 0.000 claims abstract description 11
- 230000036210 malignancy Effects 0.000 claims abstract description 10
- 230000000903 blocking effect Effects 0.000 claims abstract description 9
- 210000004369 blood Anatomy 0.000 claims abstract description 8
- 239000008280 blood Substances 0.000 claims abstract description 8
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims abstract description 7
- 239000012916 chromogenic reagent Substances 0.000 claims abstract description 6
- 238000011156 evaluation Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 33
- 102000004190 Enzymes Human genes 0.000 claims description 22
- 108090000790 Enzymes Proteins 0.000 claims description 22
- 238000002965 ELISA Methods 0.000 claims description 18
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 11
- 239000000439 tumor marker Substances 0.000 claims description 10
- 241000283707 Capra Species 0.000 claims description 8
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 8
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- 239000012089 stop solution Substances 0.000 claims description 5
- 241001494479 Pecora Species 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 239000003085 diluting agent Substances 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 238000012795 verification Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 4
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- 102000012406 Carcinoembryonic Antigen Human genes 0.000 description 15
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 15
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- 238000012216 screening Methods 0.000 description 3
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- 108010021309 integrin beta6 Proteins 0.000 description 2
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- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000001574 biopsy Methods 0.000 description 1
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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Abstract
本发明公开了ITGB6在胃癌肿瘤标记物检测中的应用，并公开了一种胃癌肿瘤标记物检测的ELISA试剂盒，所述试剂盒包括酶标板、兔抗ITGB6血清抗体、酶标羊抗ITGB6血清抗体、洗涤液、PBST封闭液、TMB底物显色试剂、反应终止液；同时公开了该试剂盒在胃癌检测、评估肿瘤恶性程度及受试者预后中的应用及具体应用方法。本发明首次将ITGB6的血清表达水平与胃癌的发生发展相联系，通过检测受试者血液中ITGB6的表达量，结合CEA水平测定，判断受试者是否患有胃癌，并预测受试者胃癌恶性程度及预后情况，从而指导临床医师给受试者提供治疗方案。本发明的方法更具有敏感性、特异性和无创性。The invention discloses the application of ITGB6 in the detection of gastric cancer tumor markers, and discloses an ELISA kit for the detection of gastric cancer tumor markers. Serum antibody, washing solution, PBST blocking solution, TMB substrate chromogenic reagent, and reaction termination solution; meanwhile, the application and specific application method of the kit in gastric cancer detection, evaluation of tumor malignancy and prognosis of subjects are disclosed. The invention links the serum expression level of ITGB6 with the occurrence and development of gastric cancer for the first time. By detecting the expression level of ITGB6 in the blood of the subject and measuring the level of CEA, it is possible to determine whether the subject has gastric cancer and predict the malignant tumor of the subject. degree and prognosis, so as to guide clinicians to provide treatment options to subjects. The method of the present invention is more sensitive, specific and non-invasive.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to an ELISA kit for detecting gastric cancer tumor markers.
Background
Gastric cancer is a common tumor of the digestive tract, with mortality rates secondary to all cancers. The burden of gastric cancer is severe, and the prevention and treatment of gastric cancer is very important. Early discovery and early treatment of gastric cancer is critical to the prognosis and survival rate of the subject. However, the current diagnosis of gastric cancer mainly depends on endoscopic biopsy and is not suitable for large-scale screening. In addition, some tumor markers commonly used in early screening have low detection specificity, such as the most commonly used traditional tumor-associated carcinoembryonic antigen (CEA) at present, and the increase thereof may also be related to rectal cancer, pancreatic cancer, liver cancer, breast cancer, etc., even some benign diseases. It can be said that there is no biomarker which is satisfactory in both sensitivity and specificity and is used for diagnosis and treatment of gastric cancer.
Disclosure of Invention
In order to solve the technical problems, the invention provides the ELISA kit for detecting the gastric cancer tumor marker, which can prompt the malignancy degree of gastric cancer, can effectively predict the prognosis of a testee, can be combined with imaging and pathology in clinic to improve the evaluation efficiency and accuracy of the malignancy degree of gastric cancer, and has important significance for early discovery and early treatment of gastric cancer.
In order to achieve the purpose, the technical scheme of the invention is as follows:
application of ITGB6 in detection of gastric cancer tumor markers.
An ELISA kit for detecting gastric cancer tumor markers comprises an ELISA plate, a rabbit anti-ITGB 6 serum antibody, an enzyme-labeled goat anti-ITGB 6 serum antibody, a washing solution, a PBST blocking solution, a TMB substrate chromogenic reagent and a reaction stop solution.
In the scheme, the concentrations of the rabbit anti-ITGB 6 serum antibody and the enzyme-labeled goat anti-ITGB 6 serum antibody are both 2 ug/ml.
In the above protocol, the washing solution was PBS containing 0.05% Tween20 by mass, and the pH was 7.5.
In the above embodiment, the PBST blocking solution contains BSA at a mass concentration of 3%.
In the above scheme, the enzyme is HRP.
In the scheme, the TMB substrate color development reagent comprises a TMB substrate solution and hydrogen peroxide which are separately packaged.
In the above scheme, the reaction termination solution is H2SO4At a concentration of 2mol/L。
An ELISA kit for detecting gastric cancer tumor markers is applied to gastric cancer detection, tumor malignancy evaluation and prognosis of a subject.
In the scheme, the application of the kit comprises the following steps:
(1) coating the ELISA plate with rabbit anti-ITGB 6 serum antibody, washing the ELISA plate with washing liquid for 2-5 times, sealing with sealing liquid at 200 ul/hole, and operating at 37 deg.C; washing the ELISA plate for 2-5 times with washing liquid;
(2) taking a blood sample of a subject, diluting the blood sample by 1000 times with purified water, adding a diluent into a coated enzyme label plate, incubating the enzyme label plate at 37 ℃ for 2-4h, and washing the enzyme label plate for 2-5 times with a washing solution;
(3) adding an enzyme-labeled goat anti-ITGB 6 serum antibody solution, 100 ul/hole, and incubating at 37 ℃ for 1-2 h; washing the ELISA plate with a washing solution for 2-5 times, and drying;
(4) adding a TMB substrate color reagent into an enzyme label plate, reacting for 3-8min at 100 ul/hole; stopping the reaction by using a reaction stopping solution, and observing the color of each hole;
(5) and (3) judging: if the basic color is colorless, the judgment is negative, and if the color is yellow, the judgment is positive; by standard curve determination and X-tile verification, ITGB6>0.5ng/ml is defined as high expression.
By the technical scheme, the ELISA kit for detecting the gastric cancer tumor marker provided by the invention has the following beneficial effects:
the novel serum tumor marker integrin beta 6(ITGB6) adopted by the invention is easy to obtain, has high specificity to gastric cancer, has important significance for early discovery and early treatment of gastric cancer, has obvious screening effect on gastric cancer by combining CEA and improves specificity. In addition, the ITGB6 can indicate the malignancy degree of the gastric cancer, can effectively predict the prognosis of a subject, and can be combined with imaging and pathology in clinic to improve the evaluation efficiency and accuracy of the malignancy degree of the gastric cancer.
The invention firstly relates the serum expression level of ITGB6 with the occurrence and development of gastric cancer, judges whether a subject has the gastric cancer by detecting the expression level of ITGB6 in the blood of the subject and combining with the CEA level measurement, and predicts the gastric cancer malignancy degree and prognosis of the subject, thereby guiding a clinician to provide a treatment scheme for the subject. Compared with the traditional detection and treatment means, the method has the advantages of sensitivity, specificity and non-invasiveness, can complete detection only by collecting blood of a subject, does not need biopsy, can relieve the pain of the subject and reduces the medical cost.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1A is a comparison of serum ITGB6 levels in patients with gastric cancer compared to normal;
FIG. 1B is a comparison of the ITGB6 levels in serum for patients with gastric carcinoma who have lymph node metastasis;
FIG. 1C is a comparison of serum ITGB6 levels for each N stage of gastric cancer patients;
FIG. 1D is a comparison of serum ITGB6 levels at various stages in gastric cancer patients;
FIG. 2 is a survival analysis of K-M of a patient with high-low expression gastric cancer with serum ITGB 6;
FIG. 3 is ROC curve for comparing survival time of patients with serum ITGB6 high-low expression gastric cancer;
FIG. 4 is the survival analysis of K-M of gastric cancer patients with high expression of both serum ITGB6 and CEA and single-index high expression of ITGB6 or CEA/ITGB 6 and low expression of CEA;
FIG. 5 is ROC curve comparing the survival time of gastric cancer patients with high expression of both serum ITGB6 and CEA and high expression of ITGB6 or single-index high expression of CEA/ITGB 6 and low expression of CEA.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
The invention provides application of ITGB6 in detection of gastric cancer tumor markers.
Firstly, constructing recombinant escherichia coli expressing ITGB6, expressing and purifying ITGB6, immunizing rabbits and sheep with the purified protein respectively, separating to obtain rabbit anti-ITGB 6 serum antibody and sheep anti-ITGB 6 serum antibody, and marking the sheep anti-ITGB 6 serum antibody to generate HRP.
Then, preparing an ELISA kit for detecting gastric cancer tumor markers, wherein the kit comprises an ELISA plate, a rabbit anti-ITGB 6 serum antibody, an enzyme-labeled goat anti-ITGB 6 serum antibody, a washing solution, a PBST blocking solution, a TMB substrate chromogenic reagent and a reaction stop solution.
Wherein, the concentrations of the rabbit anti-ITGB 6 serum antibody and the enzyme-labeled goat anti-ITGB 6 serum antibody are both 2 ug/ml; the washing solution is PBS containing Tween20 with mass concentration of 0.05%, and the pH value is 7.5; the PBST blocking solution contains BSA with the mass concentration of 3%; the enzyme is HRP; the TMB substrate color developing reagent comprises a TMB substrate solution and hydrogen peroxide which are separately packaged; the reaction terminating solution is H2SO4The concentration is 2 mol/L.
Finally, the application of the ELISA kit for detecting the gastric cancer tumor marker in gastric cancer detection, tumor malignancy evaluation and prognosis of a subject is provided.
The specific detection method comprises the following steps:
(1) coating the ELISA plate with rabbit anti-ITGB 6 serum antibody, washing the ELISA plate with washing liquid for 2-5 times, sealing with sealing liquid at 200 ul/hole, and operating at 37 deg.C; washing the ELISA plate for 2-5 times with washing liquid;
(2) taking a blood sample of a subject, diluting the blood sample by 1000 times with purified water, adding a diluent into a coated enzyme label plate, incubating the enzyme label plate at 37 ℃ for 2-4h, and washing the enzyme label plate for 2-5 times with a washing solution;
(3) adding an enzyme-labeled goat anti-ITGB 6 serum antibody solution, 100 ul/hole, and incubating at 37 ℃ for 1-2 h; washing the ELISA plate with a washing solution for 2-5 times, and patting to dry (namely putting absorbent paper on a laboratory bench, reversely buckling the ELISA plate on the absorbent paper, and patting to dry with a large force);
(4) adding a TMB substrate chromogenic reagent (after mixing a TMB substrate solution and hydrogen peroxide according to the volume ratio of 1: 1) into an enzyme label plate, reacting for 3-8min at 100 ul/hole; stopping the reaction by using a reaction stopping solution, and observing the color of each hole;
(5) and (3) judging: if the basic color is colorless, the judgment is negative, and if the color is yellow, the judgment is positive; by standard curve determination and X-tile verification, ITGB6>0.5ng/ml is defined as high expression.
The expression of integrin beta 6(ITGB6) in the serum of 135 gastric cancer subjects was investigated by the above-described detection method. The positive rate of expression of ITGB6 was found to be 87/135, and compared with 32 healthy volunteers, the expression of ITGB6 in the serum of gastric cancer subjects was significantly increased (shown in FIG. 1A), the degree of increase was closely related to the lymph node metastasis of the subjects (shown in FIG. 1B), and the expression was gradually increased with the increase of N stages and pathological grading (shown in FIG. 1C and FIG. 1D).
Taking 0.5ng/ml as cut-off value, namely taking serum ITGB6 concentration of 0.5ng/ml as high-low expression cut-off point, and taking ITGB6>0.5ng/ml as high expression, otherwise, taking low expression. As shown in fig. 2, the abscissa represents the survival time of the patient, and the ordinate represents the survival rate of the patient, and it can be seen that the patient with low-expression gastric cancer ITGB6 survives better than the patient with high-expression gastric cancer (P ═ 0.00025). Therefore, subjects with high ITGB6 expression had poor prognosis-the AUC for assessing a prediction of poor prognosis in subjects was 0.685 (shown in fig. 3).
As shown in fig. 4, DN, ITGB6 were simultaneously under-expressed with CEA; SP, ITGB6 or CEA low expression; DP, ITGB6 are highly expressed simultaneously with CEA. The abscissa represents the survival time of the patient, the ordinate represents the survival rate of the patient, and DN or SP gastric cancer patient can be seen to have better survival than DP patient (P < 0.0001). Therefore, combining serum high-expression ITGB6 and high-expression CEA can significantly assess the subject prognosis, with an AUC of 0.756 (shown in fig. 5).
The results prove that the serum ITGB6 can be used as a potential gastric cancer serum tumor marker for evaluating the tumor malignancy degree of a subject and the prognosis of the subject, and can increase the specificity of a traditional tumor marker CEA.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
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Application publication date: 20211207 |