CN113030457B - Use of PCNA autoantibodies in cancer prediction, diagnosis and prognosis evaluation - Google Patents
Use of PCNA autoantibodies in cancer prediction, diagnosis and prognosis evaluation Download PDFInfo
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Abstract
The invention provides application of a PCNA autoantibody serving as a marker in preparation of a product for cancer prediction, diagnosis or prognosis evaluation, wherein the PCNA autoantibody is an IgG antibody, and the cancer is nasopharyngeal carcinoma. The detection of the invention by an ELISA method shows that the concentration of PCNA IgG in the blood of a nasopharyngeal carcinoma patient is 1.72 times that of a healthy person, and the difference has obvious statistical significance.
Description
Technical Field
The invention relates to the field of biomedical detection, in particular to application of a PCNA autoantibody in cancer prediction, diagnosis and prognosis evaluation, and particularly relates to application of a PCNA autoantibody IgG in nasopharyngeal carcinoma prediction, diagnosis and prognosis evaluation.
Background
Nasopharyngeal carcinoma is a malignant tumor with low differentiation and high metastasis, because the nasopharynx is special in position, hidden in the middle of the skull, and has a plurality of important organs beside the nasopharynx, which are difficult to be excised by operation, the treatment means is single at present, and radiotherapy and chemotherapy are mainly used, so that patients can bear serious psychological pressure, and the life quality of the patients is influenced. There is a growing body of evidence in recent years that autoantibodies have important value for the detection of nasopharyngeal carcinoma, but only a few autoantibodies have been discovered and studied due to limitations in detection analysis techniques. Because the pathogenesis is still unclear, the continuous discovery of the low-invasiveness specific biomarker molecules which can be used for the early screening, the curative effect prediction and the prognosis judgment of the nasopharyngeal darcinoma can help to improve the curative effect and the survival rate of the nasopharyngeal darcinoma patients.
Autoantibodies are antibodies directed against tissues, organs, cells and cellular components of the body and are important weapons of the humoral immune system of the body to defend against pathogenic bacterial invasion and to maintain the balance of growth of the body. The research result shows that the human immune system can sensitively detect the tumor antigen expressed at a low level, and the level of the generated autoantibody is closely related to the development of the tumor, which shows that the tumor autoantibody can be used as a biomarker molecule for early detection, typing, prognosis and prediction of treatment effect of the tumor, and the like.
PCNA is a proliferating cell nuclear antigen, is found in serum of systemic lupus erythematosus patients in 1978, only exists in normal proliferating cells and tumor cells, and is found to have close relationship with cell DNA synthesis and play an important role in the initiation of cell proliferation.
Patent CN105527435A discloses a protein chip, which comprises 6 lung cancer self-antigen proteins of P53 antigen fragment, SOX2 antigen fragment, COPB1 antigen fragment, EFHD2 antigen fragment, EIF4G3 antigen fragment, and PCNA antigen fragment, and can be used for lung cancer diagnosis.
Patent CN1955737A discloses a protein chip for screening and diagnosing human esophageal squamous carcinoma precancerous lesions, which comprises at least one of 16 polypeptides or antigenic fragments thereof, wherein one is a PCNA polypeptide.
In addition, the non-patent literature, "the expression and clinical significance of SHH and PCNA proteins in a Hedgehog signal pathway (J.Tu.Okagaku (1): 17-20)" refers to the detection of S HH and PCNA in nasopharyngeal carcinoma tissues, which is beneficial to judging the infiltration and growth degree and the risk of metastasis of the primary focus of nasopharyngeal carcinoma and provides reference for judging the prognosis of nasopharyngeal carcinoma.
Non-patent literature, "study on CT images spreading deeply around nasopharyngeal carcinoma and correlation between the images and PCNA expression (large thesis in river-south university, 2001)" studies on the relationship between clinical CT and Proliferating Cell Nuclear Antigen (PCNA) of nasopharyngeal carcinoma, and suggests that the PCNA is highly expressed and the survival rate of non-tumor cells is low 3 years after radiotherapy because the soft tissue in the stem before radiotherapy is thickened and dense; while NPC affects the posterior nostril and nasal cavity, PCNA is mostly high-expressed and is related to the strong proliferation activity of tumor, which leads to the easy development of tumor.
Therefore, although the expression condition of PCNA in nasopharyngeal carcinoma tissues is reported, PCNA autoantibodies in serum are not reported, and the invention surprisingly discovers that the anti-PCNA autoantibodies, particularly IgG antibodies in human serum have obvious correlation with whether nasopharyngeal carcinoma exists or not and can be used as a serum marker of the nasopharyngeal carcinoma.
Disclosure of Invention
An object of the present invention is to provide the use of PCNA autoantibodies as markers for the preparation of products for the prediction, diagnosis or prognosis of cancer.
Further, the PCNA autoantibody is an IgG antibody.
Further, the cancer is selected from nasopharyngeal carcinoma, digestive system cancer (such as gastric cancer, esophageal cancer, colorectal cancer) or lung cancer, preferably, the cancer is nasopharyngeal carcinoma.
Further, the PCNA autoantibody is a PCNA autoantibody in blood, serum or plasma, and preferably, the PCNA autoantibody is a PCNA autoantibody in serum.
Further, the cancer prediction, diagnosis or prognosis evaluation comprises: detecting a PCNA autoantibody of a sample to be detected; and comparing the expression level of the PCNA autoantibody of the sample to be detected with that of the healthy human.
Further, the method for detecting the PCNA autoantibody is one or a combination of two or more selected from ELISA (enzyme-linked immunosorbent assay), immunoblotting, indirect immunofluorescence, enzyme immunospot assay, and immunofluorescence, and preferably, the method for detecting the PCNA autoantibody is ELISA.
Further, the ELISA method comprises adding a sample to be tested to a solid support and detecting, wherein the solid support is coated with PCNA (proliferating cell nuclear antigen) antigen or a fragment thereof.
Preferably, the coating includes direct coating and indirect coating, wherein the direct coating is to directly coat the antigen or the fragment thereof on the solid-phase carrier, and the indirect coating is to indirectly coat the antigen or the fragment thereof on the solid-phase carrier through a specific reaction between biotin and streptavidin.
Preferably, the solid phase carrier comprises an enzyme-labeled microporous plate, microparticles, microspheres, an affinity membrane, a liquid phase chip, a glass slide, a test strip and plastic balls.
In one embodiment of the invention, the solid support is an enzyme-labeled microplate.
Preferably, the ELISA detection method comprises an ultraviolet-visible light color development method, a chemiluminescence method or a fluorescence method.
Preferably, the ELISA method may further comprise adding any reagent suitable for ELISA method detection, such as one or more of a labeled secondary antibody, a dilution buffer, a coating buffer, a blocking buffer, a washing buffer, a chromogenic substrate, or a stop buffer.
In one embodiment of the invention, the labeled secondary antibody is horseradish peroxidase-labeled secondary IgG.
In one embodiment of the invention, the dilution buffer is PBST buffer.
In one embodiment of the present invention, the coating buffer is a carbonate buffer.
Specifically, the coating buffer is 0.05M carbonate buffer at pH 9.6.
In one embodiment of the invention, the blocking buffer is PBST buffer.
In one embodiment of the invention, the wash buffer is PBST.
In one embodiment of the invention, the chromogenic substrate is tetramethylbenzidine.
In one embodiment of the present invention, the stop solution is H 2 SO 4 。
Further, comparing the expression level of the PCNA autoantibody of the sample to be detected with that of the healthy human PCNA comprises comparing the concentration of the PCNA autoantibody of the sample to be detected with that of the healthy human PCNA and/or calculating the ratio of the expression level of the PCNA autoantibody of the sample to be detected with that of the healthy human PCNA.
In one embodiment of the present invention, when the ratio of the concentration of PCNA autoantibody in the test sample to that in the healthy human is higher than 1.72, it indicates that the test sample is derived from a nasopharyngeal carcinoma patient.
In a second aspect, the present invention provides a tumor marker, which is PCNA autoantibody IgG.
Further, the tumor marker is a nasopharyngeal carcinoma tumor marker.
The third aspect of the invention provides an application of a reagent or a kit for detecting PCNA autoantibody in preparing products for cancer prediction, diagnosis or prognosis evaluation.
Further, the PCNA autoantibody is an IgG antibody.
Further, the cancer is selected from nasopharyngeal carcinoma, cancer of digestive system (such as gastric cancer, esophageal cancer, colorectal cancer, etc.), lung cancer, etc., preferably, the cancer is nasopharyngeal carcinoma.
Further, the PCNA autoantibody is a PCNA autoantibody in blood, serum or plasma, and preferably, the PCNA autoantibody is a PCNA autoantibody in serum.
The reagent or kit for detecting the PCNA autoantibody is selected from one or a combination of more than two of an ELISA (enzyme-linked immunosorbent assay) detection reagent or kit, an immunoblotting detection reagent or kit, an indirect immunofluorescence assay detection reagent or kit, an enzyme-linked immunospot assay detection reagent or kit or an immunofluorescence assay detection reagent or kit, and preferably, the reagent or kit for detecting the PCNA autoantibody is the ELISA detection reagent or kit.
Furthermore, the ELISA detection reagent or kit comprises a solid phase carrier, wherein the solid phase carrier is coated with PCNA (proliferating cell nuclear antigen) antigen or a fragment thereof.
Preferably, the coating includes direct coating and indirect coating, wherein the direct coating is to directly coat the antigen or the fragment thereof on the solid-phase carrier, and the indirect coating is to indirectly coat the antigen or the fragment thereof on the solid-phase carrier through a specific reaction between biotin and streptavidin.
Preferably, the solid phase carrier comprises an enzyme-labeled microporous plate, microparticles, microspheres, an affinity membrane, a liquid phase chip, a glass slide, a test strip and plastic balls.
In one embodiment of the present invention, the solid support is an enzyme-labeled microplate.
Preferably, the reagent or kit for ELISA detection further comprises any reagent suitable for ELISA detection, such as one or more of a labeled secondary antibody, a dilution buffer, a coating buffer, a blocking buffer, a washing buffer, a chromogenic substrate or a stop buffer.
In one embodiment of the invention, the labeled secondary antibody is horseradish peroxidase-labeled secondary IgG.
In one embodiment of the invention, the dilution buffer is PBST buffer.
In one embodiment of the invention, the coating buffer is a carbonate buffer.
Specifically, the coating buffer is 0.05M carbonate buffer at pH 9.6.
In one embodiment of the invention, the blocking buffer is PBST buffer.
In one embodiment of the invention, the wash buffer is PBST.
In one embodiment of the invention, the chromogenic substrate is tetramethylbenzidine.
In one embodiment of the present invention, the stop solution is H 2 SO 4 。
In a fourth aspect, the present invention provides a method for building a model for cancer prediction, diagnosis or prognosis evaluation, the method comprising: detecting PCNA autoantibodies in blood, serum or plasma of healthy humans and cancer patients; comparing the expression levels of PCNA autoantibodies in healthy humans and cancer patients; a threshold value is determined.
A fifth aspect of the invention provides a method of cancer prediction, diagnosis or prognosis evaluation, the method comprising: detecting a PCNA autoantibody of a sample to be detected; and (3) comparing the expression level of the PCNA autoantibody of the sample to be detected with that of a healthy person, or comparing the expression level of the PCNA autoantibody of the sample to be detected with a threshold value to obtain a result.
Further, the PCNA autoantibody is an IgG antibody.
Further, the cancer is selected from nasopharyngeal carcinoma, cancer of digestive system (such as gastric cancer, esophageal cancer, colorectal cancer, etc.), lung cancer, etc., preferably, the cancer is nasopharyngeal carcinoma.
Further, the PCNA autoantibody is a PCNA autoantibody in blood, serum or plasma, and preferably, the PCNA autoantibody is a PCNA autoantibody in serum.
In one embodiment of the invention, the threshold is a ratio of the concentration of PCNA autoantibodies in nasopharyngeal carcinoma patients to that in healthy humans of greater than 1.72.
The invention surprisingly discovers that the PCNA autoantibody IgG is obviously related to whether the nasopharyngeal darcinoma exists, the expression of the PCNA autoantibody IgG in the blood of the nasopharyngeal darcinoma can be detected to be used as a biomarker for detecting the occurrence and the development of the nasopharyngeal darcinoma, particularly, the concentration of the PCNA autoantibody IgG in the blood of the nasopharyngeal darcinoma is 1.72 times of that of a healthy person through the detection of a double-antibody sandwich enzyme-linked immunosorbent assay, and the difference has obvious statistical significance.
The TMB refers to tetramethyl benzidine.
The PSBT of the invention refers to phosphate Tween buffer solution.
The prognostic evaluation described herein refers to prognostic judgment of the course and/or outcome of a cancer patient.
The prediction of the invention refers to the evaluation of the cancer incidence of the tested person.
The diagnosis in the invention refers to the judgment of whether a person to be tested is ill or not.
Drawings
FIG. 1: ELISA method detects the content comparison of PCNA autoantibody IgG in the serum of healthy people and nasopharyngeal carcinoma patients.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Establishment of PCNA autoantibody IgG expression difference verification and diagnosis model in serum of healthy person and nasopharyngeal carcinoma patient
1. Sample collection
Serum samples were collected at the center for tumor prevention and treatment at Zhongshan university, the mean age of nasopharyngeal carcinoma patients was 45.35 years, the male-female ratio was 2, and informed consent was obtained from all patients, and the diagnosis of nasopharyngeal carcinoma was confirmed by nasopharyngoscope exploration.
Meanwhile, 30 healthy volunteers (average age of 43 years, male-female ratio of 1.
2. The detection method comprises the following steps:
enzyme-linked immunosorbent assay (ELISA) was performed to assess the concentration of PCNA IgG in serum, as follows:
(1) PCNA antigen (Yinqiao 12131-HO 7B) was coated in 96-well plates at 100ng per well overnight at 4 ℃ (50 ul coating solution per well, the coating solution was 0.05M carbonate buffer at pH 9.6);
(2) After overnight, blocking with 5% skim milk powder 50 ul/well for 1 hour at 37 ℃ during which time serum samples were diluted in dilution buffer at 1: diluting at a ratio of 300 for standby;
(3) Then the diluted sample 50u l/hole added to 96 well plate 37 degrees C continued temperature 1 hours;
(4) Adding horseradish peroxidase-labeled IgG (China fir Jinqiao ZB-2304, concentration 1: 8000) and incubating at 37 deg.C for 1 hr, adding 50ul of TMB (Kangshiji CW 0050S) for color development for 30min after the lapse of time, adding 25ul of ELISA stop solution (Beijing Solibao C1058) at the same sample adding speed to stop the reaction,
(5) The signal was determined by measuring the absorbance at 450nm using a microplate reader.
3. Statistical analysis
Differences in the variables between groups were compared using the Mann-Whitney U Test, and P <0.05 was considered statistically significant.
4. Results of the experiment
Serum samples from nasopharyngeal carcinoma patients and healthy individuals were analyzed for PCNA IgG antibody expression levels using the Elisa method, expressed as absorbance at 450 nm.
Results referring to fig. 1, serum PCNA IgG absorbance values (0.1153, n = 60) were significantly higher in nasopharyngeal carcinoma patients than in healthy individuals (0.0657, n =30, p = 0.00092).
Example 2
Application of PCNA autoantibody IgG serving as nasopharyngeal carcinoma diagnosis marker
Taking serum of a person to be tested; PCNA antigen (12131-HO 7B, yiqiao Shenzhou) was coated in 96-well plates at 100ng per well overnight at 4 ℃ (50 ul coating solution per well, the coating solution was mainly composed of 0.05M carbonate buffer solution with pH of 9.6); after overnight, the test patient sera were blocked with 5% skim milk powder at 50 ul/well for 1 hour at 37 ℃ during which time the test patient sera were diluted in dilution buffer at 1: diluting at a ratio of 300 to prepare for later use; adding 50. Mu.l/well of the diluted sample to a 96-well plate, and continuing incubation for 1 hour at 37 ℃; adding horseradish peroxidase-labeled IgG (China fir Jinqiao ZB-2304, concentration 1; the ratio of the absorbance values of the 3-time averages to the absorbance value of the healthy individual obtained in example 1 (0.0657) was compared with a threshold value of 1.72 to obtain a detection result.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (11)
- Use of a PCNA autoantibody as a marker in the manufacture of a product for the prediction, diagnosis or prognostic assessment of cancer, said cancer being nasopharyngeal carcinoma, said prediction, diagnosis or prognostic assessment of cancer comprising: detecting the PCNA autoantibody of the sample to be detected, comparing the expression levels of the PCNA autoantibody of the sample to be detected and the healthy human, and when the concentration ratio of the PCNA autoantibody of the sample to be detected and the healthy human is higher than 1.72, indicating that the sample to be detected is from the nasopharyngeal carcinoma patient.
- 2. The use of claim 1, wherein the PCNA autoantibody is an IgG antibody.
- 3. The use of claim 2, wherein the PCNA autoantibody is a PCNA autoantibody in blood, serum or plasma.
- 4. The use of claim 3, wherein the PCNA autoantibody is a serum PCNA autoantibody.
- 5. The use according to any one of claims 1 to 4, wherein the method for detecting PCNA autoantibodies in a test sample is selected from ELISA, immunoblotting, indirect immunofluorescence, enzyme immuno-spotting or immuno-luminescence.
- 6. The use of claim 5, wherein the method for detecting PCNA autoantibodies in a test sample is an ELISA method.
- 7. The use of claim 5 or 6, wherein the ELISA comprises adding the sample to be tested to a solid support and detecting the sample, wherein the solid support is coated with the PCNA antigen or fragment thereof.
- 8. The application of a reagent or a kit for detecting PCNA autoantibody in preparing a product for cancer prediction, diagnosis or prognosis evaluation, wherein the cancer is nasopharyngeal carcinoma, the expression levels of the PCNA autoantibody of a sample to be detected and a healthy person are compared, and when the ratio of the concentration of the PCNA autoantibody of the sample to be detected to the concentration of the PCNA autoantibody of the healthy person is higher than 1.72, the sample to be detected is from a nasopharyngeal carcinoma patient.
- 9. The use of claim 8, wherein the reagent or kit is an ELISA detection reagent or kit comprising a solid support coated with PCNA antigen or a fragment thereof.
- 10. The use of claim 9, wherein the ELISA detection reagent or kit further comprises one or more of a labeled secondary antibody, a dilution buffer, a coating buffer, a blocking buffer, a washing buffer, a chromogenic substrate, or a stop buffer.
- 11. A method of modeling a cancer prediction, diagnosis or prognosis evaluation, said method comprising: detecting PCNA autoantibodies in blood, serum or plasma of healthy humans and cancer patients; comparing the PCNA autoantibody expression levels of healthy and cancer patients; determining a threshold value, wherein the cancer is nasopharyngeal cancer and the threshold value is that the ratio of the nasopharyngeal cancer patient to the healthy human PCNA autoantibody concentration is higher than 1.72.
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| CN1648666A (en) * | 2004-11-12 | 2005-08-03 | 中山大学肿瘤防治中心 | Nasopharyngeal carcinoma serological diagnostic kit |
| US20080254481A1 (en) * | 2006-11-13 | 2008-10-16 | Invitrogen Corporation | Methods and kits for detecting prostate cancer biomarkers |
| CN103333226B (en) * | 2013-05-30 | 2014-12-24 | 北京正旦国际科技有限责任公司 | Polypeptide marker SPG04 for nasopharyngeal darcinoma radiosensitivity, and ELISA kit prepared by same |
| IT201700117860A1 (en) * | 2017-10-18 | 2019-04-18 | Molipharma Srl | TEST AND KIT FOR DIAGNOSIS OF OVARIAN CARCINOMA |
| CN111308090B (en) * | 2020-02-27 | 2020-11-06 | 郑州大学第一附属医院 | Esophageal cancer multi-joint rapid detection ELISA kit |
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