CN116735879B - Application of polypeptide in diagnosis of lung cancer - Google Patents
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- CN116735879B CN116735879B CN202310663207.6A CN202310663207A CN116735879B CN 116735879 B CN116735879 B CN 116735879B CN 202310663207 A CN202310663207 A CN 202310663207A CN 116735879 B CN116735879 B CN 116735879B
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Abstract
The invention relates to the technical field of in-vitro diagnosis and protein detection, and particularly provides application of a polypeptide in diagnosing lung cancer, wherein an autoantibody combined with the polypeptide can be used as a diagnosis marker of lung cancer, and experiments prove that the specificity and the sensitivity are very high.
Description
Technical Field
The invention relates to the technical fields of in-vitro diagnosis and protein detection, in particular to application of polypeptide in diagnosing lung cancer or preparing a product for diagnosing lung cancer.
Background
Prognosis of cancer and stage of cancer development are closely related. For example, non-small cell lung cancer can provide a good prognosis if found early on by surgical excision. The 5-year survival rate of small local tumors (stage I) is 70-90%. However, most patients (about 75%) have advanced at the time of diagnosis (stage III/IV), and despite significant progress in oncology management of advanced lung cancer in recent years, survival rates remain low. Small cell lung cancer is more aggressive than non-small cell lung cancer, with worse prognosis and overall 5-year survival of about 5%. Over 90% of patients have locally advanced or distant metastasis (stage III/IV), and the window of curative treatment is very narrow. In rare cases, however, the patient is identified as a stage I disease and surgery is still beneficial. One study reported that the 5-year survival rate of small cell lung cancer resected alone was 40% with an adjuvant chemotherapy or radiotherapy of 52%. Thus, diagnosis at an early stage of cancer occurrence, which allows curative treatment, is the most effective way to increase survival. Currently, low Dose Computed Tomography (LDCT) is the preferred mode of lung cancer screening. A foreign study shows that the LDCT screening has better early screening potential for lung cancer and can reduce the death rate of high risk groups of lung cancer. However, LDCT has a high false positive rate, which may lead to excessive diagnosis and invasive diagnosis to some extent, and also has a problem that radiation may cause potential health hazard to human body. Thus, the discovery of non-invasive, non-invasive serological markers for early cancer diagnosis would greatly facilitate the prevention and early treatment of cancer. The use of conventional standard screening following initial biomarker-based screening or the combination of biomarker testing with conventional standard screening methods would be a better diagnostic tool for cancer.
Disclosure of Invention
In a first aspect, the present invention provides the use of a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or 2.
In a second aspect of the invention there is provided the use of an autoantibody binding to a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or 2.
Preferably, the length of the polypeptide is 15-30aa, preferably 15-20aa. For example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30aa.
Preferably, the polypeptide may comprise a modification or a label, such as biotin or the like.
Preferably, the diagnosis or auxiliary diagnosis or prognosis of lung cancer comprises detection of autoantibodies using polypeptides. Preferably, the presence or amount of autoantibodies is detected. Preferably, autoantibodies in serum or plasma are detected.
Preferably, the autoantibody is lgG and/or lgA.
Preferably, the subject for diagnosis or diagnosis-aid or prognostic evaluation may be a human or non-human animal.
Preferably, the diagnosis or auxiliary diagnosis or prognosis of lung cancer comprises contacting the polypeptide with a sample to be tested, and detecting the amount of autoantibodies bound to the polypeptide.
Specifically, the polypeptide is coated on a solid-phase carrier, then a sample to be detected and a marker (preferably a secondary antibody marked by anti-human IgA cyanine dye (Cy 5) or anti-human IgG cyanine dye (Cy 3)) are added, and the signal molecular intensity of the marker is detected. The detection method of the signal molecule intensity comprises a visible light color development method, a chemiluminescence method and a fluorescence method. For example by a microarray scanner. Wherein the intensity of the signal is positively correlated with the amount of autoantibodies bound to the polypeptide.
Preferably, the sample to be detected is serum or plasma.
Preferably, the product comprises a polypeptide and a solid support;
preferably, the solid phase carrier is selected from one or more than two of enzyme-labeled microwell plates, microparticles, microspheres, affinity membranes, liquid phase chips, glass slides, test strips and plastic balls;
further preferably, the product is a kit or chip.
Preferably, the product further comprises an agent, such as a secondary antibody with a label, a blocking buffer and/or a wash buffer.
Preferably, the lung cancer comprises one or a combination of more than two of lung adenocarcinoma, lung squamous cell carcinoma, large cell lung carcinoma or small cell lung carcinoma.
In a third aspect of the invention, there is provided a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or 2.
Preferably, the length of the polypeptide is 15-30aa, preferably 15-20aa. For example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30aa.
Preferably, the polypeptide is SEQ ID NO:1 or 2.
Preferably, the polypeptide may comprise a modification or a label, such as biotin or the like.
Preferably, the polypeptide may bind to an autoantibody. Further preferred are binding to autoantibodies in lung cancer patients (e.g. serum or plasma).
Preferably, the polypeptides may be used to detect autoantibodies.
Preferably, the polypeptides may be used for diagnosis or aiding in diagnosis or prognosis of lung cancer.
Preferably, the polypeptide or an autoantibody binding to the polypeptide can be used as a marker for lung cancer diagnosis or to aid in diagnosis or prognosis evaluation. For example, lung cancer can be assessed by detecting the presence or amount of autoantibodies that bind the polypeptide. Such as diagnosis, auxiliary diagnosis, prognostic assessment or clinical typing.
The polypeptides may be prepared by methods conventional in the art. Such as chemical synthesis or prokaryotic or eukaryotic expression, etc.
In a fourth aspect of the invention, there is provided a nucleic acid encoding the above polypeptide.
In a fifth aspect of the invention, there is provided a vector comprising the nucleic acid described above.
In a sixth aspect of the invention, there is provided a cell comprising a nucleic acid as described above or a vector as described above.
In a seventh aspect of the invention, there is provided a kit or chip comprising a polypeptide, nucleic acid, vector or cell as described above.
Preferably, the polypeptide comprises a modification or a label, such as biotin.
Preferably, the kit or chip further comprises a solid support.
Preferably, the solid phase carrier is selected from one or more than two of enzyme-labeled microwell plates, microparticles, microspheres, affinity membranes, liquid phase chips, glass slides, test strips and plastic balls.
Preferably, the kit or chip further comprises reagents, such as a secondary antibody with a label, a blocking buffer and/or a wash buffer.
According to an eighth aspect of the present invention, there is provided a method for preparing a kit or chip, the method comprising coating the above-described polypeptide on a solid support.
Preferably, the coating can be a direct coating method or an indirect coating method. The direct coating method is to fix the polypeptide directly on the solid carrier, and the indirect coating method is to couple the polypeptide with irrelevant protein such as Bovine Serum Albumin (BSA) and the like, and indirectly bind to the surface of the solid carrier by means of the adsorption of the conjugate and the solid carrier.
In a ninth aspect of the present invention, there is provided a biomarker for lung cancer, wherein the biomarker for lung cancer is a polypeptide or an autoantibody binding to the polypeptide.
Preferably, the autoantibody is lgG and/or lgA.
Preferably, the autoantibody is derived from serum or plasma.
In a tenth aspect of the invention, there is provided a method of detecting an autoantibody, said method comprising using a polypeptide as described above or a kit or chip as described above.
Preferably, comprising contacting the polypeptide with a sample to be tested, and detecting the amount of autoantibodies bound to the polypeptide.
Specifically, the polypeptide is coated on a solid-phase carrier, then a sample to be detected and a marker (preferably a secondary antibody marked by anti-human IgA cyanine dye (Cy 5) or anti-human IgG cyanine dye (Cy 3)) are added, and the signal molecular intensity of the marker is detected. The detection method of the signal molecule intensity comprises a visible light color development method, a chemiluminescence method and a fluorescence method. For example by a microarray scanner. Wherein the intensity of the signal is positively correlated with the amount of autoantibodies bound to the polypeptide.
Preferably, the sample to be detected is serum or plasma.
In an eleventh aspect of the present invention, there is provided a method for diagnosing lung cancer, the method comprising using the above polypeptide or the above kit or chip.
Preferably, comprising contacting the polypeptide with a sample to be tested, and detecting the amount of autoantibodies bound to the polypeptide.
Specifically, the polypeptide is coated on a solid-phase carrier, then a sample to be detected and a marker (preferably a secondary antibody marked by anti-human IgA cyanine dye (Cy 5) or anti-human IgG cyanine dye (Cy 3)) are added, and the signal molecular intensity of the marker is detected. The detection method of the signal molecule intensity comprises a visible light color development method, a chemiluminescence method and a fluorescence method. For example by a microarray scanner. Wherein the intensity of the signal is positively correlated with the amount of autoantibodies bound to the polypeptide.
Preferably, the sample to be detected is serum or plasma.
"diagnosis" as used herein refers to ascertaining whether a patient has a disease or condition in the past, at the time of diagnosis, or in the future, or to ascertaining the progression or likely progression of a disease in the future. The subject of the diagnosis may be a human or a non-human animal, wherein the non-human animal is preferably a non-human mammal. Further preferably, the non-human mammal may be a wild animal, zoo animal, economic animal, pet animal, laboratory animal, or the like. Preferably, the non-human mammal includes, but is not limited to, a pig, cow, sheep, horse, donkey, fox, raccoon dog, marten, camel, dog, cat, rabbit, mouse (e.g., rat, mouse, guinea pig, hamster, gerbil, dragon cat, squirrel) or monkey, and the like.
The term "diagnosis-aid" as used herein is meant to include the methods of the present application as well as methods or steps conventional in the art.
As used herein, "prognostic evaluation" refers to assessing a patient's response to treatment, as well as the risk of future disease.
The term "comprising" or "including" as used herein is an open reading frame, and when used to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.
Drawings
Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
fig. 1: the expression level of IgG autoantibodies binding to polypeptide P003 in plasma, where SNR is signal to noise ratio, N is healthy, LC is lung cancer patient, EC is esophageal cancer patient, GC is gastric cancer patient, and DC is other 7 cancer patients.
Fig. 2: expression level of lgA-type autoantibodies binding to polypeptide P003 in plasma, wherein SNR is signal to noise ratio, N is healthy human, LC is lung cancer patient, EC is esophageal cancer patient, GC is gastric cancer patient, DC is other 7 cancer patients.
Fig. 3: the expression level of lgG-type autoantibodies binding to polypeptide P004 in plasma, wherein SNR is signal to noise ratio, N is healthy human, LC is lung cancer patient, EC is esophageal cancer patient, GC is gastric cancer patient, and DC is other 7 cancer patients.
Fig. 4: the expression level of the lgA-type autoantibody binding to polypeptide P004 in plasma, wherein SNR is signal to noise ratio, N is healthy human, LC is lung cancer patient, EC is esophageal cancer patient, GC is gastric cancer patient, DC is other 7 cancer patients.
Fig. 5: lung cancer patients and healthy groups of IgA autoantibody ROC curves against polypeptide P003.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The reagent formulations used in the examples are as follows:
phosphate Buffer (PBS): 137mM NaCl, 2.7mM KCl, 10mM Na 2 HPO 4 、2mM KH 2 PO 4 ，pH 7.4。
Wash buffer (PBST): 1 XPBST at pH 7.4.
Blocking buffer: 1 XPBST with 5% BSA.
Example 1
1. The sequences of the screened polypeptide P003 and the control polypeptide P004 are shown in Table 1.
Table 1: polypeptide sequence
| Numbering device | SEQ ID NO：x | P53 polypeptide sequences | Position information |
| P003 | 1 | E-P-P-L-S-Q-E-T-F-S-D-L-W-K-L | 11-25 |
| P004 | 2 | Q-E-T-F-S-D-L-W-K-L-L-P-E-N-N | 16-30 |
2. Clinical samples are shown in Table 2.
Table 2: clinical samples
Table 3: lung adenocarcinoma sample patient condition
Table 4: lung squamous cell carcinoma sample patient condition
Table 5: conditions of large cell lung cancer sample patient
Table 6: sample patient condition of small cell lung cancer
Table 7: normal physical examination sample patient condition
3. Detection step
1) Printing a P53 polypeptide array: diluting the P53 polypeptide in table 1 with PBS, and then repeatedly printing the P53 polypeptide on the surface of a glass slide four times by using an Arrayjet microarray spotter, wherein the prepared P53 polypeptide microarray is stored at-20 ℃ until the microarray is ready for use;
2) And (3) rewarming: taking the P53 polypeptide glass slide out of the refrigerator at the temperature of minus 20 ℃ and balancing to the room temperature;
3) Closing: assembling the P53 polypeptide glass slide into an incubation tray, adding 400 mu L of a blocking buffer, standing for 1h at room temperature, and slightly shaking;
4) Serum sample preparation: serum samples were thawed at 4℃and centrifuged at 14,000Xg for 10min. Serum samples were prepared by adding 4 μl of serum to 400 μl of blocking buffer at a ratio of 1:100;
5) Serum samples were added: removing the blocking buffer, adding 400 mu L of diluted serum, then incubating for 2.5 hours at room temperature, and gently shaking; after incubation is complete, the slides are washed 3 times for 10 minutes with 400 μl PBST;
6) Adding a fluorescent labeling antibody: the secondary antibody was diluted with blocking buffer (working concentration 4. Mu.g/mL). 400 μl of diluted secondary antibody was added, incubated for 1h at room temperature, and gently shaken. The slide was washed 3 times with 400 μl of PBST for 10 minutes each, and twice with 400 μl of distilled water for 2 minutes each;
7) Drying the slide: removing the remaining liquid, evacuating the incubation tray with a vacuum pump or pipette, and then drying the slide;
8) Reading on a machine: all arrays were scanned using a GenePix 4300A microarray scanner at 532nm or 635nm wavelength. Median fluorescence signal intensities were extracted using GenePix Pro7 software and data saved.
4. Test results
The results of the expression levels of autoantibodies binding to the P003 or P004 polypeptides in Table 1 are shown in FIGS. 1 to 4 and Table 8. Specifically, autoantibodies that bind polypeptides have significant statistical differences between lung cancer patients and healthy controls, representing their ability to be specific markers of lung cancer.
Table 8: exemplary significance of expression levels of several polypeptides in different cancers
In addition, the results of the ROC analysis of autoantibodies of binding polypeptides from lung cancer patients and healthy groups are shown in FIG. 5. Specific:
for the lgA-type autoantibody, the AUC of polypeptide P003 was 0.6752. The specificity of the polypeptide P003 was 73.68% and the sensitivity was 61.36% when the Cutoff value was 1.234 as determined by about the Density index.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (6)
1. The use of a polypeptide for the manufacture of a product for diagnosing lung cancer by detecting IgA autoantibodies, wherein said polypeptide is SEQ ID NO:1.
2. the use of claim 1, wherein diagnosing lung cancer comprises detecting the presence or amount of autoantibodies.
3. The use of claim 1, wherein diagnosing lung cancer comprises detecting autoantibodies in serum or plasma.
4. The use of claim 1, wherein the product comprises a polypeptide and a solid support.
5. The method of claim 4, wherein the solid support is selected from the group consisting of an enzyme-labeled microplate, a microparticle, an affinity membrane, a liquid-phase chip, a slide glass, a test strip, and a plastic pellet.
6. The use according to claim 1, wherein the product is a kit or chip.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110376378A (en) * | 2019-07-05 | 2019-10-25 | 中国医学科学院肿瘤医院 | It can be used for the markers in detecting model of pulmonary cancer diagnosis |
| CN111273012A (en) * | 2020-03-04 | 2020-06-12 | 北京三品医疗科技有限公司 | Method for combined detection of serum autoantibodies |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110376378A (en) * | 2019-07-05 | 2019-10-25 | 中国医学科学院肿瘤医院 | It can be used for the markers in detecting model of pulmonary cancer diagnosis |
| CN111273012A (en) * | 2020-03-04 | 2020-06-12 | 北京三品医疗科技有限公司 | Method for combined detection of serum autoantibodies |
Non-Patent Citations (5)
| Title |
|---|
| Analysis of p53 Antibodies in Patients with Various Cancers Define B-Cell Epitopes of Human p53: Distribution on Primary Structure and Exposure on Protein Surface.;R.Lubin, et al.;《Cancer Research》;19931215;摘要，第5873页左栏第1-4段，第5873页右栏第2-4段，图2，图3 * |
| Jack A.Roth等著.《肺癌》.世界图书出版公司,2011,第156、157页. * |
| R.Lubin, et al..Analysis of p53 Antibodies in Patients with Various Cancers Define B-Cell Epitopes of Human p53: Distribution on Primary Structure and Exposure on Protein Surface..《Cancer Research》.1993,摘要，第5873页左栏第1-4段，第5873页右栏第2-4段，图2，图3. * |
| 唐金海主编.《医疗机构医务人员三基指南 肿瘤科》.东南大学出版社,2007,第127-128页. * |
| 张承江主编.《医学数据仓库与数据挖掘》.中国中医药出版社,2008,第104、105页. * |
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