CN117368479A - Biomarker and detection kit for lung adenocarcinoma diagnosis - Google Patents
Biomarker and detection kit for lung adenocarcinoma diagnosis Download PDFInfo
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Abstract
The invention discloses a biomarker for diagnosing lung adenocarcinoma and a detection kit. The biomarker is IgG and IgM autoantibodies of PRTN 3; the kit contains reagents for detecting the aforementioned biomarkers. The invention can effectively distinguish lung adenocarcinoma patients from healthy people and lung adenocarcinoma patients from benign lung nodule patients by detecting the expression levels of the two biomarkers in human blood plasma, and the combination of the two autoantibodies and the tumor marker CEA can improve the differential diagnosis efficiency of the lung adenocarcinoma patients, thereby having important significance for the management and identification of the lung adenocarcinoma patients.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly discloses a biomarker and a detection kit for lung adenocarcinoma diagnosis.
Background
The international cancer research institute (International Agency for Research on Cancer, IARC) of the world health organization showed that the incidence of Lung cancer (Lung cancer, LC) in the world is the second to the malignancy and mortality is the leading factor in cancer death. In China, lung cancer is the first cancer onset and death in China. Non-small cell lung cancer (Non-small cell lung carcinoma, NSCLC) accounts for about 85% of all lung cancer cases, with lung adenocarcinoma (lung adenocarcinoma, LUAD) accounting for about 70% of NSCLC, a type of pathology with highest incidence in lung cancer. Clinical researches show that the early lung adenocarcinoma diagnosed is treated by combining surgery with radiotherapy and chemotherapy, and the five-year survival rate can reach 90 percent. And metastatic lung adenocarcinoma is not treated by surgery, so that the survival rate is greatly reduced. Because of the early metastasis of small adenocarcinomas, more than half of lung adenocarcinoma patients show local or distant metastasis at the time of initial diagnosis, losing optimal treatment opportunity. Therefore, the perfection of the early diagnosis strategy of lung adenocarcinoma is a key for reducing the death rate of lung cancer, and the screening of the lung adenocarcinoma tumor markers with high tissue specificity and sensitivity has important significance for early diagnosis, prognosis judgment and accurate treatment of lung adenocarcinoma.
The existing image examination is a main means for finding and locating tumors, but many imaging technologies have the problems of high cost, dependence on operator skills and potential jurisdiction exposure, and a large number of lung adenocarcinoma patients cannot be diagnosed early, such as Low-dose computed tomography (Low-dose compute tomography, LDCT) widely applied to screening of lung cancer of high-risk groups, which significantly reduces the mortality of lung cancer, but is still possible to suffer from lung cancer of non-high-risk groups, and about 50% of new cases of lung cancer do not meet the screening conditions; and most pulmonary nodule patients cannot obtain important information from routine examination, namely, the benign and malignant patients cannot be effectively identified, and the false positive rate of the patients diagnosed with LC by LDCT is as high as 21.8% -26.6%. Overdiagnosis causes unnecessary harm to both the patient's body and the economy. Thus, there remains a need for further research and development and validation of noninvasive biological markers that are effective for lung adenocarcinoma diagnosis.
Blood tumor related marker detection is the most accepted diagnostic method for patients, protein markers are relatively stable, and the detection method is simple. Biomarkers for biological macromolecules in humans have been studied in a large number, including Circulating Tumor Cells (CTCs), circulating tumor cell DNA (ctDNA), micrornas, and the like in tumor "liquid biopsies," and tumor-associated proteins such as carcinoembryonic antigen (CEA), carcinoantigen 125 (CA 125), neuron-specific enolase (NSE), and the like, which have been routinely detected clinically. Although all the above biomarkers show a certain application prospect, most of the biomarkers are difficult to enter clinical practice. The CTC or ctDNA content in the blood circulation of early cancer patients is extremely tiny, RNA markers require tissue specimens or fresh blood specimens, the tissue specimens are not easy to obtain, the blood specimens frozen for a long time are not suitable for RNA detection, and the traditional tumor markers are generally not raised in early lung cancer patients, so that the detection sensitivity is low, and meanwhile, CA125 and CEA can be raised in various tumors, and the tumor specificity is lacking. Therefore, there is a need to find new minimally invasive blood molecular markers for detection of biomarkers associated with lung cancer diagnosis that can improve lung cancer risk assessment and facilitate early detection in lung cancer patients.
Tumor cells may release abnormal proteins including new antigens due to abnormal gene mutation, protein structure or modification or abnormal expression level, thereby stimulating immune response of the body, and autoantibodies are generated through clonal proliferation of immune B cells. Compared with autoantigens, tumor-associated autoantibodies (Auto antibodies against tumor-associate dantigens, TAAbs) in blood are more stable and can be easily detected after immune reaction amplification, and the TAAbs can stably exist in blood, have high expression level and long half-life period and show higher level in very early cancer stage, and have been reported in research that the TAAbs can detect the existence of the TAAbs in the blood 17-47 months before cancer diagnosis. TAAbs are thus very potential biomarkers for development. However, very few TAAbs are currently studied and there have been few reports of other types of antibody studies focusing on lgG type antibody studies. The study aims at screening and identifying lung cancer antigens of IgG and lgM humoral immunity through plasma samples of lung adenocarcinoma patients and healthy people. Through further verification, the plasma protein marker with better differential diagnosis value or the combination thereof is used as a potential tool for clinically managing and differential diagnosing lung adenocarcinoma patients.
Disclosure of Invention
The invention aims to: the invention aims to solve the technical problem of providing a lung adenocarcinoma marker aiming at the defects of the prior art.
The invention also solves the technical problem of providing an application of an autoantibody serving as a marker in preparing a lung adenocarcinoma diagnosis product.
The invention also solves the technical problem of providing an application of an autoantibody serving as a marker in preparing a product for identifying lung adenocarcinoma and healthy people and/or lung adenocarcinoma and benign lung nodule.
The invention also solves the technical problem of providing a lung adenocarcinoma diagnosis product.
The invention also solves the technical problem of providing a product for identifying lung adenocarcinoma and healthy people and/or lung adenocarcinoma and benign lung nodule.
In order to solve the first technical problem, the invention discloses a biomarker for diagnosing lung adenocarcinoma, wherein the lung adenocarcinoma marker is IgG and IgM type autoantibodies of an anti-tumor related antigen PRTN3 or the combination of the IgG and IgM type autoantibodies of the anti-tumor related antigen PRTN3 and a traditional tumor marker CEA.
Wherein the autoantibody is used as a marker for identifying lung adenocarcinoma and healthy people, and the autoantibody is used as a marker for identifying lung adenocarcinoma and benign lung nodule.
In order to solve the second technical problem, the invention discloses application of an autoantibody serving as a marker in preparing a lung adenocarcinoma diagnosis product, wherein the autoantibody is an IgG (immunoglobulin G) and IgM (immunoglobulin G) type autoantibody of an anti-tumor related antigen PRTN 3.
Wherein the diagnosis of lung adenocarcinoma comprises detecting an autoantibody, preferably an expression level of an autoantibody, preferably by ELISA.
Wherein, the diagnosis of lung adenocarcinoma comprises detection of autoantibodies and detection of a tumor marker CEA, preferably detection of the expression level of autoantibodies and detection of a tumor marker CEA by ELISA.
Wherein the product further comprises a traditional tumor marker CEA.
Wherein the product comprises a kit and a preparation.
Wherein the kit is an ELISA detection kit.
The ELISA detection kit comprises PRTN3 protein, and further comprises any one or more of coating liquid, sealing liquid, plasma sample diluent, enzyme-labeled secondary antibody, antibody diluent, washing liquid, color development liquid, stop solution and quality control plasma.
In order to solve the third technical problem, the invention discloses application of an autoantibody serving as a marker in preparing products for identifying lung adenocarcinoma and healthy people and/or lung adenocarcinoma and benign lung nodule, wherein the autoantibody is an IgG (immunoglobulin G) and IgM (immunoglobulin M) type autoantibody of an anti-tumor related antigen PRTN 3.
Wherein said identifying lung adenocarcinoma with healthy humans or said identifying lung adenocarcinoma with benign lung nodules comprises detecting an autoantibody, preferably an expression level of an autoantibody, preferably by ELISA.
Wherein the identification of lung adenocarcinoma and healthy people or the identification of lung adenocarcinoma and benign lung nodule comprises detection of autoantibodies and detection of systemic tumor markers CEA, preferably detection of the expression levels of autoantibodies and detection of systemic tumor markers CEA by ELISA.
Wherein the product further comprises a traditional tumor marker CEA.
Wherein the product comprises a kit and a preparation.
Wherein the kit is an ELISA detection kit.
The ELISA detection kit comprises PRTN3 protein, and further comprises any one or more of coating liquid, sealing liquid, plasma sample diluent, enzyme-labeled secondary antibody, antibody diluent, washing liquid, color development liquid, stop solution and quality control plasma.
In order to solve the fourth technical problem, the invention discloses a product for diagnosing lung adenocarcinoma, which comprises a reagent for detecting lung adenocarcinoma markers; the lung adenocarcinoma marker comprises IgG and IgM autoantibodies of an anti-tumor associated antigen PRTN 3.
Wherein the diagnosis of lung adenocarcinoma comprises detecting an autoantibody, preferably an expression level of an autoantibody, preferably by ELISA.
Wherein, the diagnosis of lung adenocarcinoma comprises detection of autoantibodies and detection of a tumor marker CEA, preferably detection of the expression level of autoantibodies and detection of a tumor marker CEA by ELISA.
Wherein the product further comprises a traditional tumor marker CEA.
Wherein the product comprises a kit and a preparation.
Wherein the kit is an ELISA detection kit.
The ELISA detection kit comprises PRTN3 protein, and further comprises any one or more of coating liquid, sealing liquid, plasma sample diluent, enzyme-labeled secondary antibody, antibody diluent, washing liquid, color development liquid, stop solution and quality control plasma.
To solve the fifth technical problem, the present invention discloses a product for identifying lung adenocarcinoma and healthy people and/or lung adenocarcinoma and benign lung nodule, comprising a reagent for detecting lung adenocarcinoma markers; the lung adenocarcinoma marker comprises IgG and IgM autoantibodies of an anti-tumor associated antigen PRTN 3.
Wherein said identifying lung adenocarcinoma with healthy humans or said identifying lung adenocarcinoma with benign lung nodules comprises detecting an autoantibody, preferably an expression level of an autoantibody, preferably by ELISA.
Wherein the identification of lung adenocarcinoma and healthy people or the identification of lung adenocarcinoma and benign lung nodule comprises detection of autoantibodies and detection of systemic tumor markers CEA, preferably detection of the expression levels of autoantibodies and detection of systemic tumor markers CEA by ELISA.
Wherein the product further comprises a traditional tumor marker CEA.
Wherein the product comprises a kit and a preparation.
Wherein the kit is an ELISA detection kit.
The ELISA detection kit comprises PRTN3 protein, and further comprises any one or more of coating liquid, sealing liquid, plasma sample diluent, enzyme-labeled secondary antibody, antibody diluent, washing liquid, color development liquid, stop solution and quality control plasma.
The autoantibodies in the invention are autoantibodies in peripheral blood, such as serum and plasma, and are in the specific form of IgG and/or IgM type autoantibodies.
The screening method for identifying the protein markers of lung adenocarcinoma, healthy people and benign lung nodules comprises the following steps: (1) Collecting blood samples of lung adenocarcinoma patients, healthy people and benign lung nodule patients, and separating to obtain plasma samples; (2) Screening plasma protein markers for identifying lung adenocarcinoma patients and healthy people, benign lung nodule patients; the method specifically comprises the following steps: differential plasma protein ELISA validation of lung adenocarcinoma patients and healthy population, benign lung nodule patients; the differential plasma protein ELISA assay comprises: optimal protein coating concentration and secondary antibody optimal dilution concentration of PRTN 3; ELISA detection is carried out on the plasma sample according to the reagent kit; and carrying out statistical analysis on the obtained data.
The beneficial effects are that:
the invention screens and identifies novel plasma biomarkers (IgG and IgM autoantibodies of the anti-tumor related antigen PRTN 3) which can be used for differential diagnosis of lung adenocarcinoma patients and healthy people and benign lung nodule patients through an ELISA. IgG and IgM autoantibodies of an anti-tumor related antigen PRTN3 and a tumor marker CEA are combined to construct a differential diagnosis model of lung adenocarcinoma patients and healthy people and benign lung nodule patients, and the model has good differential diagnosis efficacy for the lung adenocarcinoma patients and healthy people and benign lung nodule patients and provides assistance for better clinical management of the lung adenocarcinoma patients. The detection sample of the kit is blood plasma, so that invasive diagnosis can be avoided, the risk of lung adenocarcinoma can be obtained at an early stage by taking the blood plasma for detection in a minimally invasive mode, the blood quantity is less, the pain of a detected person is less, and the compliance is high; moreover, the operation is simple, the detection result time is short, and the method has wide market prospect and social benefit.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 is a scatter plot of SBI of IgG and IgM autoantibodies against the tumor associated antigen PRTN3 in lung adenocarcinoma, benign lung nodules, and healthy controls in experimental examples.
FIG. 2 is a ROC graph of an IgG and IgM autoantibody against the tumor associated antigen PRTN3 in experimental examples for diagnosing lung adenocarcinoma with benign lung nodules and healthy controls.
FIG. 3 is a ROC graph showing the combined diagnosis of lung adenocarcinoma, benign lung nodules and healthy controls by IgG and IgM autoantibodies against the tumor associated antigen PRTN3 and the traditional tumor marker CEA in experimental examples.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
The basic information of the tumor associated antigen PRTN3 in the invention is as follows: protease3 (PRTN 3) is known as Protease3, PRTN3 is one of the serine proteases secreted by neutrophils and regulates the immune response to participate in the progression of the associated disease. PRTN3 also acts as an inflammatory factor, and processes cell receptor-mediated signaling, thus playing a role in the inflammatory response. There have been few studies on the function of PRTN3 in tumors, and PRTN3 is reported to be an acute myeloid leukemia-associated antigen, a predictor of early metastasis of triple negative breast cancer, and to be closely related to bone metastasis of tumors. The protein sequence number of PRTN3 in NCBI is: np_002768.3.
Examples: ELISA (enzyme-Linked immuno sorbent assay) for detecting plasma expression level of autoantibody of anti-tumor associated antigen PRTN3
1. Experimental samples:
the study included 773 study specimens, including 275 lung adenocarcinoma patients, 223 benign lung nodule patients, and 275 healthy people. All samples were collected in a three-dimensional hospital from 11 months 2019 to 5 months 2022, henan province. All subjects' basic information, pathological stage, and clinical traditional tumor marker levels were consolidated by the intra-hospital system with patient consent, institutional review board, and hospital ethics board approval.
(1) Patient sample and information collection procedure
The CT detects suspicious lung nodules and patients hospitalized with the cause of the lung nodules, collect blood samples of the patients when the patients do not receive any treatment on the first day or the next day of hospitalization (partial lung cancer patients collect blood samples after treatment), number, split charging and register the basic information and compile the blood samples into a blood sample library.
After the diagnosis of the patient, the information of hospitalization number, sex, age and the like of the study object is used for consulting and collecting relevant clinical information in a hospital patient management system, wherein the information comprises smoking history, drinking history, nodule diameter, pathological diagnosis, pathological type, traditional tumor markers (detection values and cut-off values of CEA, CYFRA211 and the like), TNM stage, lymph node metastasis and remote metastasis, immunohistochemical information and the like of the lung adenocarcinoma patient and benign lung nodule patient, and a patient clinical information base is constructed.
(2) Health control sample and information collection flow
The method comprises the steps of collecting physical examination crowd in a certain three-hospital in Henan province, consulting and collecting relevant clinical information in a hospital patient management system through physical examination numbers, sexes, ages and the like of study objects, collecting blood samples, numbering and split charging, registering in a blood sample database, and then compiling into the blood sample database.
(3) Inclusion criteria and exclusion criteria for subjects
Inclusion criteria for lung adenocarcinoma patients included: 1) No chemotherapy, radiation therapy, surgery, and other interventions; 2) Has definite histopathological results, and can be diagnosed into lung adenocarcinoma according to the diagnosis and treatment guidelines of the primary lung cancer; 3) No history of other malignancy; 4) A complete medical history; 5) No autoimmune disease; 6) All are new cases; 7) The patient age was >18 years.
Benign lung nodule patient inclusion criteria include: 1) Has definite histopathological biopsy to confirm benign and malignant, and pathological histology type; 2) No history of malignancy in the lung or elsewhere; 3) All are new disease cases, and are not treated at all; 4) Age >18 years.
Healthy control inclusion criteria included: 1) No history of malignancy in the lung or elsewhere; 2) No autoimmune disease; 3) Age >18 years.
Exclusion criteria: study subjects that did not meet the inclusion criteria described above.
(4) Plasma collection:
5ml of peripheral blood of the collected study object in a fasting state is placed in a blood collection tube without an anticoagulant, kept stand for 1h at room temperature, placed in a centrifuge, and centrifuged at 3000rpm for 10min at 4 ℃. And then sucking out the plasma on the upper layer of the blood collection tube, sub-packaging the plasma into an EP tube with the volume of 1.5ml, marking sample numbers on the top and the side surface of the EP tube, putting the EP tube in a refrigerator with the temperature of-80 ℃ for freezing preservation, and recording the blood collection date and the storage position. Before use, the blood plasma is taken out, thawed and packaged in a refrigerator at 4 ℃ to avoid repeated freezing and thawing of the blood plasma.
The basic information of the specific study object is shown in table 1.
Table 1 subject basic characteristics
2. Experimental materials and reagents:
(1) PRTN3 protein purchased from Wuhan cloud clone technology service Co., ltd;
(2) HRP anti-human IgG mab purchased from wuhan aokobo biotechnology limited;
(3) Anti-human IgM (μ -chain specific) goat antibodies purchased from merck biosome;
(4) 96-well ELISA plates (8 rows by 12 columns);
(5) Coating liquid: contains 0.15% sodium carbonate (Na 2 CO 3 ) And 0.29% sodium bicarbonate (NaHCO) 3 ) Is an aqueous solution of (a);
(6) Sealing liquid: PBST buffer containing 0.2% (v/v) Tween 20 of 2% (v/v) Bovine Serum Albumin (BSA);
(7) Plasma sample dilution: PBST buffer containing 1% (w/v) BSA;
(8) Antibody dilution: PBST buffer containing 1% (w/v) BSA;
(9) Washing liquid: PBST buffer containing 0.2% (v/v) Tween 20;
(10) Color development liquid: the color development liquid consists of color development liquid A and color development liquid B, wherein the color development liquid A is 20 percent of tetramethyl benzidine dihydrochloride aqueous solution, and the color development liquid B is 3.7 percent of Na 2 HPO 4 ·12H 2 O, 0.92% citric acid and 0.75% (v/v) aqueous urea hydrogen peroxide; when in use, the color development liquid A and the color development liquid B are uniformly mixed according to the equal volume of 1:1, and are prepared at present;
(11) Quality control plasma: quality Control (QC) was prepared by mixing 100 normal control blood samples in equal amounts to Quality control blood samples for eliminating errors between individual elisa plates and representing the general level of the population. Quality control sample CV <20% between all ELISA plates made in the same day is considered as data available for subsequent analysis;
(12) Stop solution: 10% sulfuric acid.
3. The experimental method comprises the following steps:
(1) Protein pretreatment and determination of protein concentration: protein concentration was confirmed by SDS-PAGE electrophoresis after diluting the protein to an appropriate concentration using the protein dilutions suggested in the protein specification.
(2) Pre-experiments explored the optimal protein coating concentration of PRTN3 and the dilution concentration of secondary anti IgG, igM: and randomly selecting plasma of 4 lung adenocarcinoma patients and 4 healthy controls for pre-experiment, setting 2 concentration gradients for protein coating concentration and secondary antibody dilution concentration, and selecting an optimal multiple by combining a sample OD value and a standard OD value after the experiment is finished, so that the color development time of the sample and the standard is consistent, and the sample OD value is between the highest value and the lowest value of the standard OD value.
(3) And (3) cloth plate: according to the layout of the 96-well ELISA plate, the sample number, the sample amount, the protein amount, the sample layout and the like are calculated in advance. Calculating the total amount of the reagents required subsequently according to the sample size, and preparing in advance. Blank wells were 1% BSA and the loading was 50. Mu.L/well. The sample layout table is shown in table 2.
TABLE 2 sample layout (first plate for example)
| LUAD1 | LUAD2 | LUAD4 | LUAD5 | LUAD7 | LUAD8 | LUAD11 | LUAD12 | LUAD15 | LUAD16 | LUAD18 | Quality control |
| LUAD19 | LUAD20 | LUAD21 | LUAD22 | LUAD23 | LUAD24 | LUAD25 | LUAD27 | LUAD28 | LUAD29 | LUAD30 | Quality control |
| LUAD36 | LUAD38 | LUAD39 | LUAD40 | LUAD41 | LUAD42 | LUAD44 | LUAD45 | LUAD46 | LUAD47 | LUAD48 | Blank space |
| BPN1 | BPN2 | BPN3 | BPN4 | BPN5 | BPN6 | BPN7 | BPN8 | BPN9 | BPN10 | BPN11 | Blank space |
| BPN12 | BPN13 | BPN14 | BPN15 | BPN16 | BPN17 | BPN18 | BPN19 | BPN20 | BPN21 | BPN22 | Blank space |
| NC1 | NC2 | NC3 | NC4 | NC5 | NC6 | NC7 | NC8 | NC9 | NC10 | NC11 | Blank space |
| NC12 | NC13 | NC14 | NC15 | NC16 | NC17 | NC18 | NC19 | NC20 | NC21 | NC22 | Blank space |
| NC23 | NC24 | NC25 | NC26 | NC27 | NC28 | NC29 | NC30 | NC31 | NC32 | NC33 | Blank space |
(4) Split charging a sample and diluting the sample: after the required experimental samples are selected from the sample library, the samples are slowly melted in a refrigerator at 4 ℃ the day before the experiment. The next day, samples are correspondingly loaded into a 96 Kong Jiande PCR plate according to the layout of the previous plate and the required sample amount, then the samples are added into antibody diluent according to the sample dilution ratio required by experiments in a 96-hole deep well plate according to the corresponding layout, and after the film covering surface is subjected to film sealing operation, the film covering surface is placed in a refrigerator at 4 ℃ and is left for standby.
(5) Protein coating: diluting the protein to the optimal coating concentration (0.125 mug/mL) determined by a pre-experiment by using a coating liquid, adding the prepared PRTN3 protein solution into the 1 st-12 th row reaction holes of the 96-hole ELISA plate after shaking, wherein the sample adding amount is 50 mug/hole, sealing the preservative film to prevent volatilization, and coating at 4 ℃ overnight.
(6) Closing: adding a sealing liquid into each reaction hole of the coated 96-hole ELISA plate, sealing for 2 hours in a water bath at 37 ℃ with the sample adding amount of 100 mu L/hole, removing the sealing liquid, washing for 3 times by using a washing liquid, and beating to dry to obtain the ELISA plate coated with the tumor-associated antigen PRTN 3.
(7) Primary anti (plasma) response: diluting a plasma sample to be detected by using a plasma sample diluent according to the volume ratio of 1:100, and then correspondingly adding the diluted plasma sample into an ELISA plate according to the layout of a plate and the specification of 50 mu L/hole, wherein the sample adding amount is 50 mu L/hole; blank wells also correspond to 50 μl/well of antibody dilution (without any plasma samples) added. The 96-well enzyme label was then incubated in a 37℃water bath for 1h, after which the reaction wells were discarded, washed 5 times with wash solution (300. Mu.L/well) and dried by pipetting.
(8) And (3) incubation of enzyme-labeled secondary antibodies: the secondary antibody reaction dilutes HRP anti-human IgG monoclonal antibody and HRP anti-human IgM monoclonal antibody with an antibody diluent according to the ratio of the optimal dilution concentration of 1:5000 (v/v) determined by a pre-experiment, then the corresponding IgG and IgM secondary antibody diluent is added into each reaction hole of a 96-hole ELISA plate, the sample adding amount is 50 mu l/hole, the reaction holes are incubated for 1h in a 37 ℃ water bath, then the liquid in the reaction holes is discarded, and the reaction holes are washed with a washing liquid (the sample adding amount is 300 mu l/hole) for 5 times and are patted dry.
(9) Color development and termination reaction: uniformly mixing the color development liquid A and the color development liquid B according to the equal volume of 1:1, then rapidly adding the mixed color development liquid into the reaction holes of the 96-hole ELISA plate, wherein the sample adding amount is 50 mu l/hole, observing the color development condition at any time in the color development process at room temperature in a dark manner, and after full color development, adding 25 mu l of stop solution into each reaction hole to terminate the color development reaction; the absorbance (OD) at wavelengths of 450nm and 620nm was then read using a microplate reader, wherein the absorbance value at wavelength 620nm was used as background value, the difference between the absorbance at wavelengths of 450nm and 620nm was used as absorbance value for subsequent analysis, and a blank control Kong Diaoling was used.
4. Data processing
The statistical analysis method of the invention uses SPSS26.0, graphPad Prism 8.0 software to carry out statistical analysis and result visualization on experimental data. Differences in expression levels of anti-PRTN 3 autoantibodies between the different groups were analyzed by the Mann-Whitney U test, and differences in positive rates were analyzed by the chi-square test; the diagnostic value indexes such as AUC, specificity, sensitivity and 95% confidence interval (95% ci) of the anti-PRTN 3 autoantibody were calculated from ROC curves using SBI values corresponding to the maximum mount Index (YI) as cut-off values (cutoff values). All statistical analyses used a two-sided test, and differences were considered statistically significant when P < 0.05.
Analysis of results
(1) ELISA Pre-experiments explored the optimal protein coating concentration of PRTN3 and the optimal dilution concentration of secondary anti-IgG, igM: a small number of samples (4 lung adenocarcinomas and 4 benign pulmonary nodule patient plasma) were pre-experimentally analyzed using a double antibody sandwich ELISA assay, and the optimal protein coating concentration of PRTN3 and optimal dilution concentrations of secondary anti-IgG, igM in this assay were explored. The optimal protein coating concentration of PRTN3 and optimal dilution factors of secondary anti-IgG and IgM are shown in Table 3.
TABLE 3ELISA Pre-experiments to explore optimal protein coating concentration of PRTN3 and optimal dilution of secondary anti-IgG, igM
(2) ELISA experiments preliminary verification of the differential diagnostic efficacy of autoantibodies of the IgG and IgM types against the tumor associated antigen PRTN3
The expression levels of IgG, igM autoantibodies against the tumor associated antigen PRTN3 in plasma samples of lung adenocarcinoma group, healthy control group and benign lung nodule group are shown in fig. 1. As can be seen from fig. 1, the expression level of autoantibodies against the tumor-associated antigen PRTN3 in the lung adenocarcinoma group plasma samples was significantly higher than that in the healthy control group (P < 0.0001) and the benign lung nodule group (P < 0.0001).
As can be seen from fig. 2, according to the analysis of the working characteristics (Receiver operating characteristic, ROC) curves of the subjects, when the IgG type autoantibody of the anti-tumor related antigen PRTN3 is used for diagnosing and identifying the lung adenocarcinoma and the healthy control group, the AUC of the ROC curve is 0.70 (95% ci: 0.65-0.74), and when the cut-off value is 0.716, the sensitivity of the IgG type autoantibody of the anti-tumor related antigen PRTN3 for diagnosing and identifying the lung adenocarcinoma and the healthy control group is 82.5%, and the specificity is 42.5%; moreover, the area under the curve of the diagnosis and identification of the lung adenocarcinoma group and the benign lung nodule group by adopting the IgG type autoantibody of the anti-tumor related antigen PRTN3 is 0.70 (95% CI: 0.65-0.74), and when the cut-off value is 0.716, the sensitivity reaches 85.5%, and the specificity reaches 42.6%; the AUC of ROC curves for diagnosing and identifying lung adenocarcinoma groups and healthy control groups by adopting IgM type autoantibodies of an anti-tumor related antigen PRTN3 is 0.69 (95% CI: 0.64-0.73), and when the cut-off value is 0.789, the sensitivity reaches 77.1%, and the specificity reaches 51.6%; moreover, the IgM type autoantibody diagnosis of the anti-tumor related antigen PRTN3 is adopted to identify the lung adenocarcinoma group and the benign lung nodule group, the area under the curve is 0.65 (95% CI: 0.60-0.70), and when the cut-off value is 0.789, the sensitivity of the IgM type autoantibody diagnosis of the anti-tumor related antigen PRTN3 to identify the lung adenocarcinoma group and the benign lung nodule group is 77.1%, and the specificity is 41.3%. This demonstrates that autoantibodies of the IgG and IgM types, both anti-tumor associated antigen PRTN3, are able to distinguish lung adenocarcinoma groups from healthy control groups and benign lung nodule groups. This also demonstrates that autoantibodies against the tumor associated antigen PRTN3 can be used for the assisted diagnosis of lung adenocarcinoma.
FIG. 3 shows that the combined detection of autoantibodies of IgG and IgM types, which are anti-tumor associated antigen PRTN3, and a traditional tumor marker CEA by using Logistic regression, the AUC of the ROC curve for identifying the lung adenocarcinoma group and the healthy control group after combination is 0.78 (95% CI: 0.73-0.84), and the area under the curve is 0.54 (95% CI: 0.47-0.61) higher than that of the curve for identifying the lung adenocarcinoma group and the healthy control group by CEA alone; the AUC of the ROC curve after combination to identify the lung adenocarcinoma group and benign lung nodule group was 0.73 (95% ci: 0.67-0.80), which is also 0.58 (95% ci: 0.51-0.65) higher than the area under the curve to identify the lung adenocarcinoma group and benign lung nodule group by CEA alone.
In summary, the invention provides a plasma biomarker for assisting in diagnosing lung adenocarcinoma, which is an autoantibody of an anti-tumor related antigen PRTN 3. The invention discovers for the first time that the expression level of IgG and IgM autoantibodies of an anti-tumor related antigen PRTN3 in the plasma of a lung adenocarcinoma patient is obviously higher than that of a healthy control patient and a benign lung nodule patient, and has obvious difference. The combined detection of IgG and IgM autoantibodies of PRTN3 and the traditional tumor marker CEA by using Logistic regression shows that the diagnosis efficiency of the combination on lung adenocarcinoma is better than that of CEA alone. CEA is the content of CEA in serum of a patient detected by using an electrochemiluminescence method clinically, and the unit is ng/mL.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A lung adenocarcinoma marker, characterized in that it is an IgG, igM type autoantibody against PRTN3, or a combination of said IgG, igM type autoantibody against PRTN3 with a classical tumor marker CEA.
2. The application of the autoantibody serving as a marker in preparing a lung adenocarcinoma diagnosis product is characterized in that the autoantibody is an IgG and IgM type autoantibody resisting PRTN 3.
3. The application of the autoantibody serving as a marker in the preparation of products for identifying lung adenocarcinoma and healthy people and/or lung adenocarcinoma and benign lung nodule is characterized in that the autoantibody is an IgG (immunoglobulin G) and IgM (immunoglobulin M) type autoantibody resisting PRTN 3.
4. A product for diagnosing lung adenocarcinoma, comprising reagents for detecting lung adenocarcinoma markers; the lung adenocarcinoma markers include IgG, igM-type autoantibodies against PRTN 3.
5. A product for identifying lung adenocarcinoma from healthy persons and/or for identifying lung adenocarcinoma from benign lung nodules, comprising reagents for detecting lung adenocarcinoma markers; the lung adenocarcinoma markers include IgG, igM-type autoantibodies against PRTN 3.
6. The marker of claim 1 or the use of claim 2 or 3 or the product of claim 4 or 5, wherein the autoantibody is of the IgG, igM type in the peripheral blood.
7. The use according to claim 2 or 3 or the product according to claim 4 or 5, characterized in that the product further comprises the classical tumour marker CEA.
8. A use according to claim 2 or 3 or a product according to claim 4 or 5, comprising a kit, formulation; preferably, the kit is an ELISA detection kit; preferably, the ELISA detection kit comprises PRTN3 protein.
9. The use according to claim 2 or 3 or the product according to claim 4 or 5, wherein said diagnosing lung adenocarcinoma or said identifying lung adenocarcinoma from healthy humans or said identifying lung adenocarcinoma from benign lung nodules comprises detecting an autoantibody, preferably an expression level of an autoantibody by ELISA.
10. The use according to claim 2 or 3 or the product according to claim 4 or 5, wherein said diagnosing lung adenocarcinoma or said identifying lung adenocarcinoma from healthy persons or said identifying lung adenocarcinoma from benign lung nodules comprises detecting autoantibodies and detecting a systemic tumour marker CEA, preferably the expression level of autoantibodies and detecting a systemic tumour marker CEA by ELISA.
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