Protein markers MIB1 and / or YBX1 for distinguishing benign and malignant pulmonary nodules and use thereof

Abstract

Protein markers MIB1 and / or YBX1 for distinguishing benign and malignant pulmonary nodules, the use of a detection reagent for detecting the protein level of protein markers MIB1 and / or YBX1 in a biological sample in the preparation of a product for diagnosing benign and malignant pulmonary nodules, and a product for diagnosing benign and malignant pulmonary nodules, which product comprises the detection reagent for detecting the protein level of protein markers MIB1 and / or YBX1 in a biological sample. The detection of the expression level of MIB 1 or YbX1 alone or the co-expression level of the two in a biological sample can distinguish benign and malignant pulmonary nodules.

Description

Protein markers MIB1 and / or YBX1 for distinguishing benign and malignant pulmonary nodules and their applications Technical Field

[0001] The present invention belongs to the field of biological detection technology, and particularly relates to protein markers MIB1 and / or YBX1 for distinguishing benign and malignant pulmonary nodules and applications thereof. Background Art

[0002] Lung cancer is a common malignant tumor with a very high morbidity and mortality rate. Because lung cancer lacks specific symptoms in its early stages, most patients present with advanced disease by the time they seek medical attention. The five-year survival rate is less than 20%, posing a serious threat to human health. However, with early detection and prompt treatment, the five-year survival rate for lung cancer patients can reach over 80%. Therefore, early diagnosis and treatment are key to improving the prognosis for lung cancer patients.

[0003] Studies have clearly established that lung cancer originates from lung nodules. With the widespread adoption of low-dose spiral CT (LDCT), the number of small lung nodules detected by chest CT has exploded. However, due to limitations in slice thickness, respiratory motion, and scanning angles, chest CT still exhibits some discrepancies in determining the specific nature of small lung nodules, resulting in a high false-positive rate. Recent studies have shown that 30%-45% of surgically resected nodules are pathologically diagnosed as benign. Therefore, developing a noninvasive method that can accurately distinguish between benign and malignant lung nodules is urgently needed. Accurately distinguishing between benign and malignant lung nodules can both enable lung cancer patients to receive treatment earlier and prevent overtreatment of patients with benign lung nodules.

[0004] Blood biomarkers are an efficient and non-invasive diagnostic tool. Currently, commonly used serum lung cancer biomarkers in clinical practice include progastrin-releasing peptide (ProGRP), neuron-specific enolase (NSE), squamous cell carcinoma antigen (SCC-Ag), cytokeratin fragment 19 (CRYFRA21-1), carcinoembryonic antigen (CEA), and human epididymis protein 4 (HE4). In recent years, with advances in science and technology and the deepening of lung cancer research, a number of liquid biopsy tests and molecular markers, such as circulating tumor cells, ctDNA, mRNA, lncRNA, and DNA methylation, have been developed to help define the nature of small lung nodules. However, existing data show that the ability of these markers to detect lung cancer remains limited. Notably, a biomarker that uses several key clinical characteristics (nodule size, morphology, and location) and two protein markers (LG3BP and C163A) to distinguish early-stage benign from malignant nodules is the only FDA-approved biomarker used in clinical diagnosis of early-stage nodules. This biomarker has a sensitivity of 97% and a specificity of only 44%. Recently, high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) revealed that a panel of six plasma protein markers, APOA4, CD14, PFN1, APOB, PLA2G7, and IGFBP2, has the ability to distinguish benign from malignant lung nodules, with a sensitivity of 96% and a specificity of only 35%. An ELISA for plasma anti-PLOD2 autoantibodies has been used to differentiate patients with early-stage non-small cell lung cancer from those with benign lung nodules, with a sensitivity and specificity of 77.2% and 70.4%, respectively, but this does not cover all lung cancer types.

[0005] In general, there is still a lack of bodily fluid markers for distinguishing benign and malignant lung nodules in clinical practice, and no simple, low-cost detection methods suitable for distinguishing benign and malignant lung nodules have been developed. This has led to a large number of challenges in the identification and clinical management of benign and malignant lung nodules. Therefore, there is an urgent need to develop markers with higher sensitivity and specificity and simple detection for distinguishing benign and malignant lung nodules. Summary of the Invention

[0006] In view of this, it is necessary for the present invention to provide a protein marker MIB1 and / or YBX1 for distinguishing benign and malignant lung nodules. By detecting the protein levels of MIB1 and / or YBX1 in the body fluids of patients with lung nodules, a new detection idea with high sensitivity and specificity is provided for the diagnosis of benign and malignant lung nodules.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides protein markers MIB1 and / or YBX1 for distinguishing benign and malignant pulmonary nodules.

[0009] A second aspect of the present invention provides the use of a detection reagent for detecting the level of protein markers MIB1 and / or YBX1 in a biological sample in the preparation of a product for diagnosing benign and malignant pulmonary nodules.

[0010] A third aspect of the present invention provides a product for diagnosing benign and malignant pulmonary nodules.

[0011] The protein markers used to distinguish between benign and malignant lung nodules described in the present invention are MIB1 or YBX1 or a combination of the two. Specifically, the present invention has found that MIB1 or YBX1 alone has high sensitivity and specificity for distinguishing between benign and malignant lung nodules. The sensitivity of serum MIB1 protein alone in distinguishing between benign and malignant lung nodules reaches 83.3%, and the specificity reaches 80.0%; the sensitivity of serum YBX1 protein alone in distinguishing between benign and malignant lung nodules reaches 91.7%, and the specificity reaches 80.0%. The combined effect of the two is even better. The combined sensitivity of the two in distinguishing between benign and malignant lung nodules reaches 87.5%, and the specificity reaches 95.0%. Therefore, in some preferred embodiments of the present invention, the protein markers used to distinguish between benign and malignant lung nodules are preferably MIB1 and YBX1.

[0012] The protein level mentioned in the present invention refers to the protein expression amount of the protein markers mentioned herein in a biological sample.

[0013] The product described in the present invention is an in vitro diagnostic product, which may be a kit or a chip, but is not limited thereto. The product contains a reagent for detecting the protein markers MIB1 and / or YBX1 protein levels in a biological sample.

[0014] The detection reagents described in the present invention refer to reagents for detecting corresponding biomarkers using conventional protein detection techniques in the art. The protein detection techniques herein may be single molecule immunoassay, enzyme-linked immunosorbent assay, immunofluorescence, radioimmunoassay, immunoprecipitation, immunoblotting, high performance liquid chromatography, capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence, colloidal gold immunoassay, fluorescence immunochromatography, surface plasmon resonance, immuno-PCR, or biotin-avidin technology, but are not limited thereto. In some specific embodiments of the present invention, the detection reagents preferably include reagents for detecting the protein markers MIB1 and / or YBX1 protein levels in a biological sample using single molecule immunoassay.

[0015] It is understandable that the detection reagent also includes other auxiliary agents compatible with the corresponding detection technology, such as buffer solutions, cleaning solutions, etc., which will not be elaborated here. The compatibility can be adjusted or selected according to the corresponding detection technology.

[0016] The biological sample described in the present invention refers to a bodily fluid from a patient with pulmonary nodules, and may be peripheral blood or bronchoalveolar lavage fluid. Preferably, because peripheral blood sample testing is non-invasive, easily accessible, and convenient for testing compared to imaging examinations, in some specific embodiments of the present invention, the biological sample is peripheral blood from a patient with pulmonary nodules, and may be serum or peripheral blood exosomes.

[0017] The product described in the present invention is actually tested by performing the following method:

[0018] Obtaining biological samples;

[0019] Detect the protein expression levels of protein markers MIB1 and / or YBX1 in biological samples.

[0020] In a further embodiment, the method further comprises the step of determining the expression amount of the protein markers MIB1 and / or YBX1 based on their protein expression levels;

[0021] The expression level judgment here is based on the threshold of the protein expression level of the protein markers MIB1 and / or YBX1. If the expression level is higher than the threshold, it is judged to be high, indicating that the lung nodule is malignant.

[0022] Preferably, in some specific embodiments of the present invention, the expression amount is judged based on the combined expression level of the protein markers MIB1 and YBX1, wherein the combined prediction factor is calculated by performing logistic regression analysis using SPSS software based on the detection results of MIB1 and YBX1 in the biological sample, and the combined prediction factor calculated in the present invention = MIB1 expression level + 2.5 × YBX1 expression level.

[0023] The threshold (cut-off value) described here was obtained by retrospectively analyzing the clinical pathological results (pathologically diagnosed as benign pulmonary nodules or malignant pulmonary nodules) and drawing the ROC curve using SPSS software to obtain the AUC value and cut-off value.

[0024] Beneficial effects of the present invention:

[0025] The present invention provides a protein marker for clinically distinguishing benign from malignant pulmonary nodules with ease, low cost, and high accuracy. By employing conventional protein detection techniques in the art to detect MIB1 and / or YBX1 in the body fluids of patients with pulmonary nodules, the present invention can accurately differentiate lung cancer from patients with pulmonary nodules, providing a new biological target and method for the development of lung cancer diagnostic kits.

[0026] The use of protein markers MIB1 and / or YBX1 protein expression levels to differentiate between benign and malignant pulmonary nodules has the following advantages:

[0027] Improved diagnostic accuracy: By using MIB1 and / or YBX1 proteins as lung cancer markers, the present invention can more accurately distinguish patients with lung cancer from patients with benign lung nodules, thereby improving diagnostic accuracy and facilitating timely detection and treatment of lung cancer.

[0028] Simple operation and low cost: The present invention uses protein detection technology to detect MIB1 and / or YBX1 proteins in the body fluids of patients with lung nodules, with high sensitivity and specificity. The simple operation and low cost facilitate widespread application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a graph showing the expression of MIB1 and YBX1 in the serum of patients with lung cancer and benign pulmonary nodules in Example 1; Figure 1A is a graph showing the expression of MIB1 in the serum of patients with lung cancer and benign pulmonary nodules, and Figure 1B is a graph showing the expression of YBX1 in the serum of patients with lung cancer and benign pulmonary nodules.

[0030] Figure 2 is a verification graph of the expression of MIB1, YBX1 and their combined predictive factors in the serum of patients with lung cancer and benign pulmonary nodules; Figure 2A is an expression graph of MIB1 in the serum of patients with lung cancer and benign pulmonary nodules in the verification group, Figure 2B is an expression graph of YBX1 in the serum of patients with lung cancer and benign pulmonary nodules in the verification group; Figure 2C is an expression graph of the combined predictive factors in the serum of patients with lung cancer and benign pulmonary nodules in the verification group.

[0031] Figure 3 is the ROC curve of YBX1, MIB1 and their combination for diagnosing benign and malignant pulmonary nodules. Modes for Carrying Out the Invention

[0032] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In addition, unless otherwise specified, methods without specific conditions or steps are conventional methods, and the reagents and materials used can be obtained from commercial sources.

[0034] The MIB1 protein is an E3 ubiquitin ligase with a molecular weight of approximately 120-130 kDa that facilitates the attachment of ubiquitin to specific substrate proteins. Ubiquitin ligation typically leads to the degradation or translocation of the ubiquitinated protein. MIB1 regulates cellular processes by interacting with other proteins. Studies have shown that MIB1 can regulate the proliferation and metastasis of various cancer cells.

[0035] YBX1 is a typical DNA / RNA binding protein that can regulate multiple targeted proteins that are highly related to tumor occurrence and metastasis.

[0036] Although the MIB1 and YBX1 genes play key roles in the development and progression of various tumors, studies on the expression of MIB1 and YBX1 proteins in the blood have been limited. This invention aims to provide new uses for MIB1 and YBX1 proteins and to offer new targets for differential diagnosis of benign and malignant pulmonary nodules.

[0037] Description of the research specimens in the examples herein:

[0038] The 60 research specimens included in this application included 32 patients with lung cancer and 28 patients with benign lung nodules.

[0039] 1. Blood samples were collected from patients with suspicious lung nodules detected by CT scan and hospitalized for this condition before any treatment. The blood was centrifuged at 4000 × rpm for 10 minutes to obtain serum, which was then stored at -80°C. The serum was numbered, packaged, and basic information was registered. (Blood sample collection was approved by the Ethics Committee of the First Affiliated Hospital of the University of Science and Technology of China, Ethics Number: 2023-RE-413).

[0040] 2. Using the subjects' hospitalization number, gender, age and other information, the postoperative lung nodule pathology results (lung cancer\benign lung nodules) were checked in the hospital patient management system, and a patient clinical information database was constructed.

[0041] 3. Inclusion and exclusion criteria of research subjects

[0042] The inclusion criteria for patients with pulmonary nodules included: (1) no chemotherapy, radiotherapy, surgery, or other interventions; (2) clear histopathological results, confirmed as lung cancer according to the primary lung cancer diagnosis and treatment guidelines, and pathological histological type (including adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, large cell carcinoma, small cell carcinoma, and mixed carcinoma) or benign disease; (3) no history of other malignant tumors; (4) complete medical history; (5) no autoimmune disease; (6) all were new cases; and (7) patients were >18 years old.

[0043] Exclusion criteria: subjects who do not meet the above inclusion criteria.

[0044] Example 1. Expression levels of MIB1 and YBX1 in serum of patients with pulmonary nodules

[0045] In this example, single-molecule immunoassay technology was used to perform preliminary detection of MIB1 and YBX1 levels (expressed as fluorescence signal counts) in the serum of 16 patients with lung nodules (8 of whom were lung cancer patients and the other 8 were benign lung nodules patients).

[0046] 1. Single molecule immunoassay of serum collected from patients with lung nodules. The specific steps are as follows:

[0047] 1. Preparation of R1 reagent (magnetic beads coated with capture antibody)

[0048] (1) Preparation of magnetic beads: Take out the magnetic bead tube and shake it for 15 seconds, then centrifuge it for 3 seconds in a handheld centrifuge;

[0049] (2) Magnetic bead washing: Take a 2 ml centrifuge tube, add 1 ml of washing buffer to each tube, take 20 μl of magnetic beads into the centrifuge tube, shake and mix for 15 seconds, centrifuge for 3 seconds, place on the magnetic stand and magnetically absorb for 1-2 minutes (the buffer becomes clear and the magnetic beads are adsorbed to the side of the magnetic stand), then remove the supernatant, and the washing is completed once;

[0050] (3) Magnetic bead washing: Add 1 ml of 50 mM MES buffer to the upper tube, shake and mix for 15 seconds, centrifuge for 3 seconds, place on a magnetic stand for 1-2 minutes, then remove the supernatant, repeat washing twice, and finally resuspend in 900 μL of 50 mM MES buffer;

[0051] (4) Activation of magnetic beads: Add 50 µL of EDC and 50 µL of NHS to each tube and activate on a rotary mixer at 25°C, 43 × rpm for 30 min. (Note: EDC should be equilibrated at room temperature for 20 min before use. EDC and NHS should be prepared and used immediately.)

[0052] (5) Washing: Place the beads on a magnetic stand for 1-2 minutes, carefully aspirate the supernatant, resuspend with 1 ml of 50 mM MES buffer, shake and mix for 15 seconds, centrifuge for 3 seconds, place on a magnetic stand for 1-2 minutes, then aspirate the supernatant, and repeat the washing process twice;

[0053] (6) Antibody coupling: Resuspend the washed magnetic beads in 500 μL of 50 mM MES magnetic bead coating buffer, then add 20 μg of capture antibody (MIB1 or YBX1) to the magnetic bead suspension, shake and mix for 15 seconds, centrifuge for 3 seconds, and incubate in a thermomixer at 25°C, 1500 rpm for 2 hours.

[0054] (7) Blocking: After incubation, add JSR blocking agent (CE210 10µL + CE510 10µL) and ethanolamine 30µL, respectively, and block in a thermomixer at 25°C, 1500 rpm for 1 hour. (Calculate the amount of blocking agent and ethanolamine, and add 50µL to each tube after mixing).

[0055] (8) Washing: Wash three times with 1 ml of washing buffer (5×PBST) each time. The washing steps are the same as (3).

[0056] (9) Storage: Resuspend each tube with 1 ml of HEPES storage solution and store at 4°C.

[0057] 2. Preparation of R2 reagent (fluorescent microspheres coated with detection antibodies)

[0058] (1) Microsphere preparation: Take out the microsphere tube and shake it for 15 seconds, then centrifuge it for 3 seconds;

[0059] (2) Microsphere cleaning: Take a 2 ml centrifuge tube, add 900 μL of 50 mM MES labeling buffer to each tube, take 100 μL (1 mg) fluorescent microspheres into the centrifuge tube, shake and mix for 15 seconds, centrifuge for 3 seconds in a handheld centrifuge, and sonicate in a water bath for 3 minutes; centrifuge at 4°C, 15,000 rpm, centrifuge for 17 minutes, and wash once;

[0060] (3) Microsphere activation: remove the supernatant, add 900 μL of labeling buffer 50 mM MES to each tube to resuspend, blow with a gun, shake and mix for 15 seconds, centrifuge for 3 seconds, sonicate in a water bath for 3 minutes, add 50 μL of EDC and 50 μL of NHS to each tube, and activate on a rotary mixer at 25°C, 43 rpm for 30 minutes;

[0061] (4) Washing: centrifuge at 4°C, 15,000 rpm for 17 min, resuspend and wash twice with 1 ml of 50 mM MES;

[0062] (5) Antibody coupling: The activated microspheres were resuspended in 500 μL of 20 mM HEPES labeling buffer, shaken for 15 seconds, centrifuged for 3 seconds in a handheld centrifuge, and sonicated in a water bath for 3 minutes. 20 μg of detection antibody (MIB1 or YBX1) was added, shaken for 15 seconds, and incubated at 25°C, 1500 rpm, for 2 hours.

[0063] (6) Blocking: Add blocking agent (CE210 10µL + CE510 10µL) and ethanolamine 30µL, respectively, at room temperature (25°C) and 1500 rpm for 1 h;

[0064] (7) Washing: centrifuge at 4°C, 15,000 rpm for 17 min; remove the supernatant, resuspend in 1 ml of 20 mM HEPS, sonicate for 3 min, and centrifuge; wash twice;

[0065] (8) Storage: Add 1 ml of No. 2 preservation solution to each tube, pipette evenly, and ultrasonicate in a water bath for 8 minutes; (Note: put ice cubes into the ultrasonicator in advance to cool it down), and store at 4°C after the end.

[0066] 3. Sample preparation

[0067] (1) Complex 1 (R1 and sample combination): Take 100 µL of R1 reagent, add 5 µL of serum sample, and then make up to 200 µL with 95 µL of 0.5Q block storage solution (Note: Serum should be centrifuged at 3500 rpm for 5 minutes before use);

[0068] (2) Incubation: 37°C, 43 rpm, 30 min;

[0069] (3) Washing: After incubation, wash three times with 1 ml of 5× PBST solution to remove unbound samples;

[0070] (4) Complex 2 (R2 and complex 1 combined): resuspend each tube with 150 μL 0.5Q block storage solution, add 50 μL R2 reagent, incubate at 37°C, 43 rpm / min, and incubate for 30 min; (Note: fluorescent microspheres were sonicated for 3 min before use);

[0071] (5) Washing: Wash 4 times with 5×PBST buffer, and then resuspend to 150µL with 0.5Q block buffer.

[0072] 4. On-machine testing

[0073] (1) Chip preparation: Take out the chip (739W-0179, Suzhou Dicotone Biotechnology Co., Ltd.), tear off the outer packaging film, place the chip face up, and stick the 3M film on the chip channel, with two circular holes on both sides of the channel;

[0074] (2) Sample loading: Take 10 µL of the test sample processed in step 3 and add it to the small hole on one side of the chip. The magnetic beads will be spread into the chip channel. Place the chip on the magnet for 5 seconds to allow the magnetic beads to sink to the bottom.

[0075] (3) Detection: The fluorescence signal count of each group was detected after calibration using a fluorescence microscope / GY-100A IVD.

[0076] The results showed that the fluorescence signal numbers of MIB1 and YBX1 in the serum of lung cancer patients were significantly higher than those in patients with benign lung nodules, and the differences were statistically significant (see Figure 1A, P=0.008 and Figure 1B, P<0.001).

[0077] Example 2. Clinical value of MIB1 and YBX1 in differentiating benign and malignant pulmonary nodules

[0078] In this example, single-molecule immunoassay technology was used to measure the expression levels of MIB1 and YBX1 proteins in the serum of 24 lung cancer patients and 20 benign pulmonary nodule patients to verify the ability and clinical value of MIB1 and YBX1 proteins in distinguishing benign from malignant pulmonary nodules. The basic clinical data of the lung cancer and benign pulmonary nodule groups are shown in Table 1.

[0079] Table 1 Basic clinical data of patients

[0080]

[0081] The specific serum collection and single molecule immunoassay methods are the same as in Example 1 and will not be further elaborated here. The test results were analyzed by ROC curve analysis, as shown in Table 2, Figures 2 and 3.

[0082] Table 2 Sensitivity and specificity of MIB1, YBX1 and their combined diagnosis

[0083] BiomarkerCut-offAUCSensitivity(%)Specificity(%)95%CIP valueYBX1>10000.9691.780.00.91-1.00<0.001MIB1>8000.8483.380.00.72-0.96=0.001Joint factor>36000.9787.595.00.93-1.00<0.001

[0084] The results showed that compared with patients with benign nodules, the expression levels of MIB1 and YBX1 in the serum of lung cancer patients were significantly upregulated (Figure 2A, Figure 2B).

[0085] Further analysis showed that the sensitivity of serum MIB1 protein alone in distinguishing benign and malignant lung nodules reached 83.3%, and the specificity reached 80.0%; the sensitivity of serum YBX1 protein alone in distinguishing benign and malignant lung nodules reached 91.7%, and the specificity reached 80.0%.

[0086] Subsequently, logistic regression analysis of the detected YBX1 and MIB1 data was performed using SPSS software, and the resulting joint predictor was MIB1 expression level + 2.5 × YBX1 expression level. It was found that the joint predictor was significantly increased in patients with lung cancer compared with patients with benign nodules (Figure 2C); its sensitivity for distinguishing benign from malignant lung nodules reached 87.5%, and its specificity reached 95.0% (Figure 3).

[0087] The above examples illustrate that the present invention provides a protein marker that is simple to operate, low in cost, highly accurate, and can be used to distinguish between benign and malignant lung nodules. When used in conjunction with protein detection technology, it has high sensitivity and specificity in distinguishing benign and malignant lung nodules, and can accurately identify lung cancer from patients with lung nodules, providing new biological targets and methods for the development of lung cancer diagnostic products (such as kits or chips).

[0088] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0089] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A protein marker for distinguishing benign and malignant pulmonary nodules, characterized in that: The protein markers are MIB1 and / or YBX1.

2. The protein marker for distinguishing benign and malignant pulmonary nodules according to claim 1, wherein The protein markers are MIB1 and YBX1.

3. Use of a detection reagent for detecting the protein markers MIB1 and / or YBX1 protein levels in a biological sample in the preparation of a product for diagnosing benign or malignant pulmonary nodules; Preferably, the product is a kit or a chip.

4. The use according to claim 3, characterized in that The biological sample is peripheral blood or bronchoalveolar lavage fluid from a patient with pulmonary nodules; Preferably, the biological sample is peripheral blood from a patient with pulmonary nodules; Preferably, the biological sample is serum from a patient with pulmonary nodules.

5. The use according to claim 3, characterized in that The detection reagent includes a reagent for detecting the protein marker MIB1 and / or YBX1 protein level in a biological sample by using a single molecule immunoassay technique.

6. A product for diagnosing benign and malignant pulmonary nodules, characterized in that: including detection reagents for detecting the protein marker MIB1 and / or YBX1 protein levels in biological samples; Preferably, the product is a kit or a chip.

7. The product according to claim 6, characterized in that The biological sample is peripheral blood or bronchoalveolar lavage fluid from a patient with pulmonary nodules; Preferably, the biological sample is peripheral blood from a patient with pulmonary nodules; Preferably, the biological sample is serum from a patient with pulmonary nodules.

8. The product according to claim 6, characterized in that The detection reagent includes a reagent for detecting the protein marker MIB1 and / or YBX1 protein level in a biological sample by using a single molecule immunoassay technique.

9. The product according to any one of claims 6 to 8, characterized in that The product performs the following method: Obtaining biological samples; Detecting the protein expression levels of protein markers MIB1 and / or YBX1 in biological samples; Preferably, the method further comprises the step of determining the expression amount of the protein markers MIB1 and / or YBX1 according to their protein expression levels; Preferably, the judgment is performed based on a threshold value of the protein expression level of the protein markers MIB1 and / or YBX1. If the expression level is higher than the threshold value, it is judged to be high, indicating that the lung nodule is malignant.

10. The product according to claim 9, characterized in that The method includes the steps of determining the expression amount of protein markers MIB1 and YBX1 according to their combined expression levels; Preferably, the combined predictor = MIB1 expression level + 2.5 × YBX1 expression level; Preferably, the combined predictor is obtained by performing logistic regression analysis on YBX1 and MIB1 data.

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