An ELISA kit for the combined detection of three serum marker proteins for early screening and diagnosis of brain glioma

Abstract

The present invention provides a method for the combined detection of the serum proteins GFAP, CNTN1, and NCAM1 for the early screening and diagnosis of gliomas. By detecting and analyzing the concentrations of CNTN1, GFAP, and NCAM1 proteins in patient serum, the results suggest that the combined use of these three serum proteins is of great significance for the early diagnosis of gliomas. The present invention also provides the use of GFAP, CNTN1, and NCAM1 in the preparation of reagents for the early screening and diagnosis of gliomas.

Description

Technical Field

[0001] The invention belongs to the fields of molecular biology and oncology, and particularly relates to the application of GFAP, CNTN1 and NCAM1 in preparing a combined detection ELISA kit for early screening and diagnosis of brain glioma. Background Art

[0002] Gliomas are the most common primary brain tumors in adults, accounting for approximately 81% of malignant brain tumors. The incidence of gliomas in my country is 5-8 per 100,000 people, ranking third among malignant tumors in terms of five-year mortality, after pancreatic cancer and lung cancer. They have a poor prognosis, are highly malignant, and are highly invasive, with a high recurrence rate. Gliomas are often accompanied by brain symptoms such as epilepsy, intracranial hypertension, and even brain herniation, posing a serious threat to patients' health and placing a significant psychological burden. Traditionally, the diagnosis of gliomas has relied heavily on imaging and pathology, but most tumors are already in the advanced stages when imaging is performed, resulting in high treatment costs and poor efficacy. Therefore, early detection and treatment of tumors are crucial for long-term survival.

[0003] In recent years, the introduction of molecular pathology has been revolutionary in glioma research and clinical diagnosis and treatment. The discovery of an increasing number of molecular markers has enhanced our understanding of the mechanisms of glioma occurrence and development, making clinical diagnosis, pathological classification, and prognostic assessment more precise and promoting individualized optimization of glioma treatment. However, these methods still rely on postoperative pathological sections and cannot escape the significant trauma and financial burden that biopsy or surgery imposes on patients. Therefore, finding simple, rapid, and cost-effective methods for early screening and diagnosis of gliomas is a challenge that urgently needs to be addressed.

[0004] Serum tumor marker testing provides an effective approach for early screening of numerous tumors. For example, alpha-fetoprotein (AFP) has become a specific serum protein marker for the clinical diagnosis of primary liver cancer, PSA is a specific marker for prostate cancer, and NSE is an important molecular marker for screening neuroendocrine tumors. However, to date, there are no established serum molecular markers for the early screening and diagnosis of gliomas.

[0005] Glial fibrillary acidic protein (GFAP) is a type III intermediate filament protein that exists as a monomer. Eight isoforms have been found in humans, with relative molecular masses ranging from (40-53)×10 3. The human GFAP gene is located on the long arm of chromosome 17, region 2, band 1, and consists of 9 exons and 8 introns. GFAP is the main component of the astrocyte cytoskeleton, has a high degree of morphological plasticity, can quickly assemble and change its aggregation state, is a marker protein for brain glial cell-derived tumors, and is often used to identify the tissue source during the pathological diagnosis of brain gliomas. In recent years, studies have found that abnormal expression of GFAP is associated with the progression of a variety of benign and malignant central nervous system diseases. For example, the plasma GFAP level of patients with early-onset Alzheimer's disease is significantly higher than that of patients with late-onset disease; GFAP expression is increased in the plasma of patients with metastatic myxopapillary ependymoma. Aida et al. detected the expression level of GFAP in the serum of glioma patients and found that GFAP was associated with IDH1 wild type, high Ki-67 proliferation index and poor progression-free survival, suggesting that it can be used as a potential biomarker for brain glioma.

[0006] The contactin-1 (CNTN1) gene is located at 12q12 and is a neural cell adhesion factor. It was originally found to be expressed on the surface of various neuronal cells and belongs to the neural contact molecule immune superfamily. At present, the research on CNTN1 mainly focuses on two aspects: (1) CNTN1 is involved in the growth and development of the nervous system and various neural cell functions, such as neural cell differentiation, migration, axon growth, synapse formation, myelination and nerve impulse conduction; (2) CNTN1 is a gene related to tumor invasion and metastasis discovered in recent years. Studies have shown that the CNTN1 gene plays an important role in the metastasis of cancers such as prostate cancer, lung cancer, and gastric cancer. In recent years, foreign scholars have confirmed that CNTN1 is also involved in the occurrence and development of astrocytic gliomas in the nervous system. Studies have shown that CNTN1 is not expressed in normal astrocytic gliomas. On the contrary, CNTN1 is highly expressed in astrocytic gliomas, regulating the invasive ability of glial cells, and CNTN1 expression is related to the malignancy of astrocytic gliomas. This suggests that CNTN1 is of great significance for the early screening of brain gliomas.

[0007] Neural cell adhesion molecule 1 (NCAM1) is a member of the immunoglobulin superfamily of cell adhesion molecules. It is primarily expressed in the nervous system, where it regulates neuronal function and plays a key role in neural migration. The human NCAM1 gene is located at 11q22-23. It is a membrane protein that includes three isoforms: NCAM-120, NCAM-140, and NCAM-180. NCAM140 significantly promotes cell proliferation, motility, and migration, while NCAM1-180 is primarily expressed in nerve fibers, NCAM1-140 is expressed both in nerve fibers and in brain glial cells, and NCAM1-120 is primarily expressed in glial cells.

[0008] It is reported that the sensitivity and specificity of serum GFAP in diagnosing solitary glioblastoma are 76% and 100%, respectively, while no relevant data on the use of serum CNTN1 and NCAM1 in screening or diagnosing brain gliomas have been retrieved. Summary of the Invention

[0009] The present invention aims to overcome the shortcomings of existing technologies by providing a combined detection method for the serum proteins GFAP, CNTN1, and NCAM1 for the early screening and diagnosis of gliomas. By detecting and analyzing the concentrations of CNTN1, GFAP, and NCAM1 proteins in patient serum, the results suggest that the combined use of these three serum proteins is important for the early diagnosis of gliomas. Based on proteomic analysis of serum and tissue samples from healthy individuals and patients with different grades of gliomas, the inventors discovered that the combined detection of GFAP, CNTN1, and NCAM1 demonstrates high specificity and sensitivity for the diagnosis of gliomas.

[0010] The present invention provides uses of GFAP, CNTN1 and NCAM1 in preparing reagents for early screening and diagnosis of brain glioma.

[0011] The present invention provides an ELISA kit for early screening and diagnosis of brain glioma. The kit contains three serum protein markers, namely GFAP, CNTN1 and NCAM1.

[0012] In some embodiments, the above ELISA kit is characterized in that the kit contains an ELISA detection kit for GFAP, CNTN1 and NCAM1.

[0013] In some embodiments, the above-mentioned ELISA kit is characterized in that the ELISA detection kits for GFAP, CNTN1 and NCAM1 are ELISAs from abcam, Novus and Cusabio respectively.

[0014] In some embodiments, the above-mentioned ELISA kit is characterized in that the GFAP detection kit is a kit of abcam company model ab223867, the CNTN1 detection kit is a kit of Novus company model NBP2-75801, and the NCAM1 ELISA detection kit is a kit of Cusabio company model Catalog No. CSB-EL015511HU.

[0015] In some embodiments, the above-mentioned ELISA kit is characterized in that the ELISA kit consists of the following parts: (1) a 96-well ELISA plate, (2) a standard, (3) a biotin-labeled antibody and a diluent, (4) a horseradish peroxidase-labeled avidin and a diluent, (5) a sample diluent, (6) a substrate solution, (7) a washing solution, (8) a color developing solution, (9) a stop solution, and (2) the standard is a standard protein of human GFAP, CNTN1, and NCAM1.

[0016] The present invention provides a method for early screening and early diagnosis of brain glioma using the aforementioned ELISA kit, characterized in that the method comprises the following steps:

[0017] (1) Preparation of serum samples to be tested: centrifuge the collected blood at 4°C, 2500 rpm for 15 minutes, keep the supernatant, discard the sediment below, take the supernatant and package it, store the specimen at -80°C, and when testing, thaw it and centrifuge it again to take the supernatant for use.

[0018] (2) Sample addition: According to the instructions of the three ELISA kits, add the standards and the serum samples to be tested to a 96-well plate, design the reference group and the experimental group, add 100 μl of CNTN1 and NCAM1 standards and serum samples to be tested to each well, add 50 μl of GFAP standards and serum samples to each well, and incubate at 37°C for 60-120 minutes;

[0019] (3) Adding enzyme-labeled antibodies: Biotin-labeled CNTN1 and NCAM1 antibodies were added to both the control and experimental groups, 100 μl per well, and biotin-labeled GFAP antibodies were added to each well, 50 μl;

[0020] (4) Cleaning the antibody solution: Wash several times with the corresponding washing solution of the kit, and then proceed to the next step after drying;

[0021] (5) For the reference group and the experimental group, the horseradish peroxidase-labeled avidin stock solution was diluted with the substrate solution in equal proportion according to the instructions to prepare the horseradish peroxidase-labeled avidin working solution. 100 μl (CNTN1, NCAM1) and 50 μl (GFAP) of horseradish peroxidase-labeled avidin working solution were added to each well and incubated at 37°C for 1 hour.

[0022] (6) Cleaning working solution: Wash the horseradish peroxidase-labeled avidin working solution 3-5 times and spin dry;

[0023] (7) Color development and termination: Add 100 μl of color development solution to a 96-well plate and develop the color at room temperature or 35-38°C for 15-30 minutes, then add the stop solution.

[0024] (8) Standard curve: Draw a standard curve based on the OD value of the standard sample, find the content of the serum sample to be tested on the standard curve and perform statistics.

[0025] The present invention provides a method for early screening and early diagnosis of brain glioma using the aforementioned ELISA kit, characterized in that based on current test results, when the GFAP concentration is higher than 13.43 ng / ml, the CNTN1 concentration is higher than 105.04 ng / ml, and the NCAM1 concentration is higher than 1522.57 ng / ml, it is judged that the subject from which the sample is derived is more likely to suffer from brain glioma.

[0026] The present invention detects the expression levels of GFAP, CNTN1 and NCAM1 in the serum of normal people, patients with brain gliomas and patients with other cancers by ELISA method, and analyzes the correlation between GFAP, CNTN1, NCAM1 and the combination of the three and brain glioma by statistical methods. It is found that serum GFAP, CNTN1 and NCAM1 are a group of good detection indicators for brain glioma. In addition, sample acquisition is simple, fast and economical, patient trauma is minimal, and the detection method is simple and easy. This invention is expected to bring new hope to the early screening and diagnosis of brain gliomas, increase the early diagnosis rate of brain gliomas, and thus improve prognosis and reduce mortality. The present invention discloses a serum molecular marker for non-invasive early screening of brain gliomas, especially the combined detection of serum proteins CNTN1, GFAP and NCAM1, which has a particularly important role and clinical application value for the early screening and early diagnosis of brain gliomas. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1: Serum GFAP levels in normal adults (22 cases), glioma patients (39 cases) and other cancer patients (12 cases) Figure 1A )、CNTN1( Figure 1B )、NCAM1( Figure 1C ) concentration scatter plots and pairwise independent sample T-test results. Graphs and statistics were performed using GraphPad Prism 8. *P < 0.05, **P < 0.01, ***P < 0.001. ns indicates no statistically significant difference.

[0028] Figure 2: ROC curves for normal adults and glioma patients ( Figure 2A GFAP single test; Figure 2B )CNTN1 single test;( Figure 2C )NCAM1 single test.

[0029] Figure 3 : Receiver operating characteristic (ROC) curves of GFAP, CNTN1 and NCAM1 triple detection in normal adults and glioma patients. DETAILED DESCRIPTION

[0030] The present invention is further described below with reference to the following examples, which do not limit the scope of protection of the present invention. Unless otherwise specified, the reagents and materials used in the following examples are commercially available products (GFAP, abcam; CNTN1, Novus; NCAM1, Cusabio).

[0031] 1. Research Subjects

[0032] Blood samples were provided by our center from 39 patients with glioma (25 males, mean age 48.0 years; 14 females, mean age 52.1 years) and 12 patients with other cancers (3 brain metastases; 3 lung cancers; 3 esophageal cancers; 3 cervical cancers) (4 males, mean age 58.5 years; 8 females, mean age 66.1 years). Twenty-two normal blood samples were collected from healthy individuals undergoing community health checkups during the same period (11 males, mean age 59.0 years; 11 females, mean age 57.8 years). The samples used for this study were collected during the same period and under identical sampling, packaging, and storage conditions.

[0033] 2. Experimental Instruments and Reagents

[0034] 1. Instruments: Infinite M200 Pro multifunctional microplate reader (Tecan, Switzerland), high-speed refrigerated centrifuge (Thermo Fisher, USA), incubator, pipette, refrigerator, timer.

[0035] 2. Reagents: ELISA detection kits for GFAP, CNTN1, and NCAM1 were obtained from abcam, Novus, and Cusabio, with models ab223867, NBP2-75801, and Catalog No. CSB-EL015511HU, respectively.

[0036] 3. The kit contains:

[0037] (1) 96-well ELISA plate

[0038] (2) Standard products

[0039] (3) Biotin-labeled antibody and diluent

[0040] (4) Horseradish peroxidase labeled avidin and diluent

[0041] (5) Sample diluent

[0042] (6) Substrate solution

[0043] (7) Washing liquid

[0044] (8) Color development solution

[0045] (9) Stop solution

[0046] Among them, (2) the standard substances are standard proteins of human GFAP, CNTN1, and NCAM1.

[0047] 3. Experimental methods:

[0048] 1. Sample Collection

[0049] After obtaining informed consent from the patients, blood samples were collected from 39 patients with glioma and 12 patients with other cancers at the Second Hospital of Hebei Medical University as the experimental group. Twenty-two normal blood samples were collected from healthy individuals undergoing community health checkups during the same period as the control group. The samples used in the study were collected at the same time and under the same sampling, packaging, and storage conditions.

[0050] 2. Sample Preparation and Sample Dilution

[0051] (1) Preparation of serum samples for testing: Centrifuge the collected blood at 4°C, 2500 rpm for 15 minutes, retain the supernatant, discard the sediment, aliquot the supernatant, and store the specimen at -80°C. Thaw and centrifuge again for use in testing. Serum samples are diluted in the following ratios: GFAP (1:5 dilution), CNTN1 (1:10 dilution), and NCAM1 (1:10 dilution). Store on ice or at 4°C.

[0052] (2) Preparation of standard samples: The standard samples in the ELISA kits for GFAP, CNTN1, and NCAM1 were dissolved in 1 ml of sample diluent, and the standard samples were diluted in multiples according to the requirements of the three kits.

[0053] 3. Experimental Procedure

[0054] (1) Rewarming: First, take out the serum sample from the -80℃ refrigerator and thaw it on ice. Then, take out the ELISA kits for GFAP, CNTN1, and NCAM1 serum proteins from the 4℃ refrigerator.

[0055] (2) Sample addition: According to the instructions of the three ELISA kits, add the standard and the test serum sample to a 96-well plate. Design a reference group (standard 16 wells / 96 wells) and an experimental group (39+12+22 test serum samples, 80 wells / 96 wells). Add 100 μl of CNTN1 and NCAM1 standards and test serum samples to each well, and add 50 μl of GFAP standards and test serum samples to each well. Incubate at 37°C for 60-120 minutes.

[0056] (3) Add enzyme-labeled antibodies: Biotin-labeled antibodies and diluents were added to both the reference group (standard 16 wells / 96 wells) and the experimental group. 100 μl of biotin-labeled CNTN1 and NCAM1 antibody solution was added to each well, and 50 μl of biotin-labeled GFAP antibody solution was added to each well.

[0057] (4) Cleaning the antibody solution: Wash several times with the corresponding washing solution of the kit, and then proceed to the next step after drying.

[0058] (5) For the reference group (standard sample 16 wells / 96 wells) and the experimental group, the horseradish peroxidase-labeled avidin stock solution was diluted with substrate solution in equal proportion according to the instructions to prepare the horseradish peroxidase-labeled avidin working solution. 100 μl (CNTN1, NCAM1) and 50 μl (GFAP) of horseradish peroxidase-labeled avidin working solution were added to each well and incubated at 37°C for 1 hour.

[0059] (6) Cleaning working solution: Wash the horseradish peroxidase-labeled avidin working solution 3-5 times and shake dry.

[0060] (7) Color development and termination: Add 100 μl of color development solution to a 96-well plate, and develop the color at an appropriate temperature for 15 to 30 minutes according to the requirements of the three kits, and then add the stop solution.

[0061] (8) Standard curve: Draw a standard curve based on the OD value of the standard sample, find the content of the serum sample to be tested on the standard curve and perform statistics.

[0062] 4. Data Processing

[0063] The data for this study included normal blood samples, blood samples from gliomas, and other cancers. Graphs and statistics were generated based on experimentally determined serum concentrations of GFAP, CNTN1, and NCAM1 using the statistical software GraphPad Prism 8. Receiver operating characteristic (ROC) curves were generated using MedCalc software, along with standard ROC curves.

[0064] 5. Result analysis:

[0065] The average concentration of CNTN1 in the serum of glioma patients (107.17 ng / ml) was higher than that in the serum of normal subjects (101.62 ng / ml) (Table 1-1), with statistical significance (P=0.0051); the average concentration of GFAP (38.55 ng / ml) was higher than that in the serum of normal subjects (12.35 ng / ml) (Table 1-2), with statistical significance (P=0.0031); the average concentration of NCAM1 (2217.90 ng / ml) was higher than that in the serum of normal subjects (1356.82 ng / ml) (Table 1-3), with statistical significance (P=0.0075).

[0066] Tables 1-1 to 1-3 below show the mean values, standard deviations, and corrected P values ​​for pairwise comparisons of serum CNTN1, GFAP, and NCAM1 in normal adults, glioma patients, and other cancer patients.

[0067] Table 1-1

[0068]

[0069] Table 1-2

[0070]

[0071] Table 1-3

[0072]

[0073] The results in Table 2 show that the specificities of single serum protein GFAP, CNTN1, and NCAM1 for glioma detection were 86.36%, 72.73%, and 81.82%, respectively, and the sensitivities were 66.67%, 71.79%, and 64.10%, respectively. The specificity of the triple test was 95.45%, and the sensitivity was 74.36%, all significantly higher than those of single tests. Furthermore, in the ROC curve for single serum protein GFAP, CNTN1, and NCAM1 detection of glioma, the AUC values ​​were 0.735, 0.744, and 0.740, respectively, with P values ​​of 0.0002, 0.0002, and 0.0001, respectively, demonstrating statistical significance. In the ROC curve for the triple test of GFAP, CNTN1, and NCAM1, the AUC value was as high as 0.889 (P < 0.0001), demonstrating statistical significance and significantly higher than that of single tests.

[0074] Table 2: Results of separate and combined detection of three serum proteins (Normal-Glioma)

[0075]

[0076] As shown in Figure 1, the scatter plots and pairwise independent sample T-tests of serum GFAP, CNTN1, and NCAM1 concentrations in normal adults (22 cases), gliomas (39 cases), and other cancers (12 cases) show that the serum concentrations of GFAP, CNTN1, and NCAM1 in glioma patients were significantly higher than those in normal controls, and there were statistical differences. However, there were no statistical differences in the serum concentrations of the three proteins between patients with other tumors and normal controls, which further demonstrates the predictive value of the three proteins in glioma screening.

[0077] From Figure 2 and Figure 3 It can be seen that when the three serum proteins were combined to detect brain glioma, the area under the ROC curve increased to 0.889, and the P value was <0.0001, which was statistically significant, indicating that the triple detection ELISA kit has a high diagnostic value for brain glioma, further proving that this method - serum protein GFAP, CNTN1 and NCAM1 triple detection can be used as a means of early screening for brain glioma.

[0078] Experimental results show that serum proteins GFAP, CNTN1 and NCAM1 are good detection indicators for brain gliomas. In particular, the combined detection of the three has early screening and diagnostic value, plays an early warning role, and has potential clinical application value.

Claims

1. An ELISA kit for early screening and diagnosis of brain glioma, the kit detecting three serum protein markers: GFAP, CNTN1, and NCAM1; the kit comprising a GFAP ELISA detection kit, a CNTN1 ELISA detection kit, and an NCAM1 ELISA detection kit; the GFAP ELISA detection kit is abcam, model ab223867; the CNTN1 ELISA detection kit is Novus, model NBP2-75801; and the NCAM1 ELISA detection kit is Cusabio, model CSB-EL015511HU.

2. The ELISA kit for early screening and diagnosis of brain glioma according to claim 1, characterized in that The GFAP ELISA detection kit, CNTN1 ELISA detection kit and NCAM1 ELISA detection kit are composed of the following parts: (1) 96-well enzyme-labeled plate, (2) standard substance, (3) biotin-labeled antibody and diluent, (4) horseradish peroxidase-labeled avidin and diluent, (5) sample diluent, (6) substrate solution, (7) washing solution, (8) color development solution, (9) stop solution, (2) standard substance is standard protein of human GFAP, CNTN1 or NCAM1.

3. The ELISA kit for early screening and diagnosis of brain glioma according to any one of claims 1 or 2, characterized in that: The method for early screening and early diagnosis of brain glioma using the ELISA kit comprises the following steps: (1) Preparation of serum samples to be tested: centrifuge the collected blood at 4°C, 2500 rpm for 15 min, retain the supernatant, discard the sediment, take the supernatant and package it, store the specimen at -80°C, and when testing, thaw it and centrifuge it again to take the supernatant for use; (2) Sample addition: According to the instructions of the GFAP ELISA test kit, CNTN1 ELISA test kit, and NCAM1 ELISA test kit, add the standard and the serum sample to be tested to a 96-well plate respectively; design the reference group and experimental group, add 100 μl of CNTN1 and NCAM1 standard and serum sample to be tested to each well, add 50 μl of GFAP standard and serum sample to each well, and incubate at 37°C for 60-120 minutes; (3) Adding biotin-labeled antibodies: biotin-labeled CNTN1 and NCAM1 antibodies were added to both the control and experimental groups, 100 μl per well, and biotin-labeled GFAP antibodies were added to each well, 50 μl; (4) Cleaning the antibody solution: Wash several times with the corresponding washing solution of the kit, and then proceed to the next step after drying; (5) For the reference group and experimental group, the horseradish peroxidase-labeled avidin stock solution was diluted with the substrate solution in equal proportion according to the instructions to prepare the horseradish peroxidase-labeled avidin working solution. For the detection of CNTN1 and NCAM1, 100 μl of the horseradish peroxidase-labeled avidin working solution was added to each well, and for the detection of GFAP, 50 μl of the horseradish peroxidase-labeled avidin working solution was added to each well. The cells were incubated at 37°C for 1 hour. (6) Cleaning working solution: Wash the horseradish peroxidase-labeled avidin working solution 3-5 times and spin dry; (7) Color development and termination: Add 100 μl of color development solution to a 96-well plate and develop the color at room temperature or 35-38°C for 15-30 minutes; then add the stop solution. (8) Standard curve: Draw a standard curve based on the OD value of the standard sample, find the content of the serum sample to be tested on the standard curve and perform statistics.

4. The ELISA kit for early screening and diagnosis of brain glioma according to claim 3, characterized in that: When the test results show that the GFAP concentration is higher than 13.43 ng / ml, the CNTN1 concentration is higher than 105.04 ng / ml, and the NCAM1 concentration is higher than 1522.57 ng / ml, it is judged that the subject from which the sample is derived is more likely to suffer from brain glioma.

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