CN116087521A - Three serum marker protein combined detection ELISA kit for early screening and diagnosis of brain glioma - Google Patents

Abstract

The invention provides a combined detection of serum proteins GFAP, CNTN1 and NCAM1 for early screening and early diagnosis of glioma. The results of detecting and analyzing the concentration of CNTN1, GFAP and NCAM1 proteins in serum of patients suggest that the combined application of three serum proteins has important significance for early diagnosis of glioma. The invention also provides the use of GFAP, CNTN1 and NCAM1 in the preparation of a reagent for early screening and diagnosis of glioma.

Description

Three serum marker protein combined detection ELISA kit for early screening and diagnosis of brain glioma

Technical Field

The invention belongs to the fields of molecular biology and oncology, and particularly relates to application of GFAP, CNTN1 and NCAM1 in preparation of a combined detection ELISA kit for early screening and diagnosis of glioma.

Background

Gliomas are the most common primary brain tumor in adults, accounting for about 81% of malignant brain tumors. The incidence rate of the glioma in China is 5-8/10 ten thousand, and the glioma is second only to pancreatic cancer and lung cancer in 5 years of death rate of malignant tumors, and is the third in the rank. The method has the characteristics of poor prognosis, high malignancy, high invasiveness and high recurrence rate. In patients with brain glioma, brain symptoms such as epilepsy, intracranial hypertension and even cerebral hernia are often accompanied, and serious threat and psychological burden are brought to the health of the patients. The diagnosis of the traditional brain glioma depends on imaging and pathology, but most of tumor imaging is found, the diagnosis enters middle and late stages, and the treatment cost is high and the curative effect is poor. Thus for early detection of tumors, early treatment is critical for patient survival for long periods of time.

In recent years, the introduction of molecular pathology has revolutionary significance in glioma research and clinical diagnosis and treatment, and the discovery of more and more molecular markers increases the understanding of the occurrence and development mechanisms of glioma, so that the clinical diagnosis, pathological typing and prognosis evaluation are more accurate, and the individual optimization of glioma treatment is promoted. However, the method still cannot leave the postoperative pathological section, and cannot get rid of the huge injury and economic pressure brought to patients by biopsy or operation. Therefore, finding a simple, rapid and economical early screening and diagnosis method for glioma is a current urgent problem to be solved.

Detection of serum tumor markers provides an effective way for early screening of numerous tumors, for example: alpha fetoprotein AFP has become a specific serum protein marker for clinical diagnosis of primary liver cancer, PSA is a specific marker for diagnosis of prostate cancer, NSE is an important molecular index for screening neuroendocrine tumor. To date, however, more mature serum molecular markers for early screening and early diagnosis of gliomas have not emerged.

Glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP) is a type iii intermediate filamin, which exists in monomeric form. Among humans, 8 kinds of them were foundThe relative molecular mass of the homologous isomer is between (40-53) x 10 ³ . The human GFAP gene is positioned on a zone 1 of a long arm 2 of a chromosome 17, consists of 9 exons and 8 introns, is a main component of an astrocyte skeleton, has high morphological plasticity, can be quickly assembled and changed in polymerization state, is a marker protein of brain glial cell-derived tumor, and is commonly used for tissue-derived identification during pathological diagnosis of brain glioma. In recent years, abnormal expression of GFAP has been found to be associated with the progression of various benign and malignant central nervous system diseases, such as those suffering from early onset Alzheimer's disease, with significantly higher levels of GFAP in plasma than those suffering from late onset Alzheimer's disease; GFAP expression is elevated in plasma of patients with metastatic papillary myxomatosis. Aida et al found by detecting the expression level of GFAP in serum of glioma patients that GFAP is related to IDH1 wild type, high Ki-67 proliferation index and poor progression-free survival, suggesting that GFAP can be used as a potential biomarker of brain glioma.

The contact protein-1 (CNTN 1) gene, which is located at 12q12, is a neural cell adhesion factor that was originally found to be expressed on the surface of various neuronal cells and belongs to one of the neurocontact molecular immune superfamily. Currently, studies on CNTN1 are mainly focused on two aspects: (1) CNTN1 is involved in the growth and development of the nervous system, such as nerve cell differentiation, migration, axon growth, synapse formation, myelination, and nerve impulse conduction, and a variety of nerve cell functions: (2) CNTN1 is a gene related to tumor invasion and metastasis capability found in recent years, and studies have shown that CNTN1 gene plays an important role in metastasis of cancers such as prostate cancer, lung cancer, stomach cancer, etc., and studies by foreign scholars in recent years confirm that CNTN1 is also involved in the occurrence and development of astrocytomas in the nervous system, and studies have shown that CNTN1 is not expressed in normal astrocytomas. This suggests that CNTN1 is of great importance for early screening of brain gliomas.

Nerve cell adhesion molecule 1 (neural cell adhesion molecule, NCAM1) is a member of cell adhesion molecules, belongs to the immunoglobulin superfamily, is mainly expressed in the nervous system, and plays a key role in regulating nerve cell functions and in the nerve migration process. The human NCAM1 gene is positioned at 11q22-23 and is a membrane protein, and comprises three subtypes of NCAM-120, NCAM-140 and NCAM-180, wherein NCAM140 significantly promotes cell proliferation, movement and migration, NCAM1-180 is mainly expressed in nerve fibers, NCAM1-140 is expressed in nerve fibers and brain glial cells, and NCAM1-120 is mainly expressed in glial cells.

The sensitivity and specificity of serum GFAP in diagnosing single glioblastoma was reported to be 76% and 100%, respectively, whereas no data was retrieved regarding the screening or diagnosis of brain glioma with serum CNTN1 and NCAM1.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a combined detection of serum proteins GFAP, CNTN1 and NCAM1 for early screening and early diagnosis of glioma. The results of detecting and analyzing the concentration of CNTN1, GFAP and NCAM1 proteins in serum of patients suggest that the combined application of three serum proteins has important significance for early diagnosis of glioma. Based on the proteomics analysis of serum and tissue samples of normal people and brain gliomas of different grades, the inventor finds that the three proteins GFAP, CNTN1 and NCAM1 are used for combined detection and diagnosis of the brain gliomas, and the specificity and the sensitivity are high.

The present invention provides the use of GFAP, CNTN1 and NCAM1 in the preparation of reagents for early screening and diagnosis of brain glioma.

The invention provides an ELISA kit for early screening and diagnosis of brain glioma, which contains 3 serum protein markers, namely GFAP, CNTN1 and NCAM1.

In some embodiments, the ELISA kit described above is characterized in that the kit contains an ELISA detection kit of GFAP, CNTN1 and NCAM1.

In some embodiments, the ELISA kit described above is characterized in that the ELISA detection kit for GFAP, CNTN1 and NCAM1 is ELISA from abcam, novus, cusabio, respectively.

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

In some embodiments, the ELISA kit described above, characterized in that it consists of: the kit comprises (1) a 96-well ELISA plate, (2) a standard substance, (3) a biotin-labeled antibody and a diluent, (4) horseradish peroxidase-labeled avidin and a diluent, (5) a sample diluent, (6) a substrate solution, (7) a washing solution, (8) a color development solution, (9) a stop solution, and (2) standard substances which are standard proteins of human GFAP, CNTN1 and NCAM1.

The invention provides a method for early screening and early diagnosing glioma by using the ELISA kit, which is characterized in that the method comprises the following steps:

(1) Preparing a serum sample to be detected: centrifuging collected blood at 4deg.C and 2500rpm for 15min, collecting supernatant, discarding the precipitate, collecting supernatant, packaging, storing at-80deg.C, detecting, thawing, centrifuging again to obtain supernatant,

(2) Sample adding: according to three ELISA kit specifications, respectively adding a standard substance and a serum sample to be detected into a 96-well plate, designing a reference group and an experimental group, adding 100ul of the standard substance of CNTN1 and NCAM1 and each hole of the serum sample to be detected, adding 50ul of the standard substance of GFAP and each hole of the serum sample to be detected, and incubating for 60-120 minutes at 37 ℃;

(3) Adding enzyme-labeled antibody: the control and experimental groups were each filled with biotin-labeled CNTN1, NCAM1 antibody solution, 100ul per well, and 50ul per well of biotin-labeled GFAP antibody solution;

(4) Cleaning the antibody liquid: respectively washing the sample with a kit corresponding to the washing liquid for a plurality of times, and performing the next operation after spin-drying;

(5) Diluting the horseradish peroxidase-labeled avidin stock solution with a substrate solution according to the same proportion of the specification to prepare a horseradish peroxidase-labeled avidin working solution, adding 100ul (CNTN 1, NCAM 1) and 50ul (GFAP) of the horseradish peroxidase-labeled avidin working solution into each hole, and incubating at 37 ℃ for 1 hour;

(6) Cleaning working solution: respectively cleaning the working solution of the avidin marked by horseradish peroxidase for 3-5 times and spin-drying;

(7) Color development and termination: adding the color developing solution into a 96-well plate with a volume of 100 ul/well, developing at room temperature or at 35-38 ℃ for 15-30 minutes, and adding a stop solution;

(8) Standard curve: and drawing a standard curve according to the OD value of the standard substance, and finding the content of the serum sample to be detected on the standard curve and carrying out statistics.

The present invention provides a method for early screening and early diagnosis of glioma using the aforementioned ELISA kit, characterized in that the probability of a subject from a sample to develop glioma is judged to be greater when the GFAP concentration is higher than 13.43ng/ml, the CNTN1 concentration is higher than 105.04ng/ml, and the NCAM1 concentration is higher than 1522.57ng/ml, based on the current detection results.

According to the invention, the expression levels of GFAP, CNTN1 and NCAM1 in serum of normal people, brain glioma patients and other cancer patients are detected by ELISA, and the correlation of GFAP, CNTN1 and NCAM1 and the combination of the three with brain glioma is analyzed by a statistical method, so that the serum GFAP, CNTN1 and NCAM1 are a group of good detection indexes of the brain glioma. And the sample is easy, quick and economical to obtain, the wound of the patient is small, and the detection method is simple and easy to implement. The invention is expected to bring new dawn for early screening and diagnosis of glioma, improve early diagnosis rate of glioma, further improve prognosis and reduce death rate. The invention discloses a serum molecular marker for noninvasive early screening of glioma, in particular to combined detection of serum proteins CNTN1, GFAP and NCAM1, which has particularly important effects and clinical application value for early screening and early diagnosis of glioma.

Drawings

Fig. 1: serum GFAP (fig. 1A), CNTN1 (fig. 1B), NCAM1 (fig. 1C) concentration scatter plots for normal adults (22 cases), brain glioma patients (39 cases) and other cancer patients (12 cases), and two-by-two independent sample T test results. Both mapping and statistics were done by statistical software GraphPad Prism 8. * P <0.05, < P <0.01, < P <0.001.ns indicates no statistical difference.

Fig. 2: ROC curve (fig. 2A) GFAP single test of normal adult versus brain glioma patient; (FIG. 2B) CNTN1 singles; (FIG. 2C) NCAM1 singles.

Fig. 3: ROC curves of normal adult versus glioma patients for GFAP, CNTN1 and NCAM1 triple assays.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. Unless otherwise indicated, reagents and materials used in the following examples were commercially available (GFAP, abcam; CNTN1, novus; NCAM1, cusabio).

1. Study object

39 patients with glioma (men: 25 cases, average age 48.0 years; women: 14 cases, average age 52.1 years) and 12 other cancer species (brain metastasis: 3 cases; lung cancer: 3 cases; esophageal cancer: 3 cases; cervical cancer: 3 cases) (men: 4 cases; average age 58.5 years; women: 8 cases, average age 66.1 years) were provided by the center, and 22 normal blood samples were healthy individuals (men: 11 cases, average age 59.0 years; women: 11 cases, average age 57.8 years) who underwent community examination in the same period. The samples for research are collected in the same period, and the sampling, sub-packaging and preserving conditions are consistent.

2. Laboratory instrument and reagent

1. Instrument: infinite M200 Pro multifunctional enzyme labeling instrument (Tecan brand, switzerland), high-speed refrigerated centrifuge (Thermo Fisher Co., USA), incubator, pipettor, refrigerator, timer.

2. Reagent: ELISA test kits for GFAP, CNTN1 and NCAM1 were used, and were obtained from abcam, novus, and Cusabio, respectively, under the respective models ab223867, NBP2-75801 and Catalog No. CSB-EL015511HU.

3. The kit is internally provided with:

(1) 96-hole ELISA 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 liquid

(8) Color development liquid

(9) Stop solution

Wherein the standard substance (2) is standard proteins of human GFAP, CNTN1 and NCAM1.

3. The experimental method comprises the following steps:

1. sample collection

After informed consent of the patients was obtained, blood samples of 39 brain glioma patients and 12 other cancers in hospital patients in the second hospital of university of Hebei medical science were collected as an experimental group, and 22 normal blood samples were provided as a control group for healthy individuals who underwent community physical examination at the same time. The samples for research are collected in the same period, and the conditions of sampling, split charging and preservation are consistent.

2. Sample preparation and sample dilution

(1) Preparing a serum sample to be detected: preparing a serum sample to be detected: centrifuging collected blood at 4deg.C and 2500rpm for 15min, collecting supernatant, discarding the precipitate, packaging the supernatant, and storing at-80deg.C. In the detection, the supernatant was collected by centrifugation again after thawing. Serum samples were diluted at GFAP (1:5 dilution), CNTN1 (1:10 dilution), NCAM1 (1:10 dilution). Temporary storage on ice or at 4 ℃.

(2) And (3) preparation of a standard substance: standards in ELISA kits of GFAP, CNTN1, NCAM1 were dissolved with 1ml of sample dilution, and were diluted by a multiple ratio according to the requirements of 3 kits, respectively.

3. Experimental procedure

(1) And (3) rewarming: firstly, taking out serum samples from a refrigerator at the temperature of minus 80 ℃ for thawing on ice, and then respectively taking out ELISA kits of GFAP, CNTN1 and NCAM1 serum proteins from the refrigerator at the temperature of 4 ℃.

(2) Sample adding: according to the specifications of three ELISA kits, respectively adding a standard substance and a serum sample to be detected into a 96-well plate. A reference group (16 wells/96 wells for standard) and an experimental group (39+12+22 samples for serum samples to be tested, 80 wells/96 wells) were designed, 100ul of the standard for CNTN1, NCAM1 and serum samples to be tested were added per well, and 50ul of the standard for gfap and serum samples to be tested were added per well. Incubation is carried out at 37℃for 60-120 min.

(3) Adding enzyme-labeled antibody: the reference group (16 wells/96 wells as standard) and the experimental group were each filled with biotin-labeled antibodies and dilutions of biotin-labeled CNTN1, NCAM1 antibody, 100ul per well, and 50ul per well of biotin-labeled GFAP antibody.

(4) Cleaning the antibody liquid: and respectively washing the sample with the corresponding washing liquid for a plurality of times by using the kit, and performing the next operation after spin-drying.

(5) The reference group (standard 16 holes/96 holes) and the experimental group are used for diluting the horseradish peroxidase-labeled avidin stock solution with a substrate solution according to the same proportion of the specification to prepare horseradish peroxidase-labeled avidin working solution, 100ul (CNTN 1, NCAM 1) and 50ul (GFAP) of horseradish peroxidase-labeled avidin working solution are added to each hole, and the mixture is incubated for 1 hour at 37 ℃.

(6) Cleaning working solution: and respectively cleaning and spin-drying the working solution of the avidin marked by the horseradish peroxidase for 3-5 times.

(7) Color development and termination: the color developing solution is added into a 96-well plate for 100 ul/well, and the color is developed for 15 to 30 minutes at a proper temperature according to the respective requirements of 3 kits, and then the stop solution is added.

(8) Standard curve: and drawing a standard curve according to the OD value of the standard substance, and finding the content of the serum sample to be detected on the standard curve and carrying out statistics.

4. Data processing

The research data comprises normal blood samples, brain glioma blood samples and other cancer blood samples, and is characterized in that the concentration values of GFAP, CNTN1 and NCAM1 in serum samples obtained through experimental detection are plotted and counted, and the statistics is completed by statistical software GraphPad Prism 8. The subject work characteristic curve (receiver operating characteristic curve, ROC) data statistics and the plotting of ROC standard curves were accomplished using MedCalc software.

5. Analysis of results:

the average concentration of CNTN1 in serum of brain glioma patients (107.17 ng/ml) was higher than that in serum of normal people (101.62 ng/ml) (Table 1-1), with significant statistical significance (P=0.0051); the average concentration of GFAP (38.55 ng/ml) was higher than that in normal human serum (12.35 ng/ml) (table 1-2) and had a significant statistical significance (p=0.0031); the mean concentration of NCAM1 (2217.90 ng/ml) was higher than the mean concentration in normal human serum (1356.82 ng/ml) (tables 1-3), with a significant statistical significance (P=0.0075).

Tables 1-1 to 1-3 below show the mean, standard deviation and corrected P values of serum CNTN1, GFAP and NCAM1 from normal adults, glioma patients and other cancer patients compared in pairs.

TABLE 1-1

TABLE 1-2

Tables 1 to 3

The results in Table 2 show that the single serum proteins of GFAP, CNTN1 and NCAM1 have the specificities of 86.36%, 72.73% and 81.82%, the sensitivities of 66.67%, 71.79% and 64.10% respectively, the triple assay has the specificities of 95.45% and the sensitivities of 74.36% respectively, which are both improved compared with the single assay. Meanwhile, in ROC curves for detecting brain glioma by single serum proteins of GFAP, CNTN1 and NCAM1, AUC values are respectively 0.735, 0.744 and 0.740, P values are respectively 0.0002, 0.0002 and 0.0001, and the method has obvious statistical significance; in the ROC curve of the three-joint test of GFAP, CNTN1 and NCAM1, the AUC value is as high as 0.889, P is less than 0.0001, and the method has obvious statistical significance and is obviously improved compared with single test.

Table 2: results of the separate and Combined detection of the three serum proteins (Normal-Glioma)

As can be seen from fig. 1, in the serum GFAP, CNTN1, NCAM1 concentration scatter diagram of normal adult (22 cases), brain glioma (39 cases) and other cancer (12 cases) patients and the T test of two-by-two independent samples, the serum concentration values of GFAP, CNTN1, NCAM1 of brain glioma patients are significantly higher than that of normal persons, and have statistical differences, while the serum concentration values of the three are not statistically different from that of normal persons in other tumors, thereby further explaining the predictive value of three proteins in brain glioma screening.

According to the results of fig. 2 and 3, when three serum proteins are combined to detect glioma, the area under the ROC curve is increased to 0.889, the P value is less than 0.0001, the statistical significance is obvious, the triple detection ELISA kit has higher diagnostic value on glioma, and the method, namely, the triple detection of serum proteins GFAP, CNTN1 and NCAM1, can be used as a means for early screening glioma.

Experimental results prove that the serum proteins GFAP, CNTN1 and NCAM1 are good detection indexes of brain glioma, and especially the combined detection of the three has early screening and diagnosis values, plays an early warning role, and has potential clinical application values.

Claims (8)

1. Use of gfap, CNTN1 and NCAM1 in the preparation of a reagent for early screening and diagnosis of brain glioma.
2. 2. An ELISA kit for early screening and diagnosis of brain glioma, said kit comprising 3 serum protein markers, GFAP, CNTN1 and NCAM1, respectively.
3. 3. The ELISA kit according to claim 2, characterized in that it contains an ELISA detection kit of GFAP, CNTN1 and NCAM1.
4. 4. The ELISA kit of claim 3, wherein the ELISA detection kit of GFAP, CNTN1 and NCAM1 is ELISA of abcam company, novus company and Cusabio company respectively.
5. 5. The ELISA kit of claim 4, wherein the GFAP detection kit is a kit of abcam model ab223867, the CNTN1 detection kit is a kit of Novus model NBP2-75801, and the NCAM1 ELISA detection kit is a kit of Cusabio model Catalog No. CSB-EL015511HU.
6. 6. The ELISA kit according to claim 2, characterized in that it consists of: the kit comprises (1) a 96-well ELISA plate, (2) a standard substance, (3) a biotin-labeled antibody and a diluent, (4) horseradish peroxidase-labeled avidin and a diluent, (5) a sample diluent, (6) a substrate solution, (7) a washing solution, (8) a color development solution, (9) a stop solution, and (2) standard substances which are standard proteins of human GFAP, CNTN1 and NCAM1.
7. 7. Method for early screening and early diagnosis of glioma using an ELISA kit according to any of claims 2 to 6, characterized in that it comprises the following steps:

   (1) Preparing a serum sample to be detected: centrifuging collected blood at 4deg.C and 2500rpm for 15min, collecting supernatant, discarding the precipitate, collecting supernatant, packaging, storing at-80deg.C, detecting, thawing, centrifuging again, and collecting supernatant;

   (2) Sample adding: according to the specifications of three ELISA kits, respectively adding a standard substance and a serum sample to be detected into a 96-well plate; designing a reference group and an experimental group, adding 100ul of a CNTN1 standard product, a NCAM1 standard product and a serum sample to be detected into each hole, adding 50ul of a GFAP standard product and a serum sample to be detected into each hole, and incubating for 60-120 minutes at 37 ℃;

   (3) Adding enzyme-labeled antibody: the control and experimental groups were each filled with biotin-labeled CNTN1, NCAM1 antibody solution, 100ul per well, and 50ul per well of biotin-labeled GFAP antibody solution;

   (4) Cleaning the antibody liquid: respectively washing the sample with a kit corresponding to the washing liquid for a plurality of times, and performing the next operation after spin-drying;

   (5) Diluting the horseradish peroxidase-labeled avidin stock solution with a substrate solution according to the same proportion of the specification to prepare a horseradish peroxidase-labeled avidin working solution, adding 100ul (CNTN 1, NCAM 1) and 50ul (GFAP) of the horseradish peroxidase-labeled avidin working solution into each hole, and incubating at 37 ℃ for 1 hour;

   (6) Cleaning working solution: respectively cleaning the working solution of the avidin marked by horseradish peroxidase for 3-5 times and spin-drying;

   (7) Color development and termination: adding the color developing solution into a 96-well plate for 100 ul/well, and developing color for 15-30 minutes at room temperature or at 35-38 ℃; then adding a stop solution into the mixture,

   (8) Standard curve: and drawing a standard curve according to the OD value of the standard substance, and finding the content of the serum sample to be detected on the standard curve and carrying out statistics.
8. 8. A method for early screening and early diagnosis of glioma using the kit according to any one of claims 2 to 6 or the method according to claim 7, characterized in that the probability of a subject of sample origin suffering from glioma is judged to be greater when the detection result is higher than 13.43ng/ml, higher than 105.04ng/ml for CNTN1 and higher than 1522.57ng/ml for NCAM1.

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