CN116183921A - Detection reagent for detecting pancreatic cancer based on oligosaccharide chain, preparation method and application - Google Patents

Abstract

The invention provides a detection reagent for detecting pancreatic cancer based on oligosaccharide chains, a preparation method and application thereof, wherein the detection reagent is prepared by mixing the following reagents: adding SDS with the mass concentration of 1-5% into ammonium bicarbonate solution with the concentration of 10mM and the pH of 8.3; reagent B: is prepared by mixing 0.05-10 units/10 mul of sugar amine acyl enzyme, 10% mass concentration NP-40 and 10mM ammonium bicarbonate solution with pH value of 8.3, wherein the pH value of the mixed solution is 5-9; reagent C: dissolving 8-aminopyrene-1, 3, 6-trisulfonic acid in DMSO to prepare an organic reducing agent with the concentration of 0.02 mM-1M; reagent D: and (5) stopping liquid. The invention provides a method for establishing a serum natural oligosaccharide N-glycome pattern model of pancreatic cancer, which is characterized in that the reagent is used for measuring the natural oligosaccharide N-glycome pattern in serum, and peak value is quantized and statistically analyzed, and the pancreatic cancer is detected by measuring the change of natural glycosylation modification of protein under physiological and pathological states.

Description

A detection reagent, preparation method and application for detecting pancreatic cancer based on oligosaccharide chains

technical field

The invention belongs to the technical field of biomedicine and relates to a method for detecting pancreatic cancer, in particular to a method for detecting pancreatic cancer based on the specific fingerprint of serum oligosaccharide chain G-Test.

Background technique

Pancreatic cancer is a highly malignant tumor of the digestive tract that is difficult to diagnose and treat. About 90% of them are ductal adenocarcinomas originating from the ductal epithelium. The early diagnosis rate of pancreatic cancer is not high, the operative mortality rate is high, and the cure rate is very low. It is also one of the malignant tumors with the worst prognosis, and the 5-year survival rate is less than 5%. The onset of pancreatic cancer is hidden, and the early symptoms are not typical. It often manifests as upper abdominal discomfort, low back pain, indigestion or diarrhea, etc. It is easy to be confused with other digestive system diseases, and most of the symptoms are in the middle and late stages. The pathological classification of pancreatic cancer is based on histological structure and cell biological characteristics. Different types of pancreatic cancer have different morphological structures and biological behaviors, as well as different epidemiological and molecular mechanisms. Therefore, the existing pancreatic cancer There are many pathological classification systems. Among all types of pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC) is the most common, accounting for 85%-90%, and the overall 5-year survival rate of patients is less than 5%, which has not improved in the past 50 years . Other types of pancreatic tumors account for about 10% -15% of pancreatic tumors. Due to the early metastasis, rapid growth and deep location of pancreatic cancer, the symptoms and signs are not typical, so it is easy to be missed or misdiagnosed clinically.

The clinical diagnosis of pancreatic cancer mainly includes imaging detection and tumor marker detection. Imaging examinations, including CT and MRI, are used as auxiliary examinations for the diagnosis of pancreatic cancer, and their specificity needs to be further improved. The currently available diagnostic kits for pancreatic cancer are designed to detect some common tumor markers, such as C19-9, carcinoembryonic antigen CEA, etc., with low sensitivity and accuracy. Single detection often has great limitations, and it is difficult to meet the requirements of rapid diagnosis. The above-mentioned diagnostic methods have their own limitations, and there is an urgent need to explore new non-invasive methods for auxiliary diagnosis of pancreatic cancer with high sensitivity and specificity.

Glycoproteins are a class of binding proteins formed by post-translational modification of proteins, namely glycosylation. Protein glycosylation (Glycosylation) is the most common post-translational modification of proteins. It is a process in which sugars are transferred to proteins and special amino acid residues on proteins to form glycosidic bonds under the action of glycosyltransferases. Most glycoproteins are secreted proteins, widely present in cell membranes, interstitial cells, plasma, and mucus. N-sugar chains on proteins regulate the structure, stability and activity of proteins through processing and modification, so the sugar chains in glycoproteins play an important role in maintaining the biological functions of the body, thus endowing glycoproteins with various biological functions. Therefore, understanding the changes in sugar chains is helpful to elucidate the molecular mechanisms of abnormal biological behaviors such as inflammation, tumor cell invasion and metastasis of surrounding tissues.

At present, abnormal changes of protein N-sugar chains have been found in various tumors, and the modification of the terminal sialic acid of N-sugar chains is one of them. Sialic acid is a kind of negatively charged 9-carbon sugar compound, which widely exists in organisms and is often located at the end of the glycan chain; under the action of sialidase, it binds to Galactose or N-acetylgalactosamine on the sugar chains are linked to form polysialic acid chains. Studies have found that the sialic acid at the end of the glycan chain plays an important role in regulating cell mutual recognition, molecular interaction, virus infection, immune response and signal transduction, and the sialic acid modification of cell surface glycoproteins has tissue and cell specificity. Many studies have shown that abnormal sialic acid modification is involved in the occurrence and development of diseases, and is closely related to various diseases such as infectious diseases and tumor invasion and metastasis. Therefore, the detection of changes in sialic acid-related oligosaccharides has potential clinical value for the diagnosis of diseases and the development of therapeutic drugs as targets.

Contents of the invention

Aiming at the problems existing in the detection of pancreatic cancer currently used clinically, such as low specificity and accuracy of blood test and imaging detection, traumatic pathological examination, poor patient dependence, etc., the present invention provides a pancreatic cancer detection reagent, Use this reagent to measure the N-glycan profile of natural oligosaccharides in serum, and then quantify the peak value and perform statistical analysis to provide a method for establishing a model of the N-glycan profile of natural oligosaccharides in serum of pancreatic cancer. Pancreatic cancer is detected by measuring changes in the natural glycosylation modification of proteins under physiological and pathological conditions.

The technical scheme that the present invention adopts is as follows:

A detection reagent for detecting pancreatic cancer based on oligosaccharide chains, consisting of the following reagents:

Reagent A: prepared by adding SDS with a mass concentration of 1 to 5% in an ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3;

Reagent B: It is prepared by mixing 0.05-10 units/10 μl of glucosamidase, NP-40 with a mass concentration of 10%, and an ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3, and the pH value of the mixed solution is 5-9;

Reagent C: Dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO to make a reducing agent with a concentration of 0.02mM～1M;

Reagent D: stop solution.

Preferably, the volume ratio of the reagent A, reagent B and reagent C is 1:1:1.

Preferably, the volumes of reagent A, reagent B and reagent C are all 5 μl.

Preferably, the reagent D is ultrapure water.

A method for preparing a reagent for detecting pancreatic cancer based on oligosaccharide chains, comprising the following steps:

Step 1 Preparation of oligosaccharide chains

Add 5 μl of reagent A to 5 μl of inactivated serum sample for denaturation, after cooling to room temperature, add 5 μl of reagent B, react at 37°C for 3 hours, and then dry;

Labeling of step two oligosaccharide chains

Add 5 μl of reagent C to the sample obtained after drying in step 1, react for 1 hour at 60°C for fluorescent labeling, then add 100 μl of reagent D to terminate the labeling reaction;

Step 3 Oligosaccharide Chain Separation Analysis

Take 10 μl of the liquid labeled with oligosaccharide chains, and use an analyzer to separate and detect N-oligosaccharide chains to obtain the N-glycan map of natural oligosaccharides;

Step 4 Data processing and analysis

Peak quantification of the N-glycan profile: divide the peak height value of each peak by the sum of the heights of all peaks to calculate the relative content of each peak.

Application of a composition in the preparation of oligosaccharide chain pancreatic cancer detection reagents, the composition is composed of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F and G2F2 in serum, and the composition passes through G2S2/G3S3 +G2S2/G2S2F values to detect pancreatic cancer.

The G1F is an isomer.

The invention provides a method for establishing a serum natural oligosaccharide N-glycan group pattern model of pancreatic cancer. Statistical analysis is carried out by measuring the serum natural oligosaccharide chain G-Test specific fingerprint.

Materials and methods:

1. Test samples: Serum from patients with pancreatic cancer and healthy people.

2. Experimental equipment: capillary electrophoresis analyzer, PCR, centrifuge.

3. Reagent preparation:

1. Reagent A: prepared by adding SDS with a mass concentration of 1 to 5% in an ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3;

2. Reagent B: It is prepared by mixing 0.05-10 units/10 μl of glucosamidase, NP-40 with a mass concentration of 10%, and ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3. The pH value of the mixed solution is 5-5 9;

3. Reagent C: Dissolve 8-aminopyrene-1,3,6-trisulfonic acid in DMSO to make a reducing agent with a concentration of 0.02mM～1M;

4. Reagent D: stop solution.

4. Detection of N-glycan profile

1. Preparation of oligosaccharide chains

Add 5 μl reagent A to the inactivated 5 μl serum sample for denaturation; after cooling to room temperature, add 5 μl reagent B, react at 37°C for 3 hours, and then dry.

2. Labeling of oligosaccharide chains

(1) Add 5 μl of reagent C to the dried sample, react at 60°C for 1 hour, and perform fluorescent labeling;

(2) After the fluorescent labeling is completed, add 100 μl of reagent D to terminate the labeling reaction.

3. Separation analysis of oligosaccharide chains

Take 10 μl of the oligosaccharide chain-labeled liquid, and perform N-oligosaccharide chain separation and detection under the ABI3500dx instrument, so as to obtain the natural oligosaccharide N-glycan map.

4. Data processing and analysis

(1) Peak quantification of the N-glycan profile: divide the peak height value of each peak by the sum of the heights of all peaks to calculate the relative content of each peak.

(2) Analysis of N-glycan group data of pancreatic cancer patients and healthy people: The N-glycan group data of pancreatic cancer patients and healthy people were compared and analyzed. Divide the peak height of each peak by the sum of all peak heights, and quantitatively calculate the relative content of each peak, that is, the peak quantification of the N-glycan group map, and then quantify the pancreatic cancer group and the healthy control group N- The 9 N-oligosaccharide chain peaks in the glycome map were compared and analyzed statistically. The composition of the N-glycan profile consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F and G2F2, where G1F is an isomer; the pancreas is detected by the composition G2S2/G3S3+G2S2/G2S2F value cancer.

Compared with prior art, the beneficial effect of the present invention:

(1) Quantify each peak of the G-Test fingerprint, and then compare and analyze the relative content of each peak in pancreatic cancer patients (152 cases) and healthy people (143 cases), and find that the N-glycan composition G2S2, G3S3 As well as G2S2F, there is a significant statistical difference between the two groups (p<0.05); therefore, the model established based on the composition G2S2/G3S3+G2S2/G2S2F can achieve an AUC value of 0.875 under the ROC curve when distinguishing pancreatic cancer patients (Figure 2) , when the cutoff value detected by the G2S2/G3S3+G2S2/G2S2F model is 4.57, the detection of pancreatic cancer has a sensitivity of 82.30% and a specificity of 81.20%, indicating that the N-glycan composition in serum is G2S2/G3S3+G2S2 /G2S2F can be used as a marker for auxiliary diagnosis of pancreatic cancer.

(2) The G-Test detection method proposed by the present invention is based on the capillary microelectrophoresis technology (DSA-FACE) of a DNA sequencer. After fluorescently labeling the N-sugar chains of glycoproteins in serum samples, they are separated by capillary microelectrophoresis. The content of glycoprotein obtained by measuring the fluorescent signal is the natural oligosaccharide N-glycan map. The present invention detects the correlation between changes in sialic acid oligosaccharide chains and disease states under physiological and pathological conditions, and can establish a predictive model of N-glycan composition based on these changes to assist in the diagnosis of pancreatic cancer.

(3) The G-Test natural N-glycan map model constructed based on the method of the present invention can allow many patients to receive routine and non-invasive detection, and help doctors and patients monitor the occurrence and disease progression of pancreatic cancer in time. The detection technology of the present invention It has the advantages of high sensitivity, simple operation, micro volume (5 μl serum), high repeatability, good stability, high throughput (96-well plate), etc., and can be popularized and used in clinical practice.

Description of drawings

Fig. 1 is the serum natural oligosaccharide N-glycan map of pancreatic cancer patients and healthy people;

Figure 2 is the ROC curve of the differential pancreatic cancer state model established based on the N-sugar composition G2S2/G3S3+G2S2/G2S2F; the total number of detection samples is 295 cases, including 152 cases of serum samples from pancreatic cancer patients and 143 cases of serum samples from healthy people. The area under the ROC curve AUC=0.875 was obtained.

Detailed ways

The present invention will be described in further detail below in conjunction with the embodiments and accompanying drawings. It should be noted that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods that do not specify specific conditions in the following examples, they are usually tested according to conventional conditions, or according to the conditions suggested by the manufacturer.

The state of pancreatic cancer is detected by measuring the N-glycan profile of serum natural oligosaccharides and performing statistical analysis. The materials and methods used are as in the following examples.

Embodiment one

1. Test samples: serum from pancreatic cancer patients and healthy people.

2. Experimental equipment: capillary electrophoresis analyzer, PCR, centrifuge.

3. Reagent preparation:

1) Reagent A: prepared by adding SDS with a mass concentration of 2.5% into ammonium bicarbonate solution with a concentration of 10 mM;

2) Reagent B: prepared by mixing 3 units/10 μl of glucosaminylase, NP-40 with a mass concentration of 10%, and ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3, and the pH value of the solution is 7;

3) Reagent C: an organic reducing agent prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO at a concentration of 10 mM;

4) Reagent D: ultrapure water.

4. N-glycan map detection:

(1) Preparation of oligosaccharide chains

Add 5 μl reagent A to the inactivated 5 μl serum sample for denaturation; after cooling to room temperature, add 5 μl reagent B, react at 37°C for 3 hours, and then dry.

(2) Labeling of oligosaccharide chains

1) Add 5 μl of reagent C to the dried sample, react at 60°C for 1 hour, and perform fluorescent labeling;

2) After the fluorescent labeling is completed, add 100 μl of reagent D to terminate the labeling reaction.

(3) Separation analysis of oligosaccharide chains

Take 10 μl of the oligosaccharide chain-labeled liquid, and perform N-oligosaccharide chain separation and detection under the ABI3500dx instrument, so as to obtain the natural oligosaccharide N-glycan map.

(4) Data processing and analysis

1) Peak quantification of the N-glycan profile: divide the peak height value of each peak by the sum of the heights of all peaks to calculate the relative content of each peak.

2) Analysis of N-glycan group data of pancreatic cancer patients and healthy people: The N-glycan group data of pancreatic cancer patients and healthy people were compared and analyzed. As shown in Figure 1, the N-glycan profile of human serum shows nearly nine peaks of N-oligosaccharide chains, and different oligosaccharide chains show different mobility due to the different charges and molecular sizes, that is, the Different peaks on the N-glycan map represent different oligosaccharide chains, and the peak heights represent the relative content of oligosaccharide chains. In Figure 1, A is the serum N-glycan profile of healthy people, and B is the serum N-glycan profile of pancreatic cancer patients. The composition of the N-glycan profile consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F, and G2F2, where G1F is an isomer; aided by calculating the composition G2S2/G3S3+G2S2/G2S2F value Determine the status of pancreatic cancer.

The model established based on the composition G2S2/G3S3+G2S2/G2S2F has an AUC value of 0.875 under the ROC curve when distinguishing patients with pancreatic cancer (Figure 2). The detection of cancer has a sensitivity of 82.30% and a specificity of 81.20%, indicating that the N-glycan composition G2S2/G3S3+G2S2/G2S2F in serum can be used as a marker for auxiliary diagnosis of pancreatic cancer.

Embodiment two

1) Reagent A: prepared by adding SDS with a mass concentration of 1% to ammonium bicarbonate solution with a concentration of 10 mM;

2) Reagent B: prepared by mixing 0.05 units/10 μl of glucosaminylase, NP-40 with a mass concentration of 10%, and ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3, and the pH value of the solution is 5;

3) Reagent C: an organic reducing agent prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO with a concentration of 0.02mM;

4) Reagent D: ultrapure water.

4. The detection of the N-glycan profile is the same as in Example 1.

Embodiment three

1) Reagent A: prepared by adding SDS with a mass concentration of 5% into ammonium bicarbonate solution with a concentration of 10 mM;

2) Reagent B: prepared by mixing 10 units/10 μl of glucosaminylase, NP-40 with a mass concentration of 10%, and ammonium bicarbonate solution with a concentration of 10 mM and a pH of 8.3, and the pH value of the solution is 9;

3) Reagent C: an organic reducing agent prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO;

4) Reagent D: ultrapure water.

4. The detection of the N-glycan profile is the same as in Example 1.

Compared with the existing technology, this detection technology detects the correlation between changes in sialic acid oligosaccharide chains and disease states under physiological and pathological conditions, and based on these changes, a prediction model of N-glycan composition can be established to assist in the judgment of pancreatic cancer state. The G-Test natural N-glycan profile model constructed based on the method of the present invention can allow many patients to receive routine and non-invasive testing, help doctors and patients monitor the occurrence and disease progression of pancreatic cancer in a timely manner, and can be popularized and used in clinical practice.

The purpose, technical solutions and beneficial effects of the present invention have been further described in detail in conjunction with the specific embodiments described in the accompanying drawings. For the limitation of the scope of protection, those skilled in the art should understand that any modification, equivalent replacement, improvement, etc. that can be made within the spirit and principles of the present invention without creative work shall be included in the protection of the present invention. within range.

Claims (7)

1. A reagent for detecting pancreatic cancer based on oligosaccharide chains, which is characterized by comprising the following reagents:

reagent A: adding SDS with the mass concentration of 1-5% into ammonium bicarbonate solution with the concentration of 10mM and the pH of 8.3;

reagent B: is prepared by mixing 0.05-10 units/10 mul of sugar amine acyl enzyme, 10% mass concentration NP-40 and 10mM ammonium bicarbonate solution with pH value of 8.3, wherein the pH value of the mixed solution is 5-9;

reagent C: dissolving 8-aminopyrene-1, 3, 6-trisulfonic acid in DMSO to prepare an organic reducing agent with the concentration of 0.02 mM-1M;

reagent D: and (5) stopping liquid.

2. The reagent for detecting pancreatic cancer based on oligosaccharide chains according to claim 1, wherein the volume ratio of reagent a, reagent B to reagent C is 1:1:1.

3. The reagent for detecting pancreatic cancer based on oligosaccharide chains according to claim 2, wherein the volumes of the reagent A, the reagent B and the reagent C are each 5. Mu.l.

4. The reagent for detecting pancreatic cancer based on oligosaccharide chains according to claim 1, wherein the reagent D is ultrapure water.

5. The method for preparing a reagent for detecting pancreatic cancer based on oligosaccharide chains according to claim 1, comprising the steps of:

step one preparation of oligosaccharide chains

Adding 5 mu l of reagent A into 5 mu l of serum sample subjected to inactivation treatment, carrying out denaturation, cooling to room temperature, adding 5 mu l of reagent B, reacting for 3 hours at 37 ℃, and then drying;

step labelling of Di-oligosaccharide chains

Adding 5 μl of reagent C into the sample obtained after drying in the first step, performing fluorescent labeling after reacting at 60 ℃ for 1h, and then adding 100 μl of reagent D to terminate the labeling reaction;

step three oligosaccharide chain separation analysis

Taking 10 mu l of liquid marked by oligosaccharide chains, and carrying out N-oligosaccharide chain separation detection by using an analyzer to obtain a natural oligosaccharide N-sugar group map;

step four data processing analysis

Peak quantification of N-glycoset profile: the peak height value of each peak is divided by the sum of the heights of all peaks, and the relative content of each peak is calculated.

6. Use of a composition consisting of G4S4, G3S3, G2S2F, G S1, G2S1F, G1F and G2F2 in serum for the preparation of an oligosaccharide chain pancreatic cancer detection reagent, said composition detecting pancreatic cancer by the values of G2S2/G3s3+g2s2/G2S 2F.

7. The use of a composition according to claim 6, wherein G1F is an isomer in the preparation of an oligosaccharide chain pancreatic cancer detection reagent.

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