Application of composition in preparation of oligosaccharide chain ovarian cancer detection reagent

Abstract

The invention provides a detection reagent for detecting ovarian cancer based on an oligosaccharide chain and a preparation method and application thereof, the detection reagent is prepared by mixing the following reagents: a reagent A, which is prepared by adding SDS with the mass concentration of 1-5% into an ammonium bicarbonate solution with the concentration of 10mM and the pH value of 8.3; the reagent B is prepared by mixing 0.05-10 units / 10 microliters of glycosamine acylase, NP-40 with the mass concentration of 10% and an ammonium bicarbonate solution with the concentration of 10 mM and the pH value of 8.3, and the pH value of the mixed solution is 5-9; the reagent C is an organic matter reducing agent with the concentration of 0.02 mM to 1M, which is prepared by dissolving 8-aminopyrene-1, 3, 6-trisulfonic acid in DMSO (Dimethylsulfoxide); and a reagent D: a stop solution. According to the present invention, the natural oligosaccharide N-glycome map in the serum is determined through the reagent, the peak value is quantified, and the statistical analysis is performed, such that the establishment method of the ovarian cancer serum natural oligosaccharide N-glycome map model is provided, and the ovarian cancer is detected by determining the change of the natural glycosylation modification of the protein under the physiological and pathological state.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a method for detecting ovarian cancer, and specifically to a method for detecting ovarian cancer based on a serum oligosaccharide chain G-Test specific fingerprint. Background Art

[0002] Ovarian cancer is a common malignant tumor in women, ranking third in morbidity and first in mortality among female reproductive system tumors. It poses a serious threat to women's health. The ovaries are located deep in the pelvic cavity, and early ovarian lesions often lack specific clinical symptoms, leading to their neglect. By the time symptoms develop and patients seek medical attention, 70% of patients are already in the advanced stage. Therefore, early screening and diagnosis of ovarian cancer are of great significance.

[0003] Commonly used clinical screening methods include serum tumor marker testing, imaging, and histopathology. However, all three screening methods have limitations. For example, the serum tumor markers carbohydrate antigen 125 (CA125) and human epididymis protein 5 (HE4) are the most valuable tumor markers for ovarian cancer and can be used as auxiliary diagnosis. However, their positive rate for diagnosing early-stage ovarian cancer is only 40% to 60%, and there are many factors that influence this. Imaging methods include ultrasound, CT scan, and magnetic resonance imaging (MRI). Ultrasound is the preferred method because it is rapid, economical, noninvasive, and reproducible, but it can be difficult to detect solid tumors when the tumor is small, leading to missed diagnoses. CT scans can localize and characterize pelvic tumors and determine the presence of metastases to the liver, lungs, and retroperitoneal lymph nodes, but cannot distinguish between primary tumors and metastases. MRI has high resolution, multi-planar imaging, and is noninvasive, making it important in the diagnosis and differential diagnosis of pelvic lesions in ovarian cancer. However, MRI is costly, and patients with intrauterine devices must have MRI removed before undergoing MRI. Histopathological examination is the gold standard for diagnosis, but it requires obtaining ovarian tissue, making it an invasive procedure and carrying the risk of secondary infection. These diagnostic methods all have their limitations, and there is an urgent need to explore new, noninvasive methods with high sensitivity and specificity for assisting in the diagnosis of ovarian cancer.

[0004] Protein glycosylation is the most common post-translational modification of proteins. It involves the transfer of sugars to the amino groups of asparagine (ASN) on protein peptide chains under the action of glycosyltransferases to form N-linked glycans or to the hydroxyl oxygen atoms of threonine / serine to form O-linked glycans, which participate in regulating protein function. Most glycoproteins are secreted proteins and are widely present in cell membranes, extracellular matrix, plasma, and mucus. N-glycan chains on proteins regulate protein structure, stability, and activity through processing and modification. Therefore, sugar chains in glycoproteins play an important role in maintaining the body's biological functions, thereby endowing glycoproteins with multiple biological functions. Therefore, understanding changes in sugar chains can help elucidate the molecular mechanisms of abnormal biological behaviors such as inflammation, tumor cell invasion and metastasis to surrounding tissues.

[0005] Abnormal changes in protein N-glycans have been found in various tumors, and alterations in terminal sialic acid modification of N-glycans are one such finding. Sialic acid is a negatively charged, nine-carbon sugar compound that is widely present in organisms and often located at the termini of glycan chains. Sialic acid, linked to galactose or N-acetylgalactosamine on glycan chains through α-2,3 or α-2,6 glycosidic bonds by sialidases, forms polysialic acid chains. Studies have shown that sialic acid at the termini of glycan chains plays an important role in regulating cell recognition, molecular interactions, viral infection, immune responses, and signal transduction. Furthermore, sialic acid modification of cell surface glycoproteins is tissue- and cell-specific. Numerous studies have shown that abnormal sialic acid modification contributes to the development and progression of various diseases, including infectious diseases, and is closely associated with tumor invasion and metastasis. Therefore, detecting alterations in sialic acid-associated oligosaccharide chains has potential clinical value as an aid in the diagnosis of ovarian cancer. Summary of the Invention

[0006] In response to the problems currently existing in clinically used ovarian cancer detection methods, such as the low sensitivity of serological markers C125 and HE4 in early-stage cancer, the risk of misdiagnosis and missed diagnosis with imaging methods, the high cost of some methods, and the invasive nature of histopathological diagnosis, which carries the risk of secondary infection and harm. The present invention provides an ovarian cancer detection reagent that measures the natural oligosaccharide N-glycome profile in serum, quantifies the peak values, and performs statistical analysis, thereby providing a method for establishing a serum natural oligosaccharide N-glycome profile model for ovarian cancer. Ovarian cancer can be detected by measuring changes in the natural glycosylation modification of proteins under physiological and pathological conditions.

[0007] The technical solution adopted in the present invention is as follows:

[0008] A detection reagent for detecting ovarian cancer based on oligosaccharide chains, comprising the following reagents:

[0009] Reagent A: Prepared by adding 1-5% SDS to a 10 mM ammonium bicarbonate solution with a pH of 8.3.

[0010] Reagent B: Prepared by mixing 0.05-10 units / 10 μl of glycosaminoglycans, 10% NP-40, and 10 mM ammonium bicarbonate solution at a pH of 8.3. The pH of the mixed solution is 5-9.

[0011] Reagent C: 8-aminopyrene-1,3,6-trisulfonic acid dissolved in DMSO to prepare an organic reducing agent with a concentration of 0.02 mM to 1 M;

[0012] Reagent D: Stop solution.

[0013] Preferably, the volume ratio of reagent A, reagent B and reagent C is 1:1:1.

[0014] Preferably, the volumes of reagent A, reagent B, and reagent C are all 5 μl.

[0015] Preferably, the reagent D is ultrapure water.

[0016] A method for preparing a detection reagent for detecting ovarian cancer based on oligosaccharide chains comprises the following steps:

[0017] Step 1 Preparation of oligosaccharide chains

[0018] 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 and react at 37°C for 3 h, then dry.

[0019] Step 2: Labeling of oligosaccharide chains

[0020] Add 5 μl of reagent C to the sample obtained after drying in step 1, react at 60°C for 1 hour, and then perform fluorescent labeling. Then, add 100 μl of reagent D to terminate the labeling reaction.

[0021] Step 3: Oligosaccharide chain separation and analysis

[0022] Take 10 μl of the oligosaccharide chain labeled liquid and use an analyzer to separate and detect the N-oligosaccharide chains to obtain the natural oligosaccharide N-glycome profile;

[0023] Step 4: Data processing and analysis

[0024] Peak quantification of the N-glycan profile; the relative content of each peak was calculated by dividing the peak height by the sum of all peak heights.

[0025] A composition is used in the preparation of an oligosaccharide chain ovarian cancer detection reagent. The composition consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F and G2F2 in serum. The composition is used to detect ovarian cancer through the value of (G3S3+G2S2F) / G2S2.

[0026] The G1F is an isomer.

[0027] The present invention provides a method for establishing a serum natural oligosaccharide N-glycome map model for ovarian cancer, and statistical analysis is performed by measuring the serum natural oligosaccharide chain G-Test specific fingerprint map.

[0028] Materials and methods:

[0029] 1. Test samples: serum from healthy individuals and ovarian cancer patients.

[0030] 2. Experimental equipment: capillary electrophoresis analyzer, PCR, centrifuge.

[0031] 3. Reagent preparation:

[0032] 1. Reagent A: Prepared by adding 1-5% SDS to a 10 mM ammonium bicarbonate solution with a pH of 8.3.

[0033] 2. Reagent B: Prepared by mixing 0.05-10 units / 10 μl of glycosaminoglycans, 10% NP-40, and 10 mM ammonium bicarbonate solution at a pH of 8.3. The pH of the mixed solution should be 5-9.

[0034] 3. Reagent C: Prepare an organic reducing agent with a concentration of 0.02 mM to 1 M by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO;

[0035] 4. Reagent D: stop solution.

[0036] 4. N-Glycan Profile Detection

[0037] 1. Preparation of oligosaccharide chains

[0038] 5 μl of reagent A was added to 5 μl of inactivated serum sample for denaturation; after cooling to room temperature, 5 μl of reagent B was added, reacted at 37°C for 3 hours, and then dried.

[0039] 2. Labeling of oligosaccharide chains

[0040] (1) Add 5 μl of reagent C to the dried sample and react at 60°C for 1 h for fluorescent labeling;

[0041] (2) After the fluorescent labeling is completed, add 100 μl of reagent D to terminate the labeling reaction.

[0042] 3. Oligosaccharide chain separation and analysis

[0043] 10 μl of the oligosaccharide chain labeled liquid was taken and N-oligosaccharide chain separation and detection was performed on the ABI3500dx instrument to obtain the natural oligosaccharide N-glycome map.

[0044] 4. Data processing and analysis

[0045] (1) Peak quantification of N-glycome profiles: The peak height of each peak was divided by the sum of the heights of all peaks to calculate the relative content of each peak.

[0046] (2) Analysis of N-glycome data of ovarian cancer patients and healthy people: The N-glycome data of ovarian cancer patients and healthy people were compared and analyzed. The peak height value of each peak was divided by the sum of the heights of all peaks to calculate the relative content of each peak, that is, the peak quantification of the N-glycome spectrum. Then, the 9 N-oligosaccharide chain peaks in the quantified N-glycome spectrum of the ovarian cancer group and the healthy control group were compared and statistically analyzed. The composition of the N-glycome spectrum consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F and G2F2, of which G1F is an isomer; ovarian cancer is detected by the composition (G3S3+G2S2F) / G2S2 value.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] (1) The peaks of the G-Test fingerprint were quantified, and then the relative content of each peak in healthy subjects (106 cases) and ovarian cancer patients (98 cases) was compared and analyzed. It was found that the N-glycan compositions G3S3, G2S2F and G2S2 were significantly different between the two groups (p < 0.05). Therefore, the model based on the composition (G3S3 + G2S2F) / G2S2 had an AUC value of 0.864 ( Figure 2 ), when the cutoff value of the (G3S3+G2S2F) / G2S2 model detection was 4.58, the sensitivity of ovarian cancer detection was 81.20% and the specificity was 76.50%, indicating that the N-glycan composition (G3S3+G2S2F) / G2S2 in serum can be used as a marker for auxiliary diagnosis of ovarian cancer.

[0049] (2) The G-Test method proposed in the present invention is based on the capillary microelectrophoresis technology (DSA-FACE) of a DNA sequencer. After fluorescently labeling the N-glycan chains of glycoproteins in serum samples, they are separated by capillary microelectrophoresis. The glycoprotein content obtained by measuring the fluorescence signal is the natural oligosaccharide N-glycome profile. By detecting the correlation between changes in sialic acid oligosaccharide chains under physiological and pathological conditions and disease status, the present invention can establish a predictive model of N-glycan composition based on these changes to assist in the diagnosis of ovarian cancer status.

[0050] (3) The G-Test natural N-glycome profile model constructed based on the method of the present invention can enable many patients to undergo routine, non-invasive testing, helping doctors and patients to timely monitor the occurrence and progression of ovarian cancer. The detection technology of the present invention has the advantages of high sensitivity, simple operation, trace amount (5μl serum), high repeatability, good stability, and high throughput (96-well plate), and can be promoted and used in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is the serum natural oligosaccharide N-glycome profile of healthy people and ovarian cancer patients;

[0052] Figure 2 This is the ROC curve of the model for identifying ovarian cancer status based on the N-sugar composition (G3S3+G2S2F) / G2S2; the total number of samples tested was 204, including 106 serum samples from healthy people and 98 serum samples from ovarian cancer patients, and the area under the ROC curve AUC = 0.864 was obtained. DETAILED DESCRIPTION

[0053] The present invention will be further described in detail below with reference to the examples and accompanying drawings. It should be noted that the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions or under conditions recommended by the manufacturer.

[0054] The status of ovarian cancer was detected by measuring the serum natural oligosaccharide N-glycome profile and performing statistical analysis. The materials and methods used were as shown in the following examples.

[0055] Example 1

[0056] 1. Test samples: sera from 98 ovarian cancer patients and 106 healthy subjects.

[0057] 2. Experimental equipment: capillary electrophoresis analyzer, PCR, centrifuge.

[0058] 3. Reagent Preparation:

[0059] 1) Reagent A: Prepared by adding 5% SDS to a 10 mM ammonium bicarbonate solution.

[0060] 2) Reagent B: Prepared by mixing 5 units / 10 μl of glycosaminoglycans, 10% NP-40, and 10 mM ammonium bicarbonate solution at a pH of 8.3. The pH of the mixed solution is 6.

[0061] 3) Reagent C: 8-aminopyrene-1,3,6-trisulfonic acid dissolved in DMSO to prepare an organic reducing agent at a concentration of 50 mM;

[0062] 4) Reagent D: ultrapure water.

[0063] 4. N-glycomic profile detection:

[0064] (1) Preparation of oligosaccharide chains

[0065] 5 μl of reagent A was added to 5 μl of inactivated serum sample for denaturation; after cooling to room temperature, 5 μl of reagent B was added, reacted at 37°C for 3 hours, and then dried.

[0066] (2) Labeling of oligosaccharide chains

[0067] 1) Add 5 μl of Reagent C to the dried sample and incubate at 60°C for 1 hour for fluorescent labeling.

[0068] 2) After the fluorescent labeling is completed, add 100 μl of reagent D to terminate the labeling reaction.

[0069] (3) Oligosaccharide chain separation and analysis

[0070] 10 μl of the oligosaccharide chain labeled liquid was taken and N-oligosaccharide chain separation and detection was performed on the ABI3500dx instrument to obtain the natural oligosaccharide N-glycome map.

[0071] (4) Data processing and analysis

[0072] 1) Peak quantification of N-glycome profiles: The relative content of each peak was calculated by dividing the peak height of each peak by the sum of the heights of all peaks.

[0073] 2) Analysis of N-glycome data of healthy subjects and ovarian cancer patients: Comparative analysis of N-glycome data of healthy subjects and ovarian cancer patients. Figure 1 As shown in the figure, the N-glycome map of human serum shows nearly 9 N-oligosaccharide chain peaks. Different oligosaccharide chains exhibit different mobility due to their different charges and molecular sizes. That is, different peaks on the N-glycome map represent different oligosaccharide chains, and the peak height represents the relative content of the oligosaccharide chains. Figure 1Figure A shows the serum N-glycan profile of a healthy individual, and Figure B shows the serum N-glycan profile of an ovarian cancer patient. The N-glycan profile consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F, and G2F2, with G1F being an isomer. The ratio (G3S3 + G2S2F) / G2S2 is calculated to aid in diagnosing ovarian cancer status.

[0074] The model based on the combination (G3S3+G2S2F) / G2S2 achieved an AUC value of 0.864 ( Figure 2 ), when the cutoff value of the (G3S3+G2S2F) / G2S2 model detection was 4.58, it had a sensitivity of 81.20% and a specificity of 76.50% for ovarian cancer detection, indicating that the N-glycan combination group (G3S3+G2S2F) / G2S2 in serum can be used as a marker for the auxiliary diagnosis of ovarian cancer.

[0075] Example 2

[0076] 1. Test samples: sera from 57 ovarian cancer patients and 78 healthy subjects.

[0077] 2. Experimental equipment: capillary electrophoresis analyzer, PCR, centrifuge.

[0078] 3. Reagent Preparation:

[0079] 1) Reagent A: Prepared by adding 1% SDS to a 10 mM ammonium bicarbonate solution.

[0080] 2) Reagent B: Prepared by mixing 0.05 units / 10 μl of glycosaminoglycans, 10% NP-40, and 10 mM ammonium bicarbonate solution at a pH of 8.3. The pH of the solution is 5.

[0081] 3) Reagent C: 8-aminopyrene-1,3,6-trisulfonic acid dissolved in DMSO to prepare an organic reducing agent at a concentration of 0.02 mM;

[0082] 4) Reagent D: ultrapure water.

[0083] 4. N-glycome profile detection:

[0084] (1) Preparation of oligosaccharide chains

[0085] 5 μl of reagent A was added to 5 μl of inactivated serum sample for denaturation; after cooling to room temperature, 5 μl of reagent B was added, reacted at 37°C for 3 h, and then dried.

[0086] (2) Labeling of oligosaccharide chains

[0087] 1) Add 5 μl of Reagent C to the dried sample and incubate at 60°C for 1 hour for fluorescent labeling.

[0088] 2) After the fluorescent labeling is completed, add 100 μl of reagent D to terminate the labeling reaction.

[0089] (3) Oligosaccharide chain separation and analysis

[0090] 10 μl of the oligosaccharide chain labeled liquid was taken and N-oligosaccharide chain separation and detection was performed on the ABI3500dx instrument to obtain the natural oligosaccharide N-glycome map.

[0091] (4) Data processing and analysis

[0092] 1) Peak quantification of N-glycome profiles: The relative content of each peak was calculated by dividing the peak height of each peak by the sum of the heights of all peaks.

[0093] 2) Analysis of N-glycome data of healthy subjects and ovarian cancer patients: Comparative analysis of N-glycome data of healthy subjects and ovarian cancer patients. Figure 1 As shown in the figure, the N-glycome map of human serum shows nearly 9 N-oligosaccharide chain peaks. Different oligosaccharide chains exhibit different mobility due to their different charges and molecular sizes. That is, different peaks on the N-glycome map represent different oligosaccharide chains, and the peak height represents the relative content of the oligosaccharide chains. Figure 1 Figure A shows the serum N-glycan profile of a healthy individual, and Figure B shows the serum N-glycan profile of an ovarian cancer patient. The N-glycan profile consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F, and G2F2, with G1F being an isomer. The ratio (G3S3 + G2S2F) / G2S2 is calculated to aid in diagnosing ovarian cancer status.

[0094] To further verify the accuracy of the model, serum normal control group sample data from 57 ovarian cancer patients and 78 healthy controls were used to verify the model established based on the combination (G3S3 + G2S2F) / G2S2. Based on Example 1, when the cutoff value was 4.58, the discrimination was good. The sample model in this example was verified, and the sensitivity for ovarian cancer detection was 80.70% and the specificity was 76.92%, indicating that the N-glycan combination group (G3S3 + G2S2F) / G2S2 in serum can be used as a marker for auxiliary diagnosis of ovarian cancer. The verification results are shown in Table 1.

[0095] Table 1 Validation results of 57 ovarian cancer patients and 78 healthy subjects

[0096] type Sensitivity Specificity Ovarian cancer 80.70％(45 / 57) - healthy - 76.92％(60 / 78)

[0097] Example 3

[0098] 1. Test samples: sera from 98 ovarian cancer patients and 106 healthy subjects.

[0099] 2. Experimental equipment: capillary electrophoresis analyzer, PCR, centrifuge.

[0100] 3. Reagent Preparation:

[0101] 1) Reagent A: Prepared by adding 3% SDS to a 10 mM ammonium bicarbonate solution.

[0102] 2) Reagent B: Prepared by mixing 10 units / 10 μl of glycosaminoglycans, 10% NP-40, and 10 mM ammonium bicarbonate solution at a pH of 8.3. The pH of the solution is 9.

[0103] 3) Reagent C: 8-aminopyrene-1,3,6-trisulfonic acid dissolved in DMSO to prepare an organic reducing agent at a concentration of 1 M;

[0104] 4) Reagent D: ultrapure water.

[0105] 4. N-glycome profile detection was the same as in Example 1.

[0106] Compared to existing technologies, this detection technology can detect changes in sialic acid oligosaccharide chains under physiological and pathological conditions and correlate them with disease status. Based on these changes, a predictive model of N-glycan composition can be established to assist in the diagnosis of ovarian cancer. The G-Test natural N-glycome profile model constructed based on the method of this invention can enable a large number of patients to undergo routine, non-invasive testing, helping doctors and patients to timely monitor the occurrence and progression of ovarian cancer, and is suitable for widespread clinical use.

[0107] The specific embodiments described above in conjunction with the accompanying drawings further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above is only a specific embodiment of the present invention, but is not a limitation on the scope of protection of the present invention. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. that are made within the spirit and principles of the present invention without creative work should be included in the scope of protection of the present invention.

Claims

1. Use of a composition in the preparation of an oligosaccharide chain ovarian cancer detection reagent, characterized in that: The composition consists of G4S4, G3S3, G2S2, G2S2F, G2S1, G2S1F, G1F and G2F2 in serum, and the composition detects ovarian cancer through the value of (G3S3+G2S2F) / G2S2.

2. Use of a composition according to claim 1 in the preparation of an oligosaccharide chain ovarian cancer detection reagent, characterized in that: The G1F is an isomer.

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