WO2023040911A1 - Réactif de détection du carcinome du rectum et son application dans la détection du carcinome du rectum - Google Patents

Réactif de détection du carcinome du rectum et son application dans la détection du carcinome du rectum Download PDF

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WO2023040911A1
WO2023040911A1 PCT/CN2022/118798 CN2022118798W WO2023040911A1 WO 2023040911 A1 WO2023040911 A1 WO 2023040911A1 CN 2022118798 W CN2022118798 W CN 2022118798W WO 2023040911 A1 WO2023040911 A1 WO 2023040911A1
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reagent
preparation
intestinal cancer
prepared
detection
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PCT/CN2022/118798
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English (en)
Chinese (zh)
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陈翠英
王蕾
谈宗男
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江苏先思达生物科技有限公司
先思达（南京）生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/38—Post-translational modifications [PTMs] in chemical analysis of biological material addition of carbohydrates, e.g. glycosylation, glycation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases

Definitions

  • the invention belongs to the technical field of biomedicine and relates to a method for detecting intestinal cancer, in particular to a method for detecting intestinal cancer based on the specific fingerprint of serum glycoprotein oligosaccharide chain detection (G-Test).
  • G-Test serum glycoprotein oligosaccharide chain detection
  • Intestinal cancer (Carcinoma ofrectum) is a common malignant tumor, including colon cancer and rectal cancer.
  • the incidence of bowel cancer from high to low is rectum, sigmoid colon, cecum, ascending colon, descending colon and transverse colon. etc. are closely related.
  • the age of onset of colorectal cancer tends to be older, and the ratio of male to female is 1.65:1.
  • the occurrence of intestinal cancer is related to high-fat and low-fiber diet, chronic inflammation of the large intestine, colorectal adenoma, genetic factors and other factors such as: schistosomiasis, pelvic radiation, environmental factors (such as lack of molybdenum in the soil), smoking and so on.
  • Colon cancer in the late stage often invades surrounding tissues and organs, such as bladder and prostate and other adjacent tissues, causing frequent urination, urgency and dysuria. Invasion of the presacral plexus, sacral coccygeal and lumbar pain. Rectal cancer can also metastasize to the liver, causing hepatomegaly, ascites, jaundice, and even cachexia. In addition, among the complications of colorectal cancer surgery, there are ureteral injury, stoma necrosis, and internal hernia.
  • the clinical diagnosis of intestinal cancer mainly includes digital bowel examination, colonoscopy, pathological examination, carcinoembryonic antigen (CEA) determination, contrast contrast contrast radiography with barium enema, B-ultrasound examination, telomerase activity detection, and computerized tomography (CT) scan for intestinal cancer. ) detection, magnetic resonance imaging (MRI) examination, etc.
  • CT computerized tomography
  • CEA Tumor marker carcinoembryonic antigen detection, although helpful for the diagnosis of tumors.
  • CEA tumor marker carcinoembryonic antigen
  • diseases in other tissues can also cause CEA to increase.
  • a high level of CEA does not indicate the presence of distant metastasis; there are a small number of patients with metastatic tumors, and CEA does not increase.
  • CEA examination has no specific diagnostic value, and has certain false positives and false negatives, so it is not suitable for general screening or early diagnosis, but it is helpful for estimating prognosis, observing curative effect and recurrence.
  • the current misdiagnosis rate of rectal cancer is as high as more than 60%, so there is an urgent need for high-sensitivity, high-specificity new biomarkers for non-invasive and rapid diagnosis methods to assist early diagnosis.
  • Protein 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. Some enzymes and hormones are glycoproteins. Glycoproteins have a variety of biological functions. Some glycoproteins such as trocollagen are structural proteins.
  • glycoproteins are glycoproteins
  • fiber Proproteins are glycoproteins
  • Lectins have the ability to aggregate cells, and sugar chains can also stabilize peptide chains. Another important function of glycoprotein is to directly or indirectly participate in various recognition phenomena on the cell surface.
  • sugar chains Due to the importance of sugar chains in glycoproteins for maintaining biological functions of the body, changes in sugar chains help to elucidate the molecular mechanisms of abnormal biobehaviors such as inflammation, tumor cell invasion and metastasis of surrounding tissues. At present, changes in N-glycan chains have been found in various tumors.
  • Sugar chains are important bioinformatics molecules that play unique roles in many physiological and pathological processes.
  • the sugar chain structure is very complex and has microscopic heterogeneity. Its analysis and structural elucidation have always been the bottleneck of glycobiology research.
  • the analysis methods of sugar chain structure are developing rapidly, mainly including (1) high performance liquid chromatography (HPLC): high resolution, fast detection speed, high repeatability, high performance liquid chromatography column can be used repeatedly, but column efficiency will vary with time
  • HPLC high performance liquid chromatography
  • MS mass spectrometry
  • mass spectrometry mass spectrometry has high sensitivity, can be It is an ideal method for the qualitative and quantitative analysis of sugar chains due to the advantages of obtaining a variety of structural information and being suitable for analyzing mixtures.
  • capillary electrophoresis capillary electrophoresis is low in cost, high in column efficiency, high in sensitivity, fast in speed, and easy to inject. The amount is small and the operation is simple, but the repeatability is not high and the stability is not as good as HPLC.
  • the G-Test detection method is based on the capillary microelectrophoresis technology (DSA-FACE) of the DNA analyzer. After the N-sugar chain of the glycoprotein in the prostatic fluid sample is fluorescently labeled, it is separated by capillary microelectrophoresis. The content of the N-oligosaccharide chain obtained by measuring the fluorescent signal is the fingerprint spectrum (G-Test spectrum for short).
  • This detection technology has the advantages of high sensitivity, simple operation, trace volume (2 ⁇ L serum), high repeatability, good stability, high throughput (96-well plate) and other sugar chain analysis technologies, and is suitable for general laboratory departments. It is expected to be used in clinical promotion.
  • the serum G-Test glycomics detection technology applied in the present invention just satisfies these conditions, and therefore has a good application prospect.
  • the purpose of the present invention is to provide a reagent for detecting intestinal cancer, by which the glycome profile in serum is measured, the peak value is quantified, and statistical analysis is performed, thereby providing a method for establishing a serum glycome profile model of intestinal cancer.
  • a colorectal cancer detection reagent consisting of the following reagents:
  • Reagent A prepared by adding SDS with a mass concentration of 0.5 to 5% in ammonium bicarbonate solution with a concentration of 10 mM;
  • Reagent B It is prepared by mixing 0.01 ⁇ 10U/10 ⁇ L glucosamidase and 0.01 ⁇ 10U/10 ⁇ L sialidase, and the pH value of the mixed solution is 4 ⁇ 9;
  • Reagent C Prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO, the concentration is 0.01mM ⁇ 1M;
  • Reagent D stop solution.
  • the volume ratio of the reagent A, reagent B and reagent C is 2:2:1.
  • a preparation method for intestinal cancer detection reagent comprising the following steps:
  • the denaturation temperature in the preparation of the step 1 oligosaccharide is not lower than 75°C, and the incubation temperature is not lower than 25°C.
  • the temperature of fluorescent labeling in the second step is 50-90°C.
  • composition detects intestinal cancer through the ratio of (NG1A2F+NA3)/NA2FB.
  • the invention provides a method for establishing a serum glycoprotein N-glycan group pattern model of intestinal cancer, and performs statistical analysis by measuring the specific fingerprint pattern of the serum glycoprotein oligosaccharide chain G-Test.
  • Test samples Serum from colorectal cancer patients and normal controls.
  • Reagent A prepared by adding SDS with a mass concentration of 0.5 to 5% in ammonium bicarbonate solution with a concentration of 10 mM;
  • Reagent B It is prepared by mixing 0.01 ⁇ 10U/10 ⁇ L glucosamidase and 0.01 ⁇ 10U/10 ⁇ L sialidase, and the pH value of the mixed solution is 4 ⁇ 9;
  • Reagent C Prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO, the concentration is 0.01mM ⁇ 1M;
  • Reagent D stop solution.
  • the stop solution is deionized water.
  • the volume ratio of the reagent A, reagent B and reagent C is 2:2:1.
  • the preparation method of the intestinal cancer detection reagent comprises the following steps:
  • the composition detects intestinal cancer by the value of (NG1A2F+NA3)/NA2FB.
  • the method of the present invention adopts the G-Test detection method with high sensitivity, simple operation, only needs a small amount of sample, high repeatability, good stability and high throughput, and establishes the G-Test with significant difference between patients with intestinal cancer and normal controls. Test-specific fingerprint model.
  • the N-glycan profile of the serum to be tested is calculated using the profile model established by this method, which can detect whether the sample is colon cancer. Compared with the prior art, it has higher specificity and accuracy, and the sensitivity and specificity of detecting intestinal cancer reach 85.3% and 87.6% respectively.
  • the G-Test specific fingerprint model constructed based on the method of the present invention can allow many patients to receive routine and non-invasive testing, and help doctors and patients to detect the occurrence and progression of intestinal cancer in a timely manner, and is expected to be popularized and used in clinical practice.
  • Figure 1 is the serum glycoprotein N-glycan map of the normal control group and colon cancer group; the abbreviations of the oligosaccharides in the map are respectively expressed as: NG1A2F, the core fucose two-antenna (Single agalacto core- ⁇ -1,6-fucosylated bisecting biantennary); NA2FB, Bigalacto core- ⁇ -1,6-fucosylated bisecting biantennary with bisected acetylglucosamine modification; NA3, Triantennary .
  • NG1A2F the core fucose two-antenna
  • NA2FB Bigalacto core- ⁇ -1,6-fucosylated bisecting biantennary with bisected acetylglucosamine modification
  • NA3, Triantennary Triantennary .
  • Test samples Serum from colorectal cancer patients and normal controls.
  • Reagent A prepared by adding SDS with a mass concentration of 0.5 to 5% in ammonium bicarbonate solution with a concentration of 10 mM;
  • Reagent B It is prepared by mixing 0.01 ⁇ 10U/10 ⁇ L glucosamidase and 0.01 ⁇ 10U/10 ⁇ L sialidase, and the pH value of the mixed solution is 4 ⁇ 9;
  • Reagent C Prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO, the concentration is 0.01mM ⁇ 1M;
  • Reagent D stop solution.
  • the preparation method of the intestinal cancer detection reagent comprises the following steps:
  • Serum samples collected from 254 patients with intestinal cancer and normal controls were processed using G-Test detection technology, including 154 samples from patients with bowel cancer and 100 serum samples from normal controls. Statistical analysis was performed on the G-Test fingerprints obtained from samples measured by G-Test detection technology.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

La présente invention concerne un réactif de détection du carcinome du rectum et son procédé de préparation. Le réactif de détection est obtenu en mélangeant les réactifs suivants : un réactif A, préparé en ajoutant du SDS avec une concentration massique de 0,5 à 5% à une solution de bicarbonate d'ammonium avec une concentration de 10 mM ; un réactif B, préparé en mélangeant 0,01 à 10 U/10μL de glycosylamine acylase et 0,01 à 10 U/10μL de sialidase, et une valeur de pH d'une solution mélangée étant de 4 à 9 ; un réactif C, préparé en dissolvant de l'acide 8-aminopyrène-1,3,6-trisulfonique dans du DMSO, la concentration étant de 0,01 mM à 1 M ; et un réactif D, qui est une solution d'arrêt. Une carte de glycome sérique est mesurée au moyen du réactif de détection, et les valeurs de pic sont quantifiées pour une analyse statistique, afin qu'un procédé permettant d'établir un modèle de carte de glycome sérique du carcinome du rectum soit établi pour détecter le carcinome du rectum.
PCT/CN2022/118798 2021-09-15 2022-09-14 Réactif de détection du carcinome du rectum et son application dans la détection du carcinome du rectum WO2023040911A1 (fr)

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