WO2021218661A1 - Dispositif de détection combiné de g-17, de pgi et d pgii et procédé de préparation associé - Google Patents

Dispositif de détection combiné de g-17, de pgi et d pgii et procédé de préparation associé Download PDF

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WO2021218661A1
WO2021218661A1 PCT/CN2021/087746 CN2021087746W WO2021218661A1 WO 2021218661 A1 WO2021218661 A1 WO 2021218661A1 CN 2021087746 W CN2021087746 W CN 2021087746W WO 2021218661 A1 WO2021218661 A1 WO 2021218661A1
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concentration
pepsinogen
colloidal gold
detection device
immune
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Chinese (zh)
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杨小军
李欣
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吉林省格瑞斯特生物技术有限公司
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Priority to KR1020227021546A priority Critical patent/KR20220104043A/ko
Priority to JP2022537172A priority patent/JP7551998B2/ja
Publication of WO2021218661A1 publication Critical patent/WO2021218661A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/595—Gastrins; Cholecystokinins [CCK]
    • 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)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96472—Aspartic endopeptidases (3.4.23)
    • G01N2333/96475—Aspartic endopeptidases (3.4.23) with definite EC number
    • G01N2333/96477—Pepsin (3.4.23.1; 3.4.23.2; 3.4.23.3)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • the present invention relates to the field of medical detection equipment, in particular to a gastrin 17, pepsin I, and pepsin II combined detection device and a preparation method thereof.
  • the method uses colloidal gold immunochromatography technology and the principle of double antibody sandwich method to quantitatively detect whole blood,
  • the detection device and preparation method of human gastrin 17 (G-17), pepsinogen I (PGI), and pepsinogen II (PGII) in serum and plasma samples can realize the sensitivity, specificity, and sensitivity of gastric cancer risk markers. Quick check.
  • Gastric mucosal lesions are caused by many factors, including drugs, alcohol, abnormal gastric acid secretion, and Helicobacter pylori infection, among which Helicobacter pylori infection is the main factor; after the gastric mucosa is damaged, its function will also change.
  • Gastric cancer is a group of progressive malignant lesions of epithelial cells involving multiple factors, mostly from chronic atrophic gastritis. About 7% of chronic atrophic gastritis eventually progress to carcinoma in situ. Abnormal cell proliferation and HP infection can accelerate the development of atrophic gastritis. China is an area with a high incidence of gastric cancer, and more than 200,000 residents die of gastric cancer each year. Although radical surgery combined with radiotherapy and chemotherapy can improve the five-year survival rate of gastric cancer patients, it has no obvious effect on reducing mortality. Studies have shown that early screening is the key to reducing the incidence and mortality of gastric cancer.
  • Gastrin is a type of gastrointestinal hormone secreted by the G cells of the gastric antrum, duodenum and upper jejunum. Edkins was first discovered in a dog's gastric antrum in 1905 and was later known. Gastrin in human serum mainly includes two isomers: gastrin-17 (gastrin-17, G-17) and gastrin-34 (gastrin-34, G-34), of which gastrin- 17 accounts for 85%-90%. Therefore, people usually choose gastrin-17 to represent the serum gastrin level. Under physiological conditions, gastrin mainly acts by affecting the secretion of gastric acid. On the one hand, gastrin can directly stimulate parietal cells to secrete gastric acid.
  • Gastric acid activates pepsinogen into active pepsin, thereby decomposing protein.
  • the decomposition products will in turn stimulate the secretion of gastrin; on the other hand, gastrin can also act on the cholecystokinic receptors on enterochromaffin-like cells to promote the secretion of histamine from enterochromaffin-like cells, and then through histamine Stimulate parietal cells to secrete gastric acid. While gastrin affects the secretion of gastric acid, it can also reduce the tension of the lower esophageal sphincter.
  • gastrin can be used as a marker of gastric mucosal function: the level of gastrin-17 in chronic atrophic gastritis is significantly higher than that of normal mucosa, and some people think that the serum level of gastrin-17 may be higher than that of serum. Total gastrin can more accurately reflect the severity of antral atrophy.
  • Pepsinogen is a protein polypeptide chain secreted by the main cells in the gastric mucosa. Human pepsinogen is mainly divided into two biochemical and immunologically different isoenzymes (PGI and PGII). Pepsinogen I is secreted by gastric gland main cells and neck cells, and pepsinogen II can also be secreted by cardia, pyloric and proximal duodenal glands. Pepsinogen itself does not have biological activity. It can be activated into digestive pepsin under the action of gastric acid, thereby decomposing the protein ingested by the body into easily absorbed small molecule peptides and amino acids.
  • pepsinogen The vast majority of pepsinogen is secreted into the gastric cavity, and only a small part can be absorbed into the blood.
  • the levels of pepsinogen I and pepsinogen II in the serum can accurately reflect the function and histological status of the gastric mucosa. Therefore, serum pepsinogen is also called "serological biopsy".
  • serum pepsinogen I and pepsinogen II combined with sophisticated endoscopy can effectively identify gastric diseases, especially early gastric mucosal lesions.
  • Gastrointestinal angiography and chest fluoroscopy are mainly used to observe gastric ulcers and advanced gastric cancer, but they have low sensitivity and accuracy for carcinoma in situ.
  • Microscopic biopsy is a traumatic operation and has many contraindications, making it difficult to carry out.
  • Gastrin 17, pepsinogen I, and pepsinogen II are digestive enzymes secreted by gastric mucosal tissues. Pathological changes in gastric mucosa such as the fundus of the stomach and antrum can cause their levels to change.
  • the combined detection of gastrin 17, pepsinogen I and II is effective in early gastric cancer screening, which can effectively improve the diagnosis rate of gastric cancer and is worthy of clinical application and promotion.
  • Colloidal gold immunochromatography assay is a solid-phase membrane immunoassay technique that combines colloidal gold labeling technology and protein chromatography technology with a microporous membrane as a carrier.
  • Colloidal gold immunochromatography technology is a commonly used immunochromatographic detection method. Because of its simple operation, time-saving, low manufacturing cost, and easy-to-interpret results, it is very suitable for on-site testing and is widely used in biology, medicine, and food. And other fields. Since the colloidal gold immunochromatography technique completes the detection in one step, there are many interference factors in the detection process, and its low sensitivity is the main factor that limits the application scope of colloidal gold immunochromatography. The detection limit of the traditional colloidal gold immunochromatography technique is higher than ELISA and other methods.
  • the protein is fixed on the nitrocellulose membrane (NC membrane) as a capture reagent for the sample to be tested. Since the detection result completely depends on whether the capture reagent achieves a good adsorption effect on the membrane, the uniform and good adsorption of the protein on the membrane is very important for the detection result of colloidal gold. If the amount of protein bound on the NC membrane is insufficient or the protein binding force is not strong enough, there will be quite a lot of problems, which are very obvious on the detection line of the test results. If the amount of protein bound on the membrane is too low, the color of the detection line will be weak and the detection sensitivity will be reduced in the result.
  • NC membrane nitrocellulose membrane
  • the protein cannot be firmly adsorbed on the NC membrane, then the protein will diffuse before it is adsorbed on the NC membrane, resulting in a wider detection line and weaker color rather than bright and clear, making the detection result difficult to interpret.
  • the physical adsorption of the protein on the NC membrane is too weak, the flowing protein detection substance and surfactant solution may wash off the fixed protein from the NC membrane, thus showing a wide or unclear detection at all Line, it is difficult to interpret the test results.
  • the invention provides a G-17, PGI and PGII combined detection device and a preparation method thereof, which solves the problems of insufficient amount of NC membrane adsorbed protein and weak binding force existing in the prior art.
  • the three-in-one combined detection device for human gastrin 17, pepsinogen I and pepsinogen II prepared by the present invention can realize the sensitive, specific and rapid detection of gastric cancer risk markers, and increase the risk of gastric cancer for patients with gastric diseases Reasonable and comprehensive judgments can quickly and accurately carry out early warning of gastric cancer and disease risk judgment.
  • the technical scheme adopted by the present invention is: a G-17, PGI and PGII combined detection device, the sample pad 1, the immune colloidal gold glass fiber membrane 2, the immune nitrocellulose membrane 3, and the absorption pad 4 are respectively pasted on the plastic plate 5.
  • the two ends of the immune nitrocellulose membrane 3 are overlapped with the absorbent pad 4 and the immune colloidal gold glass fiber membrane 2 respectively, and the other end of the immune colloidal gold glass fiber membrane 2 is overlapped with the sample pad 1; the immune nitric acid
  • the cellulose membrane 3 is provided with a first detection line T1, a second detection line T2, a third detection line T3, and a quality control line C;
  • the first detection line T1 has a solid phase with high specific gastrin 17 antibody;
  • the second detection line T2 has a high specific pepsinogen I antibody in the solid phase;
  • the third detection line T3 has a high specific pepsinogen II antibody in the solid phase;
  • the immune nitrocellulose membrane 3 The detection lines T1, T2, T3 set
  • the present invention also provides a method for preparing a G-17, PGI, and PGII combined detection device, which includes the following steps:
  • step (b) Using the colloidal gold prepared in step (a) to label gastrin 17, pepsinogen I and pepsinogen II antibodies to obtain immune colloidal gold;
  • step (c) Dilute the immune colloidal gold in step (b) with a spray gold buffer to obtain an immune colloidal gold solution, and spray the immune colloidal gold solution on a glass fiber mat to prepare an immune colloidal gold glass fiber membrane;
  • the colloidal gold particles prepared by the trisodium citrate reduction method described in step (a) of the present invention have a particle size of 20-60 nm.
  • the gold spraying buffer described in step (c) of the present invention is composed of Tris-HCl solution, sucrose, trehalose, and bovine serum albumin BSA, with a pH value of 8.5, wherein the concentration of Tris-HCl is 0.02 mol/L, and the concentration of sucrose is 5 ⁇ 20%, trehalose concentration is 1 ⁇ 5%, bovine serum albumin BSA concentration is 0.5 ⁇ 1%.
  • the pretreatment of the nitrocellulose membrane with polyethylene glycol glycerol treatment solution in step (d) of the present invention is: soak the nitrocellulose membrane with the polyethylene glycol glycerol treatment solution for 1 hour, and then shake it slowly After taking it out, wash it with distilled water 3 times, and finally dry it in a vacuum drying oven.
  • step (d) of the present invention respectively bind gastrin 17, pepsinogen I and pepsinogen II antibodies: take oleic acid-modified ZnS as a carrier, take 1 mL of gastrin 17, pepsin The original I and pepsinogen II antibody solutions were stirred for 1 hour and collected by centrifugation at 12000 rpm, 8500 rpm and 7000 rpm for 10 minutes, respectively, and washed with deionized water twice each.
  • the polyethylene glycol glycerol treatment solution described in step (d) of the present invention is composed of polyethylene glycol glycerol diluted to a concentration of 0.5%, filtered through a 0.22 ⁇ m filter membrane, and ready for use.
  • the polyethylene glycol glycerol treatment liquid described in step (d) of the present invention is composed of a mixture of polyethylene glycol glycerol and polylysine (SIGMA, 150KD ⁇ 300KD), wherein the concentration of polyethylene glycol glycerol is The concentration of polylysine is 0.5%, and the concentration of polylysine is 0.5%. It is filtered through a 0.22 ⁇ m filter membrane for use.
  • SIGMA polyethylene glycol glycerol and polylysine
  • the polyethylene glycol glycerol treatment solution described in step (d) of the present invention is composed of a mixture of polyethylene glycol glycerol, polylysine (SIGMA, 150KD ⁇ 300KD), and PEG20000, wherein polyethylene glycol propylene
  • concentration of triol is 0.5%
  • concentration of polylysine is 0.5%
  • concentration of PEG20000 is 0.1%
  • step (d) of the present invention Take 15ml of oleic acid in absolute ethanol and add 15ml of zinc acetate aqueous solution with a concentration of 0.3mol/L, stir in a water bath at 40°C, and use ammonia Adjust the pH value, and then add 15ml of sodium sulfide aqueous solution with a concentration of 0.3mol/L. After reacting for 5 minutes, add 5ml of SDS aqueous solution. After the mixture is evenly mixed, the reaction solution is poured into a 90ml hydrothermal kettle.
  • the hydrothermal kettle After the hydrothermal kettle is airtight, it is placed in a constant temperature drying box and reacted at a constant temperature for a certain time. After the reaction is over, the temperature is lowered to 50°C, and the product is taken out. Wash with acetone, deionized water, ethanol, centrifuge, and vacuum dry at 50°C for 2 hours to obtain powder ZnS, which is stored for later use.
  • the sample pad treatment liquid used in the pretreated sample pad described in step (e) of the present invention is composed of Tris-HCl solution, bovine serum albumin BSA, casein, and surfactant (alkylphenol polyoxyethylene ether), wherein
  • Tris-HCl solution is 0.1 mol/L
  • bovine serum albumin BSA is 0.5-1%
  • casein is 0.1-0.2%
  • surfactant alkylphenol polyoxyethylene ether
  • PEGG polyethylene glycol glycerol
  • the reaction of ethylene glycol and epichlorohydrin is catalyzed by alkali, and the product is neutralized with dilute hydrochloric acid, extracted with carbon tetrachloride, and distilled under reduced pressure to obtain polyethylene glycol glycerol (PEGG), which is a pale yellow viscous substance.
  • PEGG can be miscible with water in any ratio, and can also be dissolved in common organic solvents such as ethanol, acetone, tetrahydrofuran, chloroform, and has a certain surface activity.
  • the structure of polyethylene glycol glycerol contains multiple hydroxyl groups for coupling, and the activation process is simple, which facilitates the immobilization of proteins on the surface of the NC membrane. Under normal conditions, the number of antibodies bound to the NC membrane per unit area is limited. After treatment with polyethylene glycol glycerol, the number of antibodies bound to the NC membrane per unit area can be increased, thereby achieving higher
  • the ZnS modified with oleic acid/sodium lauryl sulfate not only has a nanometer-scale particle size, but also has good water solubility and biocompatibility.
  • Using the functional groups on its outer surface it can be uniformly dispersed in an aqueous medium.
  • Zinc sulfide nanoparticles have the advantages of good stability, easy preparation, good biocompatibility and low immunogenicity, etc., and they are widely studied in the field of biomedicine. However, there is no reported application in colloidal gold immunochromatography.
  • the present invention pretreated the nitrocellulose membrane with polyethylene glycol glycerol treatment solution to combine the NC-coated antibody with the zinc sulfide nanoparticles to achieve improved The purpose of the test paper sensitivity.
  • the detection device of the present invention has a simple structure and a novel concept.
  • the gastrin 17, pepsinogen I and pepsinogen II antibodies are coated on the nitrocellulose membrane, which has strong specificity and can simultaneously detect gastric secretions in specimens.
  • Peptin 17, pepsinogen I and pepsinogen II did not increase the complexity of production operations.
  • immune colloidal gold by cooperating with suitable gold spraying buffer and sample pad treatment solution, it can ensure the complete release of immune colloidal gold, and effectively improve the sensitivity of the reaction. Under the same threshold, it can also reduce The amount of immune colloidal gold saves costs.
  • the present invention pretreats the nitrocellulose membrane, modifies the antibody coating the nitrocellulose membrane, and improves the sensitivity and specificity of the detection test paper.
  • the detection device of the present invention does not require any special equipment, and the detection cost is low.
  • the detection device of the present invention is easy to operate, does not require professionals to operate, and has strong practicability.
  • FIG. 1 is a schematic diagram of the structure of the present invention.
  • the detection lines T1, T2, and T3 arranged on the immune nitrocellulose membrane are arranged longitudinally on the same immune nitrocellulose membrane;
  • Figure 2 is a cross-sectional view taken along line A-A of Figure 1;
  • FIG. 3 is a schematic diagram of another structure of the present invention.
  • the detection lines T1, T2, and T3 are respectively arranged on three immune nitrocellulose membranes, arranged side by side to form a joint detection device;
  • Figure 4 is a B-B cross-sectional view of Figure 3;
  • Figure 5 is a C-C cross-sectional view of Figure 3;
  • Fig. 6 is a cross-sectional view taken along the line D-D in Fig. 3.
  • the sample pad 1, the immune colloidal gold glass fiber membrane 2, the immune nitrocellulose membrane 3, and the absorbent pad 4 are respectively pasted on the plastic plate 5.
  • the two ends of the immune nitrocellulose membrane 3 are respectively connected to the absorbent pad 4 and the immune colloidal gold glass
  • the fiber membrane 2 is overlapped, and the other end of the immune colloidal gold glass fiber membrane 2 is overlapped with the sample pad 1;
  • the immune nitrocellulose membrane 3 is provided with a first detection line T1, a second detection line T2, and a third detection Line T3, and quality control line (C);
  • the first test line T1 has high specific gastrin 17 antibody in the solid phase;
  • the second test line T2 has high specific pepsinogen in the solid phase I antibody;
  • the third detection line T3 has a high-specificity pepsinogen II antibody in the solid phase; as shown in Figures 1 and 2
  • the detection lines T1, T2, T3 are set on the immune nitrocellulose membrane 3 It can be arranged longitudinally on the same immune nitrocellulose membrane 3
  • the preparation method of the G-17, PGI, and PGII combined detection device of the present invention includes solid-phase purified high-specificity gastrin 17, pepsinogen I and pepsinogen II antibodies (detection lines T1, T2, T3) Nitrocellulose membrane with goat anti-mouse IgG polyclonal antibody (control line), glass fiber membrane with colloidal gold-labeled gastrin 17, pepsinogen I and pepsinogen II antibodies, sample pads, absorbent paper, etc.
  • the auxiliary materials are thus made by pasting. The specific steps are as follows:
  • the spray gold buffer includes: the concentration is 20mM Tris-HCl solution, sucrose concentration is 5%, trehalose concentration is 1%, BSA concentration is 1%, pH is 8.5;
  • polyethylene glycol glycerol treatment solution the concentration of polyethylene glycol glycerol is 0.5%, filtered through a 0.22 ⁇ m filter membrane for use;
  • Pretreatment of nitrocellulose membrane with polyethylene glycol glycerol treatment solution Put the nitrocellulose membrane in the polyethylene glycol glycerol treatment solution to soak for 1 hour, shake it slowly, take it out, and wash it with distilled water 3 times. Finally, it is dried in a vacuum drying oven;
  • Preparation of oleic acid modified zinc sulfide nanoparticles Take 15ml of oleic acid in absolute ethanol and add it to 15ml of 0.3mol/L zinc acetate aqueous solution, stir in a water bath at 40°C, adjust the pH with ammonia, and then add 15ml to a concentration of 0.3 mol/L sodium sulfide aqueous solution, after reacting for 5 minutes, add 5ml SDS aqueous solution, after mixing uniformly, pour the reaction solution into a 90ml hydrothermal kettle. After the hydrothermal kettle is airtight, it is placed in a constant temperature drying box and reacted at a constant temperature for a certain time.
  • oleic acid-modified ZnS as a carrier, take 1ml of gastrin 17, pepsinogen I and pepsinogen II antibody solution, stir for 1 hour, centrifuge at 12000rpm, 8500rpm and 7000rpm for 10 minutes to collect, wash each with deionized water 2 times.
  • the sample pad treatment solution includes: Tris-HCl solution concentration of 0.1M, bovine serum albumin BSA concentration of 0.5%, casein concentration of 0.1%, and surfactant concentration of 0.5%, dried at 37°C for use, the sample pad after treatment can improve the sensitivity of the reaction;
  • the pre-treated sample pad, immune colloidal gold glass fiber membrane, immune nitrocellulose membrane, and absorbent paper are sequentially pasted on the rubber sheet, and cut to obtain a detection reagent strip, and finally the detection reagent strip is put into a plastic shell.
  • Quantitative detection The minimum detection concentration of human gastrin 17 detected by this combined detection device is 0.5pg/ml, the minimum detection concentration of pepsinogen I is 5ng/ml, and the minimum detection concentration of pepsinogen II is detected by the colloidal gold detector. It is 5ng/ml.
  • the prepared colloidal gold particles are 40nm;
  • the gold spray buffer includes: a concentration of 20mM Tris-HCl solution, a sucrose concentration of 12%, a trehalose concentration of 3%, a BSA concentration of 0.7%, and a pH of 8.5;
  • polyethylene glycol glycerol treatment solution is composed of a mixture of polyethylene glycol glycerol and polylysine (SIGMA, 150KD ⁇ 300KD), wherein the concentration of polyethylene glycol glycerol is 0.5%, and polylysine The acid concentration is 0.5%, filtered through a 0.22 ⁇ m filter membrane, ready for use;
  • SIGMA polyethylene glycol glycerol and polylysine
  • the cup pad treatment solution includes: Tris-HCl solution concentration of 0.1M, bovine serum albumin BSA concentration of 0.7%, casein concentration of 0.15%, and surfactant concentration of 0.7%;
  • the preparation of colloidal gold particles is 60nm;
  • the gold spray buffer includes: a concentration of 20mM Tris-HCl solution, a sucrose concentration of 20%, a trehalose concentration of 5%, a BSA concentration of 1%, and a pH of 8.5;
  • polyethylene glycol glycerol treatment solution it is composed of polyethylene glycol glycerol, polylysine (SIGMA, 150KD ⁇ 300KD) and PEG2000, in which the concentration of polyethylene glycol glycerol is 0.5%, The concentration of polylysine is 0.5%, and the concentration of PEG20000 is 0.1%, filtered through a 0.22 ⁇ m filter membrane for use;
  • Cup mat treatment solution includes: Tris-HCl solution concentration of 0.1M, bovine serum albumin BSA concentration of 1%, casein concentration of 0.2%, and surfactant concentration of 1%;
  • polyethylene glycol glycerol treatment liquid polyethylene glycol glycerol group, the concentration of polyethylene glycol glycerol is 0.5%; polyethylene glycol glycerol treatment liquid polylysine group , The concentration of polyethylene glycol glycerol is 0.5%, and the concentration of polylysine is 0.5%; the group of polyethylene glycol glycerol, polylysine and PEG20000, in which the concentration of polyethylene glycol glycerol is 0.5%, the concentration of polylysine is 0.5%, the concentration of PEG20000 is 0.1%, the three groups of treatment liquids are filtered through 0.22 ⁇ m filter membrane for use.
  • the nitrocellulose membrane was soaked in the polyethylene glycol glycerol treatment solution for 1 hour, and then shaken slowly. After taking it out, it was washed with distilled water 3 times, and finally dried in a vacuum drying oven.
  • the untreated and treated nitrocellulose membranes were prepared according to the process flow of the above examples to prepare gastrin 17, pepsinogen I and pepsinogen II combined detection test papers.
  • the test process refers to the test paper instructions to compare the nitrocellulose membranes. Adsorption and stability index difference after treatment and treatment.
  • Oleic acid modified zinc sulfide nanoparticles modified gastrin 17, pepsinogen I and pepsinogen II antibodies
  • oleic acid-modified ZnS as a carrier, take 1 mL of gastrin 17, pepsinogen I and pepsinogen II antibody solutions, stir for 1 hour, centrifuge at 12000 rpm, 8500 rpm and 7000 rpm for 10 minutes to collect, wash each with deionized water 2 times.
  • the gastrin 17, pepsinogen I and pepsinogen II antibodies modified by zinc sulfide nanoparticles and the unmodified gastrin 17, pepsinogen I and pepsinogen II antibodies were prepared according to the process flow of the above-mentioned embodiment.
  • the test procedure refers to the test paper instructions to compare the difference in protein adsorption and stability indexes between treatment and untreated zinc sulfide nanoparticles.

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Abstract

L'invention concerne un dispositif de détection combiné de gastrine 17 (G-17), de pepsinogène I (PGI) et de pepsinogène II (PGII) et un procédé de préparation associé. Le dispositif de détection combiné est préparé en faisant adhérer une membrane de nitrocellulose (3) avec une phase solide contenant des anticorps G-17, PGI et PGII à haute spécificité et des anticorps polyclonaux IgG anti-souris de chèvre, une membrane en fibres de verre (2) adsorbée avec des anticorps G-17, PGI et PGII marqués par de l'or colloïdal, un tampon d'échantillon (1), du papier absorbant (4) et d'autres matériaux auxiliaires. Selon le dispositif de détection combiné, la libération complète de l'or colloïdal immun est assurée, la sensibilité de la réaction est efficacement améliorée, le dosage de l'or colloïdal immun peut être réduit sous le même seuil, le coût est réduit, trois fonctions gastriques et les marqueurs de risque de cancer gastrique de la gastrine 17, du pepsinogène I et du pepsinogène II dans un échantillon peuvent être détectés en même temps, et la complexité de l'opération de production n'est pas augmentée. Le dispositif présente une sensibilité élevée, une forte spécificité, il est simple et pratique à utiliser, permet de gagner du temps et est très simple à mettre en œuvre.
PCT/CN2021/087746 2020-04-30 2021-04-16 Dispositif de détection combiné de g-17, de pgi et d pgii et procédé de préparation associé WO2021218661A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114113592A (zh) * 2021-11-22 2022-03-01 中国药科大学 一种基于巯基苯硼酸功能化金纳米颗粒的侧流层析试纸及其检测方法
CN114460310A (zh) * 2022-04-12 2022-05-10 天津康博尔生物基因技术有限公司 一种彩色乳胶微球及其制备方法与应用
CN115015543A (zh) * 2022-04-17 2022-09-06 吉林迅准生物技术有限公司 一种多项病原体联合检测装置及其制备方法
CN117250352A (zh) * 2023-11-15 2023-12-19 聚诚(北京)生物科技有限责任公司 胃蛋白酶检测试剂盒

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111638366B (zh) * 2020-04-30 2021-02-19 吉林省格瑞斯特生物技术有限公司 G-17、pgi、pgii联合检测装置及其制备方法
CN113004412B (zh) * 2021-03-23 2021-12-03 中元汇吉生物技术股份有限公司 胃蛋白酶原i单克隆抗体及其应用

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003029826A1 (fr) * 2001-09-28 2003-04-10 Biohit Oyj Procede de detection de cancer ou d'etat precancereux
WO2003062824A1 (fr) * 2002-01-23 2003-07-31 Boditech Inc. Procede et bande de dosage quantitatif en flux lateral, et dispositif associe de detection de fluorescence induite par laser
WO2008114980A1 (fr) * 2007-03-21 2008-09-25 Hdr Co., Ltd. Trousse de diagnostic simultané de trichomonas vaginalis et chlamydia trachomatis
CN102778557A (zh) * 2011-04-28 2012-11-14 美艾利尔（上海）诊断产品有限公司 生物检测试剂涂膜液
CN205176031U (zh) * 2015-11-17 2016-04-20 深圳市金准生物医学工程有限公司 一种基于免疫层析技术的胃功能联合检测试剂盒
CN105548534A (zh) * 2015-04-04 2016-05-04 吉林双正医疗科技有限公司 心肌标志物的联合检测装置及其制备方法
CN206638677U (zh) * 2017-04-26 2017-11-14 江苏扬新生物医药有限公司 一种用于术后病人监测的多指标快速检测胶体金试剂盒
CN108097340A (zh) * 2018-02-26 2018-06-01 北京华科泰生物技术有限公司 一种用于胃功能疾病筛查的联合检测微流控芯片及其制备方法和用途
CN109001463A (zh) * 2018-07-09 2018-12-14 广州华澳生物科技有限公司 一种胃癌风险标志物联合定量检测试纸及制备方法
CN111638370A (zh) * 2020-04-30 2020-09-08 吉林省格瑞斯特生物技术有限公司 胃功能及胃癌发生风险检测装置及其制备方法
CN111638366A (zh) * 2020-04-30 2020-09-08 吉林省格瑞斯特生物技术有限公司 G-17、pgi、pgii联合检测装置及其制备方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100140112A1 (en) * 2005-06-23 2010-06-10 Kyoung-Sik Seo Nanoparticle Marker, Diagnostic Methods Using the Same and Diagnostic Kit and Apparatus Using the Same
US9482616B2 (en) * 2012-06-06 2016-11-01 Board of Regents of the Nevada System of Higher Education, on behalf the University of Nevada, Reno Methods, kits and systems for signal amplification for bioassays using zinc nanoparticles
CN111638372B (zh) * 2020-04-30 2021-02-19 吉林省格瑞斯特生物技术有限公司 一种心肌标志物的联合检测装置及其制备方法

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003029826A1 (fr) * 2001-09-28 2003-04-10 Biohit Oyj Procede de detection de cancer ou d'etat precancereux
WO2003062824A1 (fr) * 2002-01-23 2003-07-31 Boditech Inc. Procede et bande de dosage quantitatif en flux lateral, et dispositif associe de detection de fluorescence induite par laser
WO2008114980A1 (fr) * 2007-03-21 2008-09-25 Hdr Co., Ltd. Trousse de diagnostic simultané de trichomonas vaginalis et chlamydia trachomatis
CN102778557A (zh) * 2011-04-28 2012-11-14 美艾利尔（上海）诊断产品有限公司 生物检测试剂涂膜液
CN105548534A (zh) * 2015-04-04 2016-05-04 吉林双正医疗科技有限公司 心肌标志物的联合检测装置及其制备方法
CN205176031U (zh) * 2015-11-17 2016-04-20 深圳市金准生物医学工程有限公司 一种基于免疫层析技术的胃功能联合检测试剂盒
CN206638677U (zh) * 2017-04-26 2017-11-14 江苏扬新生物医药有限公司 一种用于术后病人监测的多指标快速检测胶体金试剂盒
CN108097340A (zh) * 2018-02-26 2018-06-01 北京华科泰生物技术有限公司 一种用于胃功能疾病筛查的联合检测微流控芯片及其制备方法和用途
CN109001463A (zh) * 2018-07-09 2018-12-14 广州华澳生物科技有限公司 一种胃癌风险标志物联合定量检测试纸及制备方法
CN111638370A (zh) * 2020-04-30 2020-09-08 吉林省格瑞斯特生物技术有限公司 胃功能及胃癌发生风险检测装置及其制备方法
CN111638366A (zh) * 2020-04-30 2020-09-08 吉林省格瑞斯特生物技术有限公司 G-17、pgi、pgii联合检测装置及其制备方法

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114113592A (zh) * 2021-11-22 2022-03-01 中国药科大学 一种基于巯基苯硼酸功能化金纳米颗粒的侧流层析试纸及其检测方法
CN114113592B (zh) * 2021-11-22 2023-01-31 中国药科大学 一种基于巯基苯硼酸功能化金纳米颗粒的侧流层析试纸及其检测方法
WO2023087480A1 (fr) * 2021-11-22 2023-05-25 中国药科大学 Papier réactif pour chromatographie à flux latéral à base de nanoparticules d'or fonctionnalisées par acide mercaptophénylboronique, et son procédé de détection
CN114460310A (zh) * 2022-04-12 2022-05-10 天津康博尔生物基因技术有限公司 一种彩色乳胶微球及其制备方法与应用
CN115015543A (zh) * 2022-04-17 2022-09-06 吉林迅准生物技术有限公司 一种多项病原体联合检测装置及其制备方法
CN115015543B (zh) * 2022-04-17 2023-02-28 吉林迅准生物技术有限公司 一种多项病原体联合检测装置及其制备方法
CN117250352A (zh) * 2023-11-15 2023-12-19 聚诚(北京)生物科技有限责任公司 胃蛋白酶检测试剂盒
CN117250352B (zh) * 2023-11-15 2024-02-13 聚诚(北京)生物科技有限责任公司 胃蛋白酶检测试剂盒

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