WO2022048693A1 - Bandelette réactive pour détection quantitative d'anticorps contre l'hélicobacter pylori au moyen d'or colloïdal et méthode de détection - Google Patents

Bandelette réactive pour détection quantitative d'anticorps contre l'hélicobacter pylori au moyen d'or colloïdal et méthode de détection Download PDF

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Publication number
WO2022048693A1
WO2022048693A1 PCT/CN2021/123687 CN2021123687W WO2022048693A1 WO 2022048693 A1 WO2022048693 A1 WO 2022048693A1 CN 2021123687 W CN2021123687 W CN 2021123687W WO 2022048693 A1 WO2022048693 A1 WO 2022048693A1
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Prior art keywords
helicobacter pylori
colloidal gold
antibody
reagent strip
nitrocellulose membrane
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PCT/CN2021/123687
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English (en)
Chinese (zh)
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黎宏章
赵俊
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三门县人民医院
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Publication of WO2022048693A1 publication Critical patent/WO2022048693A1/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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • 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
    • G01N33/58—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

Definitions

  • the invention relates to a detection technology for Helicobacter pylori antibodies, in particular to a colloidal gold quantitative detection reagent strip for Helicobacter pylori antibodies, and a detection method for quantitatively detecting Helicobacter pylori antibodies by using the reagent strip.
  • Helicobacter pylori (H.p), by Barry It was first discovered by J. Marshall and J. Robin Warren for which they were awarded the 2005 Nobel Prize in Physiology or Medicine.
  • Helicobacter pylori is a unipolar, polyflagellar, blunt-ended, spiral-curved bacterium, 2.5-4.0 ⁇ m long and 0.5-1.0 ⁇ m wide.
  • the surface of gastric mucosal epithelial cells often presents a typical spiral or arc shape.
  • Helicobacter pylori infection is the main pathogenic factor of chronic active gastritis, peptic gastric ulcer and gastric mucosa-associated lymphoid tissue lymphoma, and is closely related to the occurrence of gastric cancer.
  • WHO/IARC World Health Organization/International Agency for Research on Cancer
  • the infection rates of juvenile Helicobacter pylori in mainland China, Vietnam, and India are 60%, 40%, and 70%, respectively.
  • the detection rate of Helicobacter pylori in gastric mucosal biopsy specimens from patients with chronic gastritis can reach 80% to 90%.
  • peptic ulcer patients are higher, up to more than 95%, or even close to 100%.
  • the detection rate of gastric cancer is different because of local epithelial cells that have undergone dissimilation.
  • Stomach infection of Helicobacter pylori is a worldwide medical problem, and its accurate diagnosis is beneficial to control the spread and prevalence of H. pylori and monitor the treatment for eradication of H. pylori infection.
  • invasive and non-invasive mainly refers to the method of taking biopsy specimens through gastroscopy, and it is a routine method in the current gastroenterology discipline. It includes bacterial isolation and culture, direct smear, rapid urease test, and drug sensitivity test.
  • Non-invasive methods mainly refer to methods for diagnosing Helicobacter pylori infection without biopsy specimens taken by gastroscope. Such methods include antibody detection, antigen detection, urea 13C/14C Breath test, etc.
  • the existing methods for antibody detection include enzyme-linked immunosorbent assay, western blotting, colloidal gold method and latex-enhanced immunoturbidimetric method.
  • Serological testing is a commonly used non-invasive test, especially in areas with high rates of Helicobacter pylori infection. Helicobacter pylori causes chronic inflammation, and the system's immune response persists, producing IgG antibodies. Commonly used serological tests include ELISA, chemiluminescence, Western blot, colloidal gold immunochromatography and other methods. Among them, colloidal gold immunochromatography has the fastest detection speed, and the results can be obtained in only 10 minutes, which is especially suitable for rapid on-site detection. However, there is no quantitative Helicobacter pylori antibody colloidal gold immunochromatographic detection reagent on the market.
  • the present invention provides a colloidal gold quantitative detection reagent strip for Helicobacter pylori antibodies.
  • the colloidal gold quantitative detection reagent strip of the Helicobacter pylori antibody of the present invention can realize the quantitative detection of the Helicobacter pylori antibody by the colloidal gold immunochromatography method.
  • the present invention also provides a method for quantitatively detecting Helicobacter pylori antibodies by using the colloidal gold quantitatively detecting Helicobacter pylori antibody reagent strip of the present invention.
  • Colloidal gold quantitative detection Helicobacter pylori antibody reagent strip includes a bottom plate, and a sample pad, colloidal gold pad, nitrocellulose membrane and water-absorbing filter paper pasted on the bottom plate; the sample pad, colloidal gold pad, nitrocellulose The plain film and the water-absorbing filter paper overlap each other for a preset length in turn; the nitrocellulose film is coated with the natural antigen of Helicobacter pylori by scribing; Loaded onto glass cellulose membrane and obtained after drying.
  • the colloidal gold pad is prepared by the following method steps: Step a. Use 0.1-0.3 mol/L K2CO3 solution to prepare the colloidal gold solution The pH of the colloidal gold solution is adjusted to 7.0-9.0; step b. The colloidal gold solution in step a is placed on a magnetic stirrer and stirred, and 0.5-2 mg of Helicobacter pylori recombinant protein antigen is added per 100 mL of colloidal gold solution to recombine Helicobacter pylori. Add the protein antigen dropwise to the colloidal gold solution, and continue to stir for 2-3 hours; step c.
  • step d Centrifuge the colloidal gold solution after sealing and labeling in step c at 12000-15000r/m, discard the supernatant, and use 0.01mol/L for the precipitate Resuspend the PBS solution, and resuspend the sediment to 1/10 of the original sediment volume to obtain a resuspended solution; step e.
  • step d Centrifuge the resuspended solution obtained in step d at 2000-3000 r/min for 30 min, and take the upper layer
  • the supernatant is added to a Sephadex chromatography column for purification to obtain a colloidal gold-labeled Helicobacter pylori recombinant protein solution with a purity of more than 90%; step f. the colloidal gold-labeled Helicobacter pylori recombinant protein obtained in step e.
  • the antigen solution was added to the glass cellulose membrane and dried for 4 to 6 hours at a temperature of 20°C to 25°C to prepare a colloidal gold pad for use.
  • the method steps for coating the natural antigen of Helicobacter pylori on the nitrocellulose membrane by scribing are as follows: Step i. Dilute the Helicobacter pylori natural antigen to 0.2-0.5 mg/mL with 0.01 mol/L PBS; step ii. Using a dot membrane apparatus, the natural antigen of Helicobacter pylori diluted in step i was streaked and coated on a nitrocellulose membrane at 1 ⁇ l/cm; step iii. After the Helicobacter pylori natural antigen coating is completed, the nitrocellulose membrane is dried at room temperature in a drying room for use.
  • the nitrocellulose membrane is also coated with mouse anti-Helicobacter pylori monoclonal antibody by streaking for quality control.
  • the method steps of coating the mouse anti-Helicobacter pylori monoclonal antibody by streaking on the nitrocellulose membrane are as follows: Step 1. Dilute the mouse anti-Helicobacter pylori monoclonal antibody to 0.2 ⁇ 0.5mg/mL with 0.01mol/L PBS; step 2.
  • step i The mouse anti-Helicobacter pylori monoclonal antibody diluted in step i was streaked and coated on a nitrocellulose membrane at 1 ⁇ l/cm with a dot film apparatus; step 3. After the mouse anti-Helicobacter pylori monoclonal antibody coating is completed, the nitrocellulose membrane is dried at room temperature in a drying room for use.
  • Step 1 Paste the coating on the center of the bottom plate. pylori natural antigen nitrocellulose membrane; step II. paste absorbent filter paper on the upper edge of the nitrocellulose membrane; step III. paste colloidal gold pad on the lower edge of the nitrocellulose membrane; step IV. paste on the lower edge of the colloidal gold pad Sample pad, after completion, cut the pasted test board into test strips with a cutting machine, and seal it in an aluminum foil bag.
  • the bottom plate may be made of PVC.
  • the colloidal gold quantitative detection reagent strip for Helicobacter pylori antibody of the present invention is a Helicobacter pylori antibody colloidal gold quantitative detection reagent strip prepared by the double antigen sandwich method of the combination of Helicobacter pylori recombinant protein antigen and natural antigen,
  • the colloidal gold detector reads the T/C value and substitutes it into the standard curve to obtain the content of Helicobacter pylori antibody (AU/mL) in the clinical sample, and realize the detection of Helicobacter pylori antibody (IgG antibody). ) rapid quantitative detection.
  • the technical scheme of the present invention is:
  • a detection method for quantitatively detecting Helicobacter pylori antibody by colloidal gold adopts the aforementioned colloidal gold quantitative detection Helicobacter pylori antibody reagent strip of the present invention to perform qualitative detection and quantitative detection of Helicobacter pylori antibody;
  • the qualitative detection process is as follows: place the Helicobacter pylori antibody detection strip horizontally, and accurately drop 1 drop of serum or plasma sample on the sample pad of the test strip. After 10 minutes of reaction at room temperature, a red band appears on the quality control line and red on the detection line. Band, the test result is Helicobacter pylori IgG antibody positive; a red band appears on the quality control line, but no red band appears on the test line, the test result is Helicobacter pylori IgG antibody negative; no red band or quality control line appears on the quality control line There is no red band on the line and detection line, and the detection of the reagent strip is invalid;
  • the quantitative detection process is as follows: place the Helicobacter pylori antibody reagent strip horizontally, accurately drop 1 drop of serum or plasma sample on the sample pad of the test strip, and react at room temperature for 10 minutes, then put the Helicobacter pylori antibody reagent strip into colloidal gold for quantification.
  • the reader read the ratio of the peak area of the T line to the C line, that is, the T/C value, and substitute it into the Helicobacter pylori antibody detection standard curve to obtain the Helicobacter pylori antibody content (AU/mL) in the sample.
  • the method for preparing the standard curve for detecting Helicobacter pylori antibodies includes the following process: using Helicobacter pylori antibody-negative serum to dilute in a gradient manner 1AU/mL Helicobacter pylori antibody detection standard, add 50 ⁇ L to the sample pad of the reagent strip, react at room temperature for 10 minutes, put the reagent strip into the colloidal gold quantitative reader, and read the peak area ratio of T line and C line That is, the T/C value, take the T/C value as the abscissa and the AU value of the Helicobacter pylori antibody detection standard as the ordinate to make a standard curve;
  • the preparation method of the 1AU/mL Helicobacter pylori antibody detection standard is as follows: collect To 20 pieces of Helicobacter pylori antibody positive serum were mixed, and purified by saturated ammonium sul
  • the significant progress achieved by the above method of the present invention is that the quantitative detection of Helicobacter pylori is realized by using colloidal gold immunochromatography.
  • Fig. 1 is the structural representation of colloidal gold quantitative detection Helicobacter pylori antibody reagent strip of the present invention
  • Labels in the drawings 1- bottom plate, 2- sample pad, 3- colloidal gold pad, 4- water-absorbing filter paper, 5- nitrocellulose membrane, 501- detection line, 502- quality control line.
  • the colloidal gold quantitative detection Helicobacter pylori antibody reagent strip of the present invention comprises a bottom plate 1 made of PVC material, and a sample pad 2, a colloidal gold pad 3, a nitrocellulose film 5 and a water absorbing material pasted on the bottom plate 1 of the PVC material.
  • filter paper 4; the sample pad 2, the colloidal gold pad 3, the nitrocellulose membrane 5 and the water-absorbing filter paper 4 are sequentially overlapped end to end for a predetermined length.
  • the nitrocellulose membrane 5 is coated with the natural antigen of Helicobacter pylori by scribing to form a detection line 501; on the plain film and obtained after drying.
  • the nitrocellulose membrane 5 can be coated with mouse anti-Helicobacter pylori monoclonal antibody by scribing to form a quality control line 502 for quality control.
  • the colloidal gold quantitative detection reagent strip for Helicobacter pylori antibody in this embodiment is prepared according to the following steps.
  • Preparation of the colloidal gold pad coated with the recombinant protein antigen of Helicobacter pylori adjust the pH of the colloidal gold solution to 8 with 0.2mol/LK 2 CO 3 . Then put the solution on a magnetic stirrer and stir slowly, add 1.25 mg of Helicobacter pylori recombinant protein antigen per 100 mL of colloidal gold solution, and slowly drop the protein into the colloidal gold solution, continue stirring for 2.5 h, and then add dropwise to a final concentration of 1% BSA and 1.5% casein were used for blocking and labeling for 60 min.
  • nitrocellulose membrane with Helicobacter pylori natural antigen Dilute the Helicobacter pylori natural antigen to 0.3mg/mL with 0.01mol/L PBS, and then use the BIODOT film spotter to coat the Helicobacter pylori natural antigen on the nitrocellulose membrane 5 was streaked and coated at 1 ⁇ l/cm to form a detection line 501; at the same time, a mouse anti-Helicobacter pylori monoclonal antibody was coated at 1 ⁇ l/cm on the nitrocellulose membrane 5, which was used for the quality control line 502 of the product, including After being completed, the nitrocellulose membrane 5 was dried at room temperature for 8 hours in a drying room for use.
  • the method for rapid quantitative detection of Helicobacter pylori antibody using the reagent card of the present invention is as follows: place the reagent strip horizontally, accurately drop 1 drop of serum or plasma sample on the sample pad 2 of the test strip, and react at room temperature for 10 minutes, according to the following method for quantitative detection.
  • the quantitative detection process is as follows: place the Helicobacter pylori antibody reagent strip horizontally, accurately drop 1 drop of serum or plasma sample on the sample pad 2 of the test strip, and after 10 minutes of reaction at room temperature, put the Helicobacter pylori antibody reagent strip into the colloid
  • the gold quantitative reader read the peak area ratio of T line and C line, that is, the T/C value, and substitute it into the standard curve of Helicobacter pylori antibody detection to obtain the content of Helicobacter pylori antibody in the sample (AU/mL).
  • the method for establishing the standard curve of Helicobacter pylori antibody detection is as follows: use Helicobacter pylori antibody negative serum to dilute 1AU/mL Helicobacter pylori antibody detection standard in a gradient, respectively take 50 ⁇ L and add them to the sample pad 2 of the reagent strip, react at room temperature for 10 minutes, Put the reagent strip into the colloidal gold quantitative reader, read the peak area ratio between the T line and the C line, that is, the T/C value, take the T/C value as the abscissa, and the Helicobacter pylori antibody detection standard AU value as the ordinate. , to create a standard curve. When the reagent detects clinical samples, the colloidal gold quantitative reader reads the T/C value and substitutes it into the standard curve to obtain the content of Helicobacter pylori antibody (AU/mL) in the sample.
  • the preparation method of the above-mentioned Helicobacter pylori antibody detection standard is as follows: 20 pieces of Helicobacter pylori antibody positive serum are collected and mixed, salted out with saturated ammonium sulfate and combined with Protein Purified by G-column affinity chromatography to obtain a final concentration of 0.5mg/mL purified Helicobacter pylori antibody, which was set as 1AU/mL.
  • Helicobacter pylori antibody detection reagent strip specificity test use Helicobacter pylori IgG-positive serum, hepatitis B virus IgG-positive serum, cytomegalovirus IgG-positive serum, herpes simplex virus IgG-positive serum, adenovirus IgG-positive serum, respiratory tract IgG-positive serum Cytovirus virus IgG positive serum and Helicobacter pylori IgG negative serum were detected by Helicobacter pylori antibody immune colloidal gold detection strips. Only the Helicobacter pylori standard IgG positive serum test results were positive, and the others were negative, indicating that the reagent strip was specific. Sex is good.
  • the reagent strip of the present invention can also be used for qualitative detection of Helicobacter pylori antibodies, and the specific method is as follows:
  • a red band appears on the quality control line 502, and a red band appears on the test line 501, which means Helicobacter pylori IgG antibody positive;
  • Negative a red band appears on the quality control line 502, and no red band appears on the test line 501, it is Helicobacter pylori Bacterial IgG antibody is negative; failure: no red band appears on the quality control line 502, and it is judged that the reagent strip is invalid (when the sample is added dropwise, the quality control substance is added dropwise).

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Abstract

L'invention concerne une bandelette réactive pour la détection quantitative d'un anticorps contre l'Helicobacter pylori au moyen d'or colloïdal. La bandelette réactive comprend une platine inférieure et un tampon d'échantillon, un tampon d'or colloïdal, une membrane en nitrocellulose et un papier filtre absorbant l'eau qui sont collés à la platine inférieure. Le tampon d'échantillon, le tampon d'or colloïdal, la membrane de nitrocellulose et le papier filtre absorbant l'eau sont successivement superposés bout à bout sur une longueur prédéfinie ; la membrane de nitrocellulose est recouverte d'antigènes naturels d'Helicobacter pylori par linéation ; le tampon d'or colloïdal est obtenu en ajoutant une solution d'antigènes protéiques recombinants d'Helicobacter pylori marqués à l'or colloïdal sur une membrane de verre et de cellulose, puis en la séchant. La bandelette réactive pour la détection quantitative d'un anticorps contre l'Helicobacter pylori au moyen d'or colloïdal de la présente invention peut permettre de détecter quantitativement l'anticorps contre l'Helicobacter pylori en utilisant une méthode d'immunochromatographie à l'or colloïdal. En conséquence, la présente invention fournit également une méthode pour détecter quantitativement l'anticorps contre l'Helicobacter pylori à l'aide de la bandelette réactive pour détecter quantitativement l'anticorps contre l'Helicobacter pylori à l'aide d'or colloïdal selon la présente invention.
PCT/CN2021/123687 2020-09-07 2021-10-14 Bandelette réactive pour détection quantitative d'anticorps contre l'hélicobacter pylori au moyen d'or colloïdal et méthode de détection WO2022048693A1 (fr)

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CN202010925995.8A CN111983229A (zh) 2020-09-07 2020-09-07 胶体金定量检测幽门螺杆菌抗体试剂条及检测方法
CN202010925995.8 2020-09-07

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CN115060904A (zh) * 2022-08-16 2022-09-16 山东康华生物医疗科技股份有限公司 一种乙肝表面抗原检测试剂盒用胶体金溶液的制备方法及试剂条、试剂盒
CN116008562A (zh) * 2022-12-26 2023-04-25 北京利德曼生化股份有限公司 磁微粒化学发光双抗原夹心法检测幽门螺杆菌抗体的方法

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CN115887641A (zh) * 2022-11-23 2023-04-04 四川大学华西医院 他米巴罗汀在制备免疫佐剂中的应用

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CN107356755A (zh) * 2017-07-25 2017-11-17 江斌 幽门螺杆菌抗体金检测试剂盒
CN111983229A (zh) * 2020-09-07 2020-11-24 三门县人民医院 胶体金定量检测幽门螺杆菌抗体试剂条及检测方法

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CN115060904A (zh) * 2022-08-16 2022-09-16 山东康华生物医疗科技股份有限公司 一种乙肝表面抗原检测试剂盒用胶体金溶液的制备方法及试剂条、试剂盒
CN115060904B (zh) * 2022-08-16 2022-11-11 山东康华生物医疗科技股份有限公司 一种乙肝表面抗原检测试剂盒用胶体金溶液的制备方法及试剂条、试剂盒
CN116008562A (zh) * 2022-12-26 2023-04-25 北京利德曼生化股份有限公司 磁微粒化学发光双抗原夹心法检测幽门螺杆菌抗体的方法

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