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WO2024066594A1 - Papier réactif et trousse de détection d'helicobacter pylori - Google Patents

Papier réactif et trousse de détection d'helicobacter pylori Download PDF

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Publication number
WO2024066594A1
WO2024066594A1 PCT/CN2023/104528 CN2023104528W WO2024066594A1 WO 2024066594 A1 WO2024066594 A1 WO 2024066594A1 CN 2023104528 W CN2023104528 W CN 2023104528W WO 2024066594 A1 WO2024066594 A1 WO 2024066594A1
Authority
WO
WIPO (PCT)
Prior art keywords
helicobacter pylori
monoclonal antibody
test paper
kit
colloidal gold
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2023/104528
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English (en)
Chinese (zh)
Inventor
王丽
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Jinwofu Bioengineering Technology Co Ltd
Original Assignee
Beijing Jinwofu Bioengineering Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Jinwofu Bioengineering Technology Co Ltd filed Critical Beijing Jinwofu Bioengineering Technology Co Ltd
Priority to GB2313918.1A priority Critical patent/GB2627550A/en
Priority to ZA2023/08365A priority patent/ZA202308365B/en
Publication of WO2024066594A1 publication Critical patent/WO2024066594A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the technical field of microbial detection, and in particular to a test paper and a kit for detecting Helicobacter pylori.
  • Helicobacter pylori is a spiral-shaped, slightly anaerobic bacterium that has very demanding growth conditions. It may cause diseases such as gastritis, peptic ulcers, lymphoproliferative gastric lymphoma and even gastric cancer.
  • the first is the bacterial culture method, which has high specificity and can perform drug sensitivity and resistance tests at the same time, but the test is time-consuming (2 to 4 days), labor-intensive, and has high conditions and technology, and is unable to detect dead bacteria and active ingredients.
  • the second is the rapid urease test, which uses mucosal biopsy tissue as the specimen. It is fast and can be used for initial screening, taking about an hour. However, it is traumatic, and invasive sampling can easily increase patient pain, requiring experienced technicians.
  • the third breath test is currently the most widely used method.
  • the method is simple and quick, takes about 30 minutes, is painless to the patient, and has high accuracy.
  • it still has the disadvantages of expensive equipment, high fees, radioactivity, and is not suitable for pregnant women, infants, and people with hypertension and heart disease.
  • the fourth PCR method has high sensitivity and has no requirements for live or dead bacteria, the specimens are easily contaminated, the false positive rate is high, the operation requirements are high, and it requires very experienced technicians. Invasive sampling increases the pain of patients.
  • the fifth enzyme-linked immunosorbent assay cannot distinguish between patients with previous infection and current infection and is mainly used in epidemiological surveys.
  • the purpose of the present invention is to provide a test paper and a kit for detecting Helicobacter pylori.
  • the detection of Helicobacter pylori using the reagent of the present invention is rapid, does not require invasive sampling of the test sample, is non-invasive, and is painless for the patient; the operation is simple, the detection cost is low, no expensive equipment is required, and the application range is wide; the specificity, sensitivity and accuracy are high.
  • the present invention selects an immunogen with good specificity.
  • Helicobacter pylori contains a large amount of highly active extracellular urease, which accounts for about 10% of the whole bacterial protein. Detection of urease can be used as a method for detecting Helicobacter pylori.
  • the present invention further designs immunogens for the antigenic epitopes of amino acids at positions 70 to 84aa and 128 to 140aa on the 29KDa fragment of urease for subsequent preparation of monoclonal antibodies, and finds that the monoclonal antibodies prepared by immunogens designed for the target have better specificity.
  • the present invention provides a Helicobacter pylori immunogen, comprising an amino acid sequence as shown in SEQ ID NO.1.
  • the present invention further provides a method for preparing a monoclonal antibody for detecting Helicobacter pylori, which is prepared by using the Helicobacter pylori immunogen as an immunogen.
  • the present invention provides a test strip for detecting Helicobacter pylori, wherein the test strip comprises a detection line, and the detection line is coated with the monoclonal antibody.
  • the concentration of the monoclonal antibody is 2 mg/mL.
  • test paper also includes an immune nitrocellulose membrane, on which the detection line and the quality control line are arranged, and the quality control line is coated with sheep anti-mouse IgG.
  • the test paper also includes an immune colloidal gold pad, which is prepared by the following method:
  • the colloidal gold can be prepared by a trisodium citrate reduction method, for example, including the following process:
  • the dosage of the sodium citrate aqueous solution is 0.65-1% (volume ratio) of the HAuCl 4 aqueous solution.
  • the preparation of the colloidal gold-monoclonal antibody complex comprises:
  • the colloidal gold particles and the monoclonal antibody are reacted at pH 7.8 for 10 to 15 minutes; the monoclonal antibody is a monoclonal antibody that can specifically bind to the Helicobacter pylori polypeptide antigen.
  • the usage ratio of the colloidal gold particles to the monoclonal antibody is 1000 mL: (20-40 mg).
  • the carrier membrane is one or more of a glass cellulose membrane, a polyester fiber membrane or a nitrocellulose membrane.
  • test paper also includes absorbent paper and a sample pad.
  • test paper can be prepared into a corresponding product in any of the following ways:
  • test paper cut the test paper into reagent strips, put them into a plastic card, and put them into an aluminum foil bag together with a desiccant, and seal and package them to obtain a single-person HP polypeptide antigen detection test paper (card type).
  • the present invention provides a kit, which includes the test paper.
  • kit also includes a buffer and instructions.
  • single-person HP polypeptide antigen detection test paper card type
  • strip type single-person HP polypeptide antigen detection test papers
  • the monoclonal antibody provided by the present invention targets a specific site on the 29KDa polypeptide, has high immunogenicity, and has high specificity and sensitivity when used for the detection of Helicobacter pylori.
  • the present invention detects HP polypeptide antigens.
  • Polypeptides are intermediate products of protein hydrolysis. Even after being degraded in the digestive tract, the retained polypeptide fragments can be detected.
  • the test sample is feces, and invasive sampling is not required. At the same time, current infections can be diagnosed.
  • the detection object is HP antigen polypeptide, dead bacteria and secreted active ingredients can be detected.
  • the test paper provided by the present invention has strong specificity for detecting HP polypeptide antigens, and is not interfered by other bacteria, viruses, H2 receptor antagonists and sampling sites in feces. It is particularly suitable for the diagnosis of HP infection in patients who are not suitable for invasive examinations (especially suitable for young children, pregnant women, the elderly and patients who are not suitable for gastroscopy). It can be used as one of the preferred detection methods to determine whether there is HP infection in the stomach, observe the efficacy of anti-HP drugs, and observe recurrence or reinfection after anti-HP treatment. It can also be used as an evaluation indicator for long-term radical treatment efficacy.
  • the kit provided by the present invention is simple and fast to detect, easy to collect specimens, relatively cheap, and a non-invasive diagnostic method that can be repeated many times.
  • the detection of antigens directly reflects the information of the current infection of pathogenic organisms, and can also diagnose acute HP infection and evaluate the therapeutic efficacy.
  • FIG1 is a schematic diagram of an HPAg detection kit (card type) provided in Example 1 of the present invention.
  • FIG2 is a schematic diagram of the HPAg detection kit (bar type) provided in Example 1 of the present invention.
  • This embodiment provides a process for preparing a kit for detecting Helicobacter pylori, which is as follows:
  • HP polypeptide antigen was used, and the amino acid sequence was shown in SEQ ID NO.1.
  • the purified HP polypeptide antigen was added with Freund's complete adjuvant and, after being fully emulsified, was injected into the back skin of BALB/c mice at multiple points. After an interval of 2 weeks, the second immunization was performed with HP purified antigen plus Freund's incomplete adjuvant, and a booster injection was given into the tail vein 3 days before fusion.
  • Mouse myeloma cells (SP2/0-Ag14) were fused with immune mouse spleen cells under the help of PEG, and purified HP antigen was used as the target of indirect ELISA to screen hybridoma cells with high titer antibodies.
  • the screened cells were cloned multiple times by limiting dilution method, expanded and preserved.
  • the expanded hybridoma cells were injected into BALB/c mice intraperitoneally, and the ascites was collected after 10 to 14 days, centrifuged, and the supernatant was HP-McAb.
  • hybridoma cells were obtained as described above, and the present invention conducted subsequent experiments on monoclonal antibodies secreted by several of these hybridoma cells.
  • the four antibodies were coated on a polystyrene 96-well plate at a dilution of 1:3000 and incubated at 37°C overnight. The next day, the coating solution was discarded, the plate was washed with PBS, and 1 ⁇ 10 6 CFU/ml of Helicobacter pylori treated with lysis buffer was added for 1 hour. The plate was washed and the four antibodies were added at a dilution of 1:3000 for 1 hour. The plate was washed and rabbit anti-mouse-HRP antibody was added for 30 minutes, and 50 ⁇ l of stop solution (2 mol/L H 2 SO 4 ) was added to terminate the reaction. The OD value at 450 nm was read using an enzyme reader to determine the reaction status.
  • HP monoclonal antibody 1, HP monoclonal antibody 2, and HP monoclonal antibody 4 can complete sandwich pairing detection targets.
  • HP monoclonal antibody 3, HP monoclonal antibody 2, and HP monoclonal antibody 4 can form sandwich pairing detection targets.
  • HP monoclonal antibody 2 and HP monoclonal antibody 4 HP monoclonal antibody 1, and HP monoclonal antibody 3 cannot form effective pairing detection targets.
  • HP monoclonal antibodies 1, 2, 3, and 4 were diluted to 1 mg/ml with PBS and coated on nitrocellulose membranes respectively. HP monoclonal antibodies 1, 2, 3, and 4 were labeled with colloidal gold and dried on glass cellulose membranes respectively. Eight combinations of 1-2, 1-4, 3-2, and 3-4 were tested respectively.
  • monoclonal antibody 4 is not suitable for use as a coating antibody, while the other three can be used for coating, and all four antibodies can be used for labeling.
  • Preparation of colloidal gold-HP monoclonal antibody 1 complex Take 1000ml colloidal gold, add 100ml of pH7.8, 0.01M phosphate buffer, and mix; under stirring, add 10ml HP monoclonal antibody 1 solution to the prepared colloidal gold solution, mix, and react at room temperature for 10 minutes; after the reaction, add 5% BSA (as a protective agent) to make the final concentration of BSA in the solution 0.01%, mix, and react at room temperature for 10 minutes; centrifuge the reaction complex at 10000rpm for purification; add pH7.8, 0.01M phosphate buffer to the original volume, centrifuge, take the precipitate, add pH7.8, 0.01M phosphate buffer to the final volume of 90ml to obtain immune colloidal gold solution.
  • the optimal HP monoclonal antibody 1 labeling amount per 1000ml colloidal gold is 20mg.
  • the nitrocellulose membrane was attached to the PVC plate.
  • the optimal coating concentration of the HP monoclonal antibody 2 fixed on the detection line (T line) was 2.0 mg/ml, and the optimal coating concentration of the goat anti-mouse IgG on the C line was 2.0 mg/ml.
  • the sprayed membrane was dried.
  • FIG. 1 is a plastic box
  • 2 is a result observation window
  • 3 is a quality control line (C line)
  • 4 is a detection line (T line)
  • 5 is a detection test paper
  • 6 is a sample addition hole.
  • 1 is the handle paper
  • 2 is the quality control line (C line)
  • 3 is the detection line (T line)
  • 4 is the detection test paper
  • 5 is the MAX line
  • 6 is the MAX glue.
  • the present invention collected 15 Helicobacter pylori positive stool samples from Anhui, Guangdong and Shandong, and compared the Helicobacter pylori test kit prepared by the present invention with the C13 breath test results. The results are shown in the following table:
  • This test example further tests the sensitivity of the test strip provided by the present invention.
  • the positive standard is configured according to the following concentrations: Helicobacter pylori (ATCC 700392). After the gastric Helicobacter pylori is cultured, counted and inactivated, the concentrations are adjusted to a series of 1 ⁇ 10 7 CFU/ml, 5 ⁇ 10 6 CFU/ml, 1 ⁇ 10 6 CFU/ml, 1 ⁇ 10 5 CFU/ml, 1 ⁇ 10 4 CFU/ml, and 5 ⁇ 10 3 CFU/ml for detection. The results are shown in the following table.
  • the antigen reagent can effectively detect Helicobacter pylori when the concentration is 1 ⁇ 10 4 CFU/ml.
  • the present invention counts and inactivates a variety of bacteria that may produce false positive results or interfere with the result product, such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enteritidis, and Candida albicans, and then adjusts the concentration value to 1 ⁇ 10 6 /ml.
  • the adenovirus and rotavirus samples are adjusted to 1 ⁇ 10 5 PFU/mL and then detected with an antigen reagent.
  • the test results are shown in the following table, and the results show that there is no cross reaction, indicating that the kit for detecting Helicobacter pylori provided by the present invention has high specificity.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
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  • Engineering & Computer Science (AREA)
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  • Urology & Nephrology (AREA)
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  • Physics & Mathematics (AREA)
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  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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Abstract

La présente invention concerne le domaine technique de la détection microbienne, en particulier un papier réactif et une trousse de détection d'Helicobacter pylori. Le papier réactif comprend une ligne d'essai, et la ligne d'essai est préparée à partir d'un anticorps monoclonal servant à détecter Helicobacter pylori. L'anticorps monoclonal est préparé à partir d'un antigène, tel que représenté dans SEQ ID NO 1 en tant qu'immunogène, tous les anticorps monoclonaux préparés à partir de l'antigène présentant une spécificité élevée. La présente invention concerne en outre un papier réactif comprenant un anticorps monoclonal préparé à partir de l'antigène et une trousse correspondante. Le papier réactif et la trousse peuvent détecter rapidement et avec précision Helicobacter pylori.
PCT/CN2023/104528 2022-09-28 2023-08-08 Papier réactif et trousse de détection d'helicobacter pylori Ceased WO2024066594A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
GB2313918.1A GB2627550A (en) 2022-09-28 2023-08-08 Test strip and kit for detecting helicobacter pylori(HP)
ZA2023/08365A ZA202308365B (en) 2022-09-28 2023-08-30 Test strip and kit for detecting helicobacter pylori (hp)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211193877.8A CN116068170B (zh) 2022-09-28 2022-09-28 一种检测幽门螺旋杆菌的试纸和试剂盒
CN202211193877.8 2022-09-28

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GB2627550A (en) * 2022-09-28 2024-08-28 Beijing Jinwofu Bioengineering Tech Co Ltd Test strip and kit for detecting helicobacter pylori(HP)
CN116068170B (zh) * 2022-09-28 2023-09-15 北京金沃夫生物工程科技有限公司 一种检测幽门螺旋杆菌的试纸和试剂盒
CN116735869B (zh) * 2023-05-11 2025-05-30 江苏默乐生物科技股份有限公司 一种幽门螺旋杆菌抗原胶体金检测试剂盒及其应用
CN119409811B (zh) * 2025-01-06 2025-05-16 天津龙晟生物科技有限公司 幽门螺旋杆菌抗体及其检测产品和应用

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