WO2022048693A1 - 胶体金定量检测幽门螺杆菌抗体试剂条及检测方法 - Google Patents

胶体金定量检测幽门螺杆菌抗体试剂条及检测方法 Download PDF

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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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helicobacter pylori
colloidal gold
antibody
reagent strip
nitrocellulose membrane
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PCT/CN2021/123687
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English (en)
French (fr)
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黎宏章
赵俊
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三门县人民医院
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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

本发明公开了一种胶体金定量检测幽门螺杆菌抗体试剂条，所述试剂条包括底板，以及粘贴于底板上的样品垫、胶体金垫、硝酸纤维素膜和吸水滤纸；所述样品垫、胶体金垫、硝酸纤维素膜和吸水滤纸依次首尾相互重叠一段预设长度；所述硝酸纤维素膜上通过划线包被幽门螺杆菌天然抗原；所述胶体金垫通过将标记胶体金的幽门螺杆菌重组蛋白抗原溶液加样到玻璃纤维素膜上，干燥后获得。本发明的胶体金定量检测幽门螺杆菌抗体试剂条可以实现用胶体金免疫层析法定量检测幽门螺杆菌抗体。对应的，本发明还提供了使用本发明的胶体金定量检测幽门螺杆菌抗体试剂条定量检测幽门螺杆菌抗体的方法。

Description

胶体金定量检测幽门螺杆菌抗体试剂条及检测方法 技术领域
本发明涉及幽门螺杆菌抗体的检测技术，具体是胶体金定量检测幽门螺杆菌抗体试剂条，以及用该试剂条定量检测幽门螺杆菌抗体的检测方法。
 背景技术
幽门螺杆菌(Helicobacter pylori,H.p)，由Barry J.Marshall和J.Robin Warren二人首次发现，此二人因此获得2005年的诺贝尔生理学或医学奖。幽门螺杆菌是一种单极、多鞭毛、末端钝圆、螺旋形弯曲的细菌，长2.5～4.0μm，宽0.5～1.0μm。在胃粘膜上皮细胞表面常呈典型的螺旋状或弧形。
幽门螺杆菌感染是慢性活动性胃炎、消化性胃溃疡以及胃黏膜相关淋巴组织淋巴瘤的主要致病因素，并且与胃癌的发生具有密切相关性。1994年世界卫生组织/国际癌症研究机构(WHO/IARC) 将幽门螺杆菌定为I 类致癌原。在亚洲地区，中国大陆、越南、印度等少年幽门螺杆菌的感染率分别60%、40%、70%，慢性胃炎患者的胃粘膜活检标本中幽门螺杆菌检出率可达80％～90％，而消化性胃溃疡患者更高，可达95％以上，甚至接近100％。胃癌由于局部上皮细胞已发生异化，因此其检出率高低报道不一。幽门螺杆菌的胃部感染已是个世界性医学问题，对其进行准确诊断有利于控制H.p传播与流行及根除H.p 感染治疗的监测。
目前对幽门螺杆菌感染的诊断已发展出了许多方法，包括有细菌学、病理学、血清学、同位素示踪、分子生物学等。但总的讲来，从标本采集角度看，可以分为侵入性和非侵入性两大类。侵入性方法主要指必需通过胃镜取活检标本检查的方法，是目前消化病学科的常规方法。它包括细菌的分离培养和直接涂片、快速尿素酶试验，药敏试验。非侵入性方法主要指不通过胃镜取活检标本诊断幽门螺杆菌标本感染的方法。这类方法包括抗体检测、抗原检测、尿素13C/14C 呼气试验等。抗体检测目前已有的方法包括酶联免疫法、免疫印迹法、胶体金法和胶乳增强免疫比浊法等。
血清学检测是一项常用的非侵入性检测方法，尤其适用于幽门螺杆菌感染率较高的地区。幽门螺杆菌引起慢性炎症，***的免疫反应会持续存在，产生 IgG抗体。常用的血清学试验包括ELISA 法、化学发光、蛋白印迹、胶体金免疫层析等方法，其中胶体金免疫层析法检测速度最快，只需要10分钟即可出结果，特别适合现场快速检测，但目前尚无定量的幽门螺杆菌抗体胶体金免疫层析法检测试剂面市。
 技术问题
为了克服现有技术中存在的不足，本发明提供了胶体金定量检测幽门螺杆菌抗体试剂条。本发明的胶体金定量检测幽门螺杆菌抗体试剂条可以实现用胶体金免疫层析法定量检测幽门螺杆菌抗体。对应的，本发明还提供了使用本发明的胶体金定量检测幽门螺杆菌抗体试剂条定量检测幽门螺杆菌抗体的方法。
 技术解决方案
对于试剂条，本发明技术方案为：
胶体金定量检测幽门螺杆菌抗体试剂条，所述试剂条包括底板，以及粘贴于底板上的样品垫、胶体金垫、硝酸纤维素膜和吸水滤纸；所述样品垫、胶体金垫、硝酸纤维素膜和吸水滤纸依次首尾相互重叠一段预设长度；所述硝酸纤维素膜上通过划线包被幽门螺杆菌天然抗原；所述胶体金垫通过将标记胶体金的幽门螺杆菌重组蛋白抗原溶液加样到玻璃纤维素膜上，干燥后获得。
作为本发明的一种优化方案，前述的胶体金定量检测幽门螺杆菌抗体试剂条中，所述胶体金垫通过以下方法步骤制备：步骤a.用0.1～0.3mol/L K2CO3溶液将胶体金溶液的pH调到7.0～9.0；步骤b.将步骤a中的胶体金溶液置于磁力搅拌器上搅拌，按每100mL胶体金溶液加入0.5～2mg幽门螺杆菌重组蛋白抗原的用量将幽门螺杆菌重组蛋白抗原滴加到胶体金溶液中，继续搅拌2～3h；步骤c.将步骤b中搅拌后的胶体金溶液加入到终浓度为1%BSA和1～2％的酪蛋白混合液中进行封闭标记60min；步骤d.将步骤c中封闭标记后的胶体金溶液在12000～15000r/m条件下离心，弃上层清液，沉淀物用0.01mol/L PBS溶液重悬，将沉淀物重悬为原沉淀物体积的1/10，得重悬液；步骤e.将步骤d制得的重悬液在2000～3000r/min条件下离心30min，取上层清液加入到葡聚糖凝胶层析柱纯化，得到纯度达90%以上的标记胶体金的幽门螺杆菌重组蛋白溶液；步骤f.将步骤e制得的标记胶体金的幽门螺杆菌重组蛋白抗原溶液加样到玻璃纤维素膜上，在温度20℃～25℃的条件下干燥4～6h，制成胶体金垫，备用。
作为本发明的一种优化方案，前述的胶体金定量检测幽门螺杆菌抗体试剂条中，在所述硝酸纤维素膜上通过划线包被幽门螺杆菌天然抗原的方法步骤如下：步骤i．用0.01mol/L的PBS将幽门螺杆菌天然抗原稀释成0.2～0.5mg/mL；步骤ii．用点膜仪将经步骤i稀释后的幽门螺杆菌天然抗原在硝酸纤维素膜上按1µl/cm进行划线包被；步骤iii．幽门螺杆菌天然抗原包被完成后，将硝酸纤维素膜在干燥间室温干燥，备用。
作为本发明的一种优化方案，前述的胶体金定量检测幽门螺杆菌抗体试剂条中，所述硝酸纤维素膜上还通过划线包被鼠抗幽门螺杆菌单克隆抗体，用于质控。进一步，在所述硝酸纤维素膜上，通过划线包被鼠抗幽门螺杆菌单克隆抗体的方法步骤如下：步骤①．用0.01mol/L的PBS将鼠抗幽门螺杆菌单克隆抗体稀释成0.2～0.5mg/mL；步骤②．用点膜仪将经步骤i稀释后的鼠抗幽门螺杆菌单克隆抗体在硝酸纤维素膜上按1µl/cm进行划线包被；步骤③．鼠抗幽门螺杆菌单克隆抗体包被完成后，将硝酸纤维素膜在干燥间室温干燥，备用。
作为本发明的一种优化方案，前述的胶体金定量检测幽门螺杆菌抗体试剂条中，所述试剂条的组装在干燥间内完成，具体步骤如下：步骤I.在底板中央贴上包被好的幽门螺杆菌天然抗原硝酸纤维素膜；步骤II.在硝酸纤维素膜上缘粘贴吸水滤纸；步骤III.在硝酸纤维素膜下缘粘贴胶体金垫；步骤IV.在胶体金垫下缘粘贴样品垫，完成后用裁切机将贴好的试纸板切成试纸条，并密封于铝箔袋中。
优选的，所述底板可以由PVC制成。
与现有技术相比，本发明的胶体金定量检测幽门螺杆菌抗体试剂条是采用幽门螺杆菌重组蛋白抗原和天然抗原组合双抗原夹心法制备出的幽门螺杆菌抗体胶体金定量检测试剂条，用该试剂条检测临床样本时，胶体金检测仪读取T/C值代入标准曲线，即可获得临床样本中幽门螺杆菌抗体的含量（AU/mL），实现对幽门螺杆菌抗体（IgG抗体）的快速定量检测。
对于检测方法，本发明的技术方案为：
胶体金定量检测幽门螺杆菌抗体的检测方法，该方法采用前述本发明的胶体金定量检测幽门螺杆菌抗体试剂条对幽门螺杆菌抗体进行定性检测和定量检测；
定性检测过程如下：将幽门螺杆菌抗体检测条水平放置，准确滴加1滴血清或血浆样本在试纸条的样品垫上，室温反应10分钟后，质控线出现红色条带，检测线出现红色条带，检测结果为幽门螺杆菌IgG抗体阳性；质控线出现红色条带，检测线不出现红色条带，检测结果为幽门螺杆菌IgG抗体阴性；质控线不出现红色条带或质控线、检测线均不出现红色条带，试剂条检测失效；
定量检测过程如下：将幽门螺杆菌抗体试剂条水平放置，准确滴加1滴血清或血浆样本在试纸条的样品垫上，室温反应10分钟后，将幽门螺杆菌抗体试剂条放入胶体金定量读数仪中，读取T线与C线的峰面积比值即T/C值，代入幽门螺杆菌抗体检测标准曲线，获得样本中幽门螺杆菌抗体的含量（AU/mL）。
作为本发明的一种优化方案，前述的胶体金定量检测幽门螺杆菌抗体的检测方法中，所述幽门螺杆菌抗体检测标准曲线的制作方法包括以下过程：用幽门螺杆菌抗体阴性血清按梯度稀释1AU/mL幽门螺杆菌抗体检测标准品，分别取50μL加到试剂条的样品垫中，室温反应10min，将试剂条放入胶体金定量读数仪中，读取T线与C线的峰面积比值即T/C值，以T/C值为横坐标、幽门螺杆菌抗体检测标准品AU值为纵坐标，制作标准曲线；所述1AU/mL幽门螺杆菌抗体检测标准品的制备方法如下：收集到20份幽门螺杆菌抗体阳性血清混合，经饱和硫酸铵盐析结合Protein G柱亲和层析纯化，得到终浓度为 0.5mg/mL纯化幽门螺杆菌抗体，定为1AU/mL。
 有益效果
与现有技术相比，本发明的上述方法取得的显著进步是：利用胶体金免疫层析法实现了对幽门螺旋杆菌的定量检测。
 附图说明
图1是本发明胶体金定量检测幽门螺杆菌抗体试剂条的结构示意图；
附图中标记：1-底板，2-样品垫，3-胶体金垫，4-吸水滤纸，5-硝酸纤维素膜，501-检测线，502-质控线。
 本发明的实施方式
以下结合具体实施例对本发明作进一步说明，但不作为限制本发明的依据。
参见图1，本发明的胶体金定量检测幽门螺杆菌抗体试剂条包括PVC材质的底板1，以及粘贴于PVC材质的底板1上的样品垫2、胶体金垫3、硝酸纤维素膜5和吸水滤纸4；所述样品垫2、胶体金垫3、硝酸纤维素膜5和吸水滤纸4依次首尾相互重叠一段预设长度。
其中，所述硝酸纤维素膜5上通过划线包被幽门螺杆菌天然抗原，形成检测线501；所述胶体金垫3通过将标记胶体金的幽门螺杆菌重组蛋白抗原溶液加样到玻璃纤维素膜上，干燥后获得。
实施本发明时，硝酸纤维素膜5上可以通过划线包被鼠抗幽门螺杆菌单克隆抗体，形成质控线502，用于质控。
实施例：
本实施例的胶体金定量检测幽门螺杆菌抗体试剂条按照如下步骤制得。
一、制备包被有幽门螺杆菌重组蛋白抗原的胶体金垫：用0.2mol/L K 2CO 3将胶体金溶液pH调到8。然后将溶液置于磁力搅拌器上缓慢搅拌，按每100mL胶体金溶液加入1.25mg幽门螺杆菌重组蛋白抗原将蛋白缓慢滴加到胶体金溶液中，继续搅拌2.5h，再滴加入到终浓度为1%BSA和1.5％的酪蛋白进行封闭标记60min，封闭标记结束后以13500r/m离心，弃上层清液，沉淀物用0.01mol/L PBS溶液重悬，将沉淀重悬为原体积的1/10，再经2500r/min离心30min，取上层清液加至葡聚糖凝胶层析柱纯化，得到纯度达90%以上的标记胶体金的幽门螺杆菌重组蛋白溶液。然后将标记胶体金的幽门螺杆菌重组蛋白抗原溶液加样于玻璃纤维素膜上，在温度22℃干燥5h，制成胶体金垫3，干燥备用。
二、幽门螺杆菌天然抗原包被硝酸纤维素膜：用0.01mol/L PBS将幽门螺杆菌天然抗原稀释成0.3mg/mL，然后用BIODOT点膜仪将幽门螺杆菌天然抗原在硝酸纤维素膜5上按1µl/cm进行划线包被，形成检测线501；同时在硝酸纤维素膜5上按1µl/cm包被鼠抗幽门螺杆菌单克隆抗体，用于产品的质控线502，包被完成后将硝酸纤维素膜5在干燥间室温干燥8h，备用。
三、试剂的组装：在干燥间内准备好PVC材质的底板1、样品垫2、胶体金垫3、硝酸纤维素膜5和吸水滤纸4，底板1中央贴上包被好的幽门螺杆菌天然抗原硝酸纤维素膜5，硝酸纤维素膜5上缘粘贴吸水滤纸4，硝酸纤维素膜5下缘粘贴包被幽门螺杆菌重组蛋白抗原胶体金垫3，胶体金垫3下缘粘贴样品垫2，完成后用裁切机将贴好的试纸板切成4 mm宽的试纸条。再将试纸条密封于铝箔袋中，完成试剂的组装，获得产品。
以下结合检测方法以及一些必要的试验对本发明作进一步的说明。
使用本发明的试剂卡快速定量检测幽门螺杆菌抗体的方法如下：将试剂条水平放置，准确滴加1滴血清或血浆样本在试纸条的样品垫2上，室温反应10分钟后，依照下列方法进行定量检测。
定量检测过程如下：将幽门螺杆菌抗体试剂条水平放置，准确滴加1滴血清或血浆样本在试纸条的样品垫2上，室温反应10分钟后，将幽门螺杆菌抗体试剂条放入胶体金定量读数仪中，读取T线与C线的峰面积比值即T/C值，代入幽门螺杆菌抗体检测标准曲线，获得样本中幽门螺杆菌抗体的含量（AU/mL）。
幽门螺杆菌抗体检测标准曲线的建立方法如下：用幽门螺杆菌抗体阴性血清按梯度稀释1AU/mL幽门螺杆菌抗体检测标准品，分别取50μL 加到试剂条的样品垫2中，室温反应10min，将试剂条放入胶体金定量读数仪中，读取T线与C线的峰面积比值即T/C值，以T/C值为横坐标、幽门螺杆菌抗体检测标准品AU值为纵坐标，制作标准曲线。该试剂检测临床样本时，胶体金定量读数仪读取T/C值代入标准曲线，获得样本中幽门螺杆菌抗体的含量（AU/mL）。
上述幽门螺杆菌抗体检测标准品的制备方法如下：收集到20份幽门螺杆菌抗体阳性血清混合，经饱和硫酸铵盐析结合Protein G柱亲和层析纯化，得到终浓度为 0.5mg/mL纯化幽门螺杆菌抗体，定为1AU/mL。
幽门螺杆菌抗体检测试试剂条特异性试验：以幽门螺杆菌IgG阳性血清、乙型肝炎病毒IgG阳性血清、巨细胞病毒IgG阳性血清、单纯疱疹病毒IgG阳性血清、腺病毒IgG阳性血清、呼吸道合胞病毒病毒IgG阳性血清和幽门螺杆菌IgG阴性血清采用幽门螺杆菌抗体免疫胶体金检测试纸条检测，只有幽门螺杆菌标准IgG阳性血清检测结果为阳性，其它均为阴性，说明该试剂条特异性良好。
幽门螺杆菌抗体免疫胶体金检测试纸条重复性试验：随机抽取20份幽门螺杆菌IgG阳性血清、阴性血清分别于不同的时间进行重复检测，变异系数均<5%，证明该试剂条具有良好的稳定性和重复性。
临床标本检测对比试验：
试纸条与芬兰BIOHIT公司试剂盒幽门螺杆菌IgG抗体ELISA检测试剂盒的比较：
对200份标本用两种试剂盒检测比较阴性与阳结果，如表1所示：
表1 与芬兰BIOHIT公司试剂盒的比较结果
Figure 486839dest_path_image001
对200份标本用两种试剂盒检测共有195例相符(97.5%)，只有5例不符（2.5%），这也表明本发明的试剂灵敏度和特异性高。
以上是以本发明的一个具体实施例进行说明的，在本发明技术方案的范围内对上述实施例中相关参数取值调整后所形成的其它具体实施例同样能够实现本发明的目的，在此不一一罗列。
本发明的试剂条还可以用于定性检测幽门螺杆菌抗体，具体方法如下：
将试剂条水平放置，准确滴加1滴血清或血浆样本在试纸条的样品垫2上，室温反应10分钟后，进行定性结果判断。
阳性：质控线502出现红色条带，检测线501出现红色条带，为幽门螺杆菌IgG抗体阳性；阴性：质控线502出现红色条带，检测线501不出现红色条带，为幽门螺杆菌IgG抗体阴性；失效：质控线502不出现红色条带，判断为试剂条失效（滴加样本时，同时滴加质控品）。
上述对本申请中涉及的发明的一般性描述和对其具体实施例的描述不应理解为是对该发明技术方案构成的限制。本领域所属技术人员根据本申请的公开，可以在不违背所涉及的发明构成要素的前提下，对上述一般性描述或/和实施例中的公开技术特征进行增加、减少或组合，形成属于本申请保护范围之内的其它的技术方案。

Claims (9)

  1. 胶体金定量检测幽门螺杆菌抗体试剂条，其特征在于：
    所述试剂条包括底板，以及粘贴于底板上的样品垫、胶体金垫、硝酸纤维素膜和吸水滤纸；所述样品垫、胶体金垫、硝酸纤维素膜和吸水滤纸依次首尾相互重叠一段预设长度；
    所述硝酸纤维素膜上通过划线包被幽门螺杆菌天然抗原；所述胶体金垫通过将标记胶体金的幽门螺杆菌重组蛋白抗原溶液加样到玻璃纤维素膜上，干燥后获得。
  2. 根据权利要求1 所述的胶体金定量检测幽门螺杆菌抗体试剂条，其特征在于，所述胶体金垫通过以下方法步骤制备：
    步骤a：用0.1～0.3mol/L K 2CO 3溶液将胶体金溶液的pH调到7.0～9.0；
    步骤b：将步骤a中的胶体金溶液置于磁力搅拌器上搅拌，按每100mL胶体金溶液加入0.5～2mg幽门螺杆菌重组蛋白抗原的用量将幽门螺杆菌重组蛋白抗原滴加到胶体金溶液中，继续搅拌2～3h；
    步骤c：将步骤b中搅拌后的胶体金溶液加入到终浓度为1%BSA和1～2％的酪蛋白混合液中进行封闭标记60min；
    步骤d：将步骤c中封闭标记后的胶体金溶液在12000～15000 r/m条件下离心，弃上层清液，沉淀物用0.01mol/L PBS溶液重悬，将沉淀物重悬为原沉淀物体积的1/10，得重悬液；
    步骤e：将步骤d制得的重悬液在2000～3000r/min条件下离心30min，取上层清液加入到葡聚糖凝胶层析柱纯化，得到纯度达90%以上的标记胶体金的幽门螺杆菌重组蛋白溶液；
    步骤f：将步骤e制得的标记胶体金的幽门螺杆菌重组蛋白抗原溶液加样到玻璃纤维素膜上，在温度20℃～25℃的条件下干燥4～6h，制成胶体金垫，备用。
  3. 根据权利要求1所述的胶体金定量检测幽门螺杆菌抗体试剂条，其特征在于，在所述硝酸纤维素膜上通过划线包被幽门螺杆菌天然抗原的方法步骤如下：
    步骤ⅰ．用0.01mol/L的PBS将幽门螺杆菌天然抗原稀释成0.2～0.5mg/mL；
    步骤ⅱ．用点膜仪将经步骤i稀释后的幽门螺杆菌天然抗原在硝酸纤维素膜上按1µl/cm进行划线包被；
    步骤ⅲ．幽门螺杆菌天然抗原包被完成后，将硝酸纤维素膜在干燥间室温干燥，备用。
  4. 根据权利要求1所述的胶体金定量检测幽门螺杆菌抗体试剂条，其特征在于，所述硝酸纤维素膜上还通过划线包被鼠抗幽门螺杆菌单克隆抗体，用于质控。
  5. 根据权利要求1所述的胶体金定量检测幽门螺杆菌抗体试剂条，其特征在于，在所述硝酸纤维素膜上，通过划线包被鼠抗幽门螺杆菌单克隆抗体的方法步骤如下：
    步骤①．用0.01mol/L的PBS将鼠抗幽门螺杆菌单克隆抗体稀释成0.2～0.5mg/mL；
    步骤②．用点膜仪将经步骤i稀释后的鼠抗幽门螺杆菌单克隆抗体在硝酸纤维素膜上按1µl/cm进行划线包被；
    步骤③．鼠抗幽门螺杆菌单克隆抗体包被完成后，将硝酸纤维素膜在干燥间室温干燥，备用。
  6. 根据权利要求1所述的胶体金定量检测幽门螺杆菌抗体试剂条，其特征在于，所述试剂条的组装在干燥间内完成，具体步骤如下：
    步骤Ⅰ在底板中央贴上包被好的幽门螺杆菌天然抗原硝酸纤维素膜；
    步骤Ⅱ在硝酸纤维素膜上缘粘贴吸水滤纸；
    步骤Ⅲ在硝酸纤维素膜下缘粘贴胶体金垫；
    步骤Ⅳ在胶体金垫下缘粘贴样品垫，完成后用裁切机将贴好的试纸板切成试纸条，并密封于铝箔袋中。
  7. 根据权利要求1-6任一权利要求所述的胶体金定量检测幽门螺杆菌抗体试剂条，其特征在于，所述底板由PVC制成。
  8. 胶体金定量检测幽门螺杆菌抗体的检测方法，其特征在于，该方法使用权利要求1-7任一权利要求的胶体金定量检测幽门螺杆菌抗体试剂条对幽门螺杆菌抗体进行定量检测；
    定量检测过程如下：将幽门螺杆菌抗体试剂条水平放置，准确滴加1滴血清或血浆样本在试纸条的样品垫上，室温反应10分钟后，将幽门螺杆菌抗体试剂条放入胶体金定量读数仪中，读取T线与C线的峰面积比值即T/C值，代入幽门螺杆菌抗体检测标准曲线，获得样本中幽门螺杆菌抗体的含量（AU/mL）。
  9. 根据权利要求8所述的胶体金定量检测幽门螺杆菌抗体的检测方法，其特征在于，所述幽门螺杆菌抗体检测标准曲线的制作方法包括以下过程：
    用幽门螺杆菌抗体阴性血清按梯度稀释1AU/mL幽门螺杆菌抗体检测标准品，分别取50μL加到试剂条的样品垫中，室温反应10min，将试剂条放入胶体金定量读数仪中，读取T线与C线的峰面积比值即T/C值，以T/C值为横坐标、幽门螺杆菌抗体检测标准品AU值为纵坐标，制作标准曲线；
    所述1AU/mL幽门螺杆菌抗体检测标准品的制备方法如下：收集到20份幽门螺杆菌抗体阳性血清混合，经饱和硫酸铵盐析结合Protein G柱亲和层析纯化，得到终浓度为 0.5mg/mL纯化幽门螺杆菌抗体，定为1AU/mL。
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