WO2022053072A1 - 荧光层析定量检测幽门螺杆菌抗体试剂卡及检测方法 - Google Patents

荧光层析定量检测幽门螺杆菌抗体试剂卡及检测方法 Download PDF

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WO2022053072A1
WO2022053072A1 PCT/CN2021/123686 CN2021123686W WO2022053072A1 WO 2022053072 A1 WO2022053072 A1 WO 2022053072A1 CN 2021123686 W CN2021123686 W CN 2021123686W WO 2022053072 A1 WO2022053072 A1 WO 2022053072A1
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helicobacter pylori
antibody
detection
reagent card
nitrocellulose membrane
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PCT/CN2021/123686
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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
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the invention relates to a detection technology for Helicobacter pylori antibodies, in particular to a reagent card for quantitative detection of Helicobacter pylori antibodies by fluorescence chromatography and a detection method.
  • 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 of H. pylori eradication.
  • 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 blotting, colloidal gold immunochromatography, lanthanide time-resolved fluorescence chromatography, etc.
  • lanthanide time-resolved fluorescence chromatography detection technology is the latest rapid detection method. The detection speed only takes 15 minutes to get the results, and the detection results can be quantitatively analyzed by the fluorescence chromatography detector, which is especially suitable for rapid quantitative detection on-site in medical institutions at all levels.
  • the present invention provides a reagent card for quantitatively detecting Helicobacter pylori antibodies by fluorescence chromatography.
  • the reagent card of the present invention is a Helicobacter pylori antibody lanthanide time-resolved fluorescence chromatography quantitative detection reagent card prepared by using the combination of Helicobacter pylori recombinant protein antigen and natural antigen by the double antigen sandwich method, which can realize the detection of Helicobacter pylori antibody ( Rapid quantitative detection of IgG antibodies).
  • the present invention also provides a detection method for quantitatively detecting Helicobacter pylori antibodies by fluorescence chromatography.
  • the technical scheme of the present invention is: a reagent card for quantitative detection of Helicobacter pylori antibody by fluorescence chromatography, comprising a cartridge case and a test strip installed in the cartridge case; the test strip includes a bottom plate, and a nitrocellulose attached to the bottom plate plain membrane, binding pad and sample pad; the sample pad, binding pad and nitrocellulose membrane overlap each other for a preset length in turn; the nitrocellulose membrane is coated with the natural antigen of Helicobacter pylori by scribing; the The binding pad is sprayed with lanthanide-labeled latex microspheres coated with the recombinant protein antigen of Helicobacter pylori.
  • the lanthanide-labeled latex microspheres coated with the Helicobacter pylori recombinant protein antigen are prepared by the following method steps: Step I. Under the temperature condition of 20°C ⁇ 25°C, the Helicobacter pylori recombinant protein antigen was coated with lanthanide-labeled latex microspheres for 4-6h, and the coating concentration was 20-50 ⁇ g Helicobacter pylori recombinant protein antigen/1mg lanthanide labeling. The latex microspheres; step II.
  • the particle size of the lanthanide-labeled latex microspheres used to coat the Helicobacter pylori recombinant protein antigen is 100-150 nm.
  • the lanthanide-labeled latex microspheres coated with the Helicobacter pylori recombinant protein antigen are sprayed on the binding pad by a spot film instrument, and dried at room temperature in a drying room after spraying is completed.
  • the method steps for coating the natural antigen of Helicobacter pylori on the nitrocellulose membrane by scribing are as follows: Step i. Dilute the natural antigen of Helicobacter pylori to 0.2 ⁇ 0.5mg/ml with 0.01mol/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 was 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. 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 nitrocellulose membrane coated with the natural antigen of Helicobacter pylori on the center of the bottom plate; Step 2, place the upper edge of the nitrocellulose membrane on the Paste and spray the binding pad with the lanthanide-labeled latex microspheres coated with the Helicobacter pylori recombinant protein antigen; step 3, paste the sample pad on the upper edge of the binding pad to obtain a test board; step 4, use a cutting machine to cut the pasted The test board is cut into test strips whose width is suitable for the card shell, and then the reagent strip is put into the card shell to complete the assembly of the reagent card.
  • the reagent card for quantitative detection of Helicobacter pylori antibody by fluorescence chromatography of the present invention is a Helicobacter pylori antibody lanthanide time-resolved fluorescence prepared by the double antigen sandwich method of the combination of Helicobacter pylori recombinant protein antigen and natural antigen.
  • the chromatographic quantitative detection reagent card is fast and convenient to use, and can be used in clinical applications to achieve rapid quantitative detection of Helicobacter pylori antibodies (IgG antibodies).
  • the technical scheme of the present invention is: a detection method for quantitatively detecting Helicobacter pylori antibody by fluorescence chromatography, which uses the aforementioned fluorescent chromatography quantitative detection Helicobacter pylori antibody reagent card of the present invention to quantitatively detect the Helicobacter pylori antibody.
  • the detection process is as follows: place the Helicobacter pylori antibody reagent card horizontally, drop 1 drop of serum or plasma sample into the sample hole of the reagent card, and react at room temperature for 15 minutes, insert the reagent card into the fluorescence chromatography detection instrument, read Take the peak area ratio of T line and C line, namely the T/C value, and substitute it into the standard curve of Helicobacter pylori antibody detection to obtain the content of Helicobacter pylori antibody (U/ml) in the sample.
  • the preparation method of the Helicobacter pylori antibody detection standard curve includes the following steps: Step A. Use the Helicobacter pylori antibody negative serum to dilute 100U/ml of the Helicobacter pylori antibody detection standard in a 5-fold gradient, and prepare a 20U/ml Helicobacter pylori antibody detection standard.
  • Step B take 100U/ml Helicobacter pylori antibody detection standard and 20U/ml, 4U/ml, 0.8U prepared in Step A, respectively 50 ⁇ L/ml of Helicobacter pylori antibody detection standard was added to the sample hole of the reagent card, and reacted at room temperature for 15 minutes;
  • Step C insert the reagent card into the fluorescence chromatography detection instrument, and read the peak area of T line and C line The ratio is the T/C value.
  • the preparation method of the 100U/ml Helicobacter pylori antibody detection standard is as follows: collect 20 pieces of Helicobacter pylori antibody positive serum, mix and purify through saturated ammonium sulfate salting out combined with Protein G column affinity chromatography to obtain a final concentration of 1mg/ml purified Helicobacter pylori antibody, set as 100U/ml.
  • the detection method for quantitatively detecting Helicobacter pylori antibodies by fluorescence chromatography of the present invention is reasonably designed, and can be used for rapid quantitative detection of Helicobacter pylori antibodies (IgG antibodies) clinically.
  • FIG. 1 is a schematic structural diagram of a reagent strip for quantitative detection of Helicobacter pylori antibody by fluorescence chromatography of the present invention
  • Labels in the drawings 1-bottom plate, 2-sample pad, 3-binding pad, 4-nitrocellulose membrane, 401-detection line, 402-quality control line.
  • the fluorescent chromatography quantitative detection of Helicobacter pylori antibody reagent card of the present invention includes a PVC bottom plate 1, and a nitrocellulose membrane 4, a binding pad 3 and a sample pad 2 pasted on the bottom plate 1; the sample pad 2, The binding pad 3 and the nitrocellulose membrane 4 are sequentially overlapped end to end for a predetermined length.
  • the nitrocellulose membrane 4 is coated with the Helicobacter pylori natural antigen by scribing to form a detection line 401; ball.
  • the reagent card for quantitative detection of Helicobacter pylori antibody by fluorescence chromatography in this embodiment is prepared according to the following steps.
  • Helicobacter pylori recombinant protein antigen-coated lanthanide-labeled latex microspheres The particle size of the latex microspheres is 100 nm. For 6 hours, the beads were coated with a concentration of 30 ⁇ g Helicobacter pylori recombinant protein antigen/1mg lanthanide-labeled latex microspheres, and centrifuged at 14,000 r/min for 30 minutes to separate the latex microspheres coated with Helicobacter pylori recombinant protein antigens.
  • step 1 Spray the lanthanide-labeled latex microspheres coated with the Helicobacter pylori recombinant protein antigen on the binding pad: use the BIODOT dot membrane instrument to spray the lanthanide-labeled latex microspheres coated with the Helicobacter pylori recombinant protein antigen (step 1). The obtained suspension) was sprayed on the bonding pad 3 made of glass fiber, and after spraying, it was dried at room temperature in a drying room for 8 hours, and used for later use.
  • Coating 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 4 is scribed and coated at 1 ⁇ l/cm to form a detection line 401 .
  • nitrocellulose membrane 4 obtained in step 4
  • binding pad 3 obtained in step 2
  • sample pad 2 and PVC base plate 1 in the drying room
  • the nitrocellulose membrane 4 coated with the natural antigen of Helicobacter pylori, the upper edge of the nitrocellulose membrane 4 is pasted and sprayed with a binding pad 3 of latex microspheres labeled with lanthanide elements coated with the recombinant protein antigen of Helicobacter pylori, and the binding pad 3 Paste the sample pad 2 on the upper edge to complete the production of the test board; use the cutting machine to cut the pasted test board into test strips with a width of 4 mm, and finally put the reagent strip into the cartridge case, complete the assembly of the reagent card, and obtain the product.
  • the method for rapid quantitative detection of Helicobacter pylori antibodies using the reagent card of the present invention is as follows: place the detection card horizontally, drop 1 drop of serum or plasma sample on the sample pad 2 of the test strip, and react at room temperature for 15 minutes, then place the detection card on the sample pad 2 of the test strip. Insert into the fluorescence chromatography detection instrument, 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 (U/ml) , that is, the quantitative detection of Helicobacter pylori antibodies is realized.
  • the method for establishing the standard curve of Helicobacter pylori antibody detection is as follows: use Helicobacter pylori antibody negative serum to dilute 100U/ml Helicobacter pylori antibody detection standard in a 5-fold gradient, and prepare 20U/ml, 4U/ml and 0.8U/ml respectively. Helicobacter pylori antibody detection standard.
  • Helicobacter pylori antibody detection standard 50 ⁇ L Take 100U/ml, 20U/ml, 4U/ml, and 0.8U/ml of Helicobacter pylori antibody detection standard 50 ⁇ L, respectively, and add them to the sample hole of the Helicobacter pylori antibody reagent card, react at room temperature for 15 minutes, and then put the card strip Insert into the fluorescence chromatography detection instrument, read the peak area ratio between T line and C line, that is, the T/C value, take the T/C value as the abscissa and the Helicobacter pylori antibody detection standard U as the ordinate, to obtain Helicobacter pylori. Antibody detection standard curve.
  • the preparation method of the above-mentioned 100U/ml Helicobacter pylori antibody detection standard is as follows: collect 20 parts of Helicobacter pylori antibody positive serum, mix and purify through saturated ammonium sulfate salting out combined with Protein G column affinity chromatography to obtain a final concentration of 1mg/ml purified Helicobacter pylori antibody, set as 100U/ml.
  • Helicobacter pylori IgG antibody positive serum Helicobacter pylori IgG antibody positive serum, hepatitis B virus IgG antibody positive serum, cytomegalovirus IgG antibody positive serum, herpes simplex virus IgG antibody positive serum, adenovirus IgG antibody positive serum, respiratory syncytial virus IgG antibody positive serum and The Helicobacter pylori IgG antibody negative serum was detected using the fluorescent chromatography quantitative detection Helicobacter pylori antibody reagent card of the above-mentioned embodiment of the present invention. Only the Helicobacter pylori IgG antibody positive serum detection result was positive, and the others were negative. Conclusion: The reagent card of the present invention has good specificity.
  • the reagent card of the present invention has good stability and repeatability.
  • the Helicobacter pylori IgG antibody ELISA detection kit from Weirun Sairun Company of Germany was used as a comparative reagent. 100 clinical specimens were respectively detected with the reagent card of the above-mentioned embodiment of the present invention and the Helicobacter pylori IgG antibody ELISA detection kit of Germany Microrun Sairun Company, and the negative and positive results were compared, as shown in Table 1.

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Abstract

本发明的荧光层析定量检测幽门螺杆菌抗体试剂卡，包括卡壳和装于卡壳内的试纸条；所述试纸条包括底板，以及粘贴于底板上的硝酸纤维素膜、结合垫和样品垫；所述样品垫、结合垫和硝酸纤维素膜依次首尾相互重叠一段预设长度；硝酸纤维素膜上通过划线包被幽门螺杆菌天然抗原；结合垫上喷涂有包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球。本发明的试剂卡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镧系元素时间分辨荧光层析定量检测试剂卡，其能够实现对幽门螺杆菌抗体（IgG抗体）的快速定量检测。对应的，本发明还提供了荧光层析定量检测幽门螺杆菌抗体的检测方法。

Description

荧光层析定量检测幽门螺杆菌抗体试剂卡及检测方法 技术领域
本发明涉及幽门螺杆菌抗体的检测技术，具体是荧光层析定量检测幽门螺杆菌抗体试剂卡及检测方法。
 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、镧系元素时间分辨荧光层析等方法，其中镧系元素时间分辨荧光层析检测技术为最新的快速检测手段，检测速度只需要15分钟即可出结果，检测结果可以通过荧光层析检测仪定量分析，特别适合各级医疗机构现场快速定量检测。荧光层析检测抗体方法目前有间接法和双抗原夹心法，其中间接法只需一种抗原，研发比较简单，但是难以使灵敏度和特异性同时保持在较好的水平，易存在部分假阳性与假阴性结果；双抗原夹心法需要配对抗原，研发难度更大一些，但是检测灵敏度和特异性显著高于间接法。目前尚无能定量检测幽门螺杆菌抗体的镧系元素时间分辨荧光层析双抗原夹心法检测试剂面市。
 技术问题
为了克服现有技术中存在的不足，本发明提供了荧光层析定量检测幽门螺杆菌抗体试剂卡。本发明的试剂卡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镧系元素时间分辨荧光层析定量检测试剂卡，其能够实现对幽门螺杆菌抗体（IgG抗体）的快速定量检测。对应的，本发明还提供了荧光层析定量检测幽门螺杆菌抗体的检测方法。
 技术解决方案
对于试剂卡，本发明的技术方案为：荧光层析定量检测幽门螺杆菌抗体试剂卡，包括卡壳和装于卡壳内的试纸条；所述试纸条包括底板，以及粘贴于底板上的硝酸纤维素膜、结合垫和样品垫；所述样品垫、结合垫和硝酸纤维素膜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；所述结合垫上喷涂有包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球。
作为优化，所述包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球通过以下方法步骤制备：步骤I．在20℃～25℃温度条件下，将幽门螺杆菌重组蛋白抗原包被镧系元素标记的乳胶微球4～6h，包被浓度为20～50μg幽门螺杆菌重组蛋白抗原/1mg镧系元素标记的乳胶微球；步骤II．12000～15000r/m条件下离心30min，分离出包被幽门螺杆菌重组蛋白抗原的的乳胶微球并用0.01mol/L的PBS洗涤3次，然后加入到终浓度为1～2%BSA和1～2％酪蛋白的封闭液中封闭包被幽门螺杆菌重组蛋白抗原的乳胶微球30min；步骤III．用0.01mol/L的PBS洗涤3次封闭后的包被幽门螺杆菌重组蛋白抗原的乳胶微球；步骤IV．用0.01mol/L的PBS重悬包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球，备用。
作为优化，所述步骤I中，用于包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球的粒径为100～150nm。
作为优化，所述包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球通过点膜仪喷涂于结合垫上，喷涂完成后在干燥间室温干燥。
作为优化，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。
作为优化，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。
作为优化，所述试剂卡的组装在干燥间内完成，具体步骤如下：步骤一、在底板中央贴上包被幽门螺杆菌天然抗原的硝酸纤维素膜；步骤二、在硝酸纤维素膜上缘粘贴喷涂有包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球的结合垫；步骤三、在结合垫上缘粘贴样品垫，获得试纸板；步骤四、用裁切机将贴好的试纸板切成宽度与卡壳适配的试纸条，然后将试剂条装入卡壳内，完成试剂卡的组装。
与现有技术相比，本发明的荧光层析定量检测幽门螺杆菌抗体试剂卡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镧系元素时间分辨荧光层析定量检测试剂卡，使用快捷方便，可以在临床上应用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：荧光层析定量检测幽门螺杆菌抗体的检测方法，该方法使用前述本发明的荧光层析定量检测幽门螺杆菌抗体试剂卡对幽门螺杆菌抗体进行定量检测；检测过程如下：将幽门螺杆菌抗体试剂卡水平放置，滴加1滴血清或血浆样本在试剂卡的加样孔中，室温反应15分钟后，将试剂卡***荧光层析检测仪器中，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（U/ml）。
作为优化，所述幽门螺杆菌抗体检测标准曲线的制作方法包括以下步骤：步骤A、用幽门螺杆菌抗体阴性血清按5倍梯度稀释100U/ml的幽门螺杆菌抗体检测标准品，制备成20U/ml、4U/ml、0.8U/ml的幽门螺杆菌抗体检测标准品；步骤B、分别取100U/ml的幽门螺杆菌抗体检测标准品以及步骤A制备的20U/ml、4U/ml、0.8U/ml的幽门螺杆菌抗体检测标准品各50μL加到试剂卡的加样孔中，室温反应15min；步骤C、将试剂卡***荧光层析检测仪器中，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U值为纵坐标，得到幽门螺杆菌抗体检测标准曲线。所述100U/ml的幽门螺杆菌抗体检测标准品的制备方法如下：收集20份幽门螺杆菌抗体阳性血清，混合后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 1mg/ml纯化幽门螺杆菌抗体，定为100U/ml。
 有益效果
本发明的上述荧光层析定量检测幽门螺杆菌抗体的检测方法设计合理，可以在临床上用于对幽门螺杆菌抗体（IgG抗体）进行快速定量检测。
 附图说明
图1为本发明荧光层析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-结合垫，4-硝酸纤维素膜，401-检测线，402-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，本发明的荧光层析定量检测幽门螺杆菌抗体试剂卡包括PVC底板1，以及粘贴于底板1上的硝酸纤维素膜4、结合垫3和样品垫2；所述样品垫2、结合垫3和硝酸纤维素膜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4上通过划线包被幽门螺杆菌天然抗原，形成检测线401；所述结合垫3上喷涂有包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球。
实施例：
本实施例的荧光层析定量检测幽门螺杆菌抗体试剂卡按照如下步骤制得。
一、幽门螺杆菌重组蛋白抗原包被镧系元素标记的乳胶微球：乳胶微球粒径为100nm，在25℃温度条件下，将幽门螺杆菌重组蛋白抗原包被镧系元素标记的乳胶微球6h，包被浓度为30μg幽门螺杆菌重组蛋白抗原/1mg镧系元素标记的乳胶微球，在14000r/min条件下离心30min，分离出包被幽门螺杆菌重组蛋白抗原的的乳胶微球，并用0.01mol/L的 PBS洗涤3次，然后加入到终浓度为终浓度为1～2%BSA和1～2％酪蛋白中，封闭包被幽门螺杆菌重组蛋白抗原的乳胶微球30min；再用0.01mol/L的 PBS洗涤3次封闭包被后的幽门螺杆菌重组蛋白抗原的乳胶微球；最后用0.01mol/L的 PBS重悬包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球，备用。
二、结合垫上喷涂包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球：用BIODOT点膜仪将包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球（步骤一制得的悬浮液）喷涂于玻璃纤维制成的结合垫3上，喷涂完毕在干燥间室温干燥8h，备用。
三、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4上按1µl/cm进行划线包被，形成检测线401。
四、鼠抗幽门螺杆菌单克隆抗体包被硝酸纤维素膜：用0.01mol/L PBS将鼠抗幽门螺杆菌单克隆抗体稀释成0.3mg/ml，然后用BIODOT点膜仪将鼠抗幽门螺杆菌单克隆抗体在已经包被幽门螺杆菌天然抗原的硝酸纤维素膜4上按1µl/cm进行划线包被；鼠抗幽门螺杆菌单克隆抗体包被完成后将硝酸纤维素膜4在干燥间室温干燥8h，备用。（硝酸纤维素膜4包被鼠抗幽门螺杆菌单克隆抗体，形成质控线402用于质控，检测临床标本时，滴加质控液，如果鼠抗幽门螺杆菌单克隆抗体检测呈阴性，说明检测体系不正常，IgG抗体检测结果不能被采信）
五、试剂卡的组装：在干燥间内准备好硝酸纤维素膜4（步骤四制得）、结合垫3（步骤二制得）、样品垫2和PVC材质的底板1；在底板1中央贴上包被幽门螺杆菌天然抗原的硝酸纤维素膜4，硝酸纤维素膜4上缘粘贴喷涂有包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球的结合垫3，结合垫3上缘粘贴样品垫2，完成试纸板的制作；用裁切机将贴好的试纸板切成4mm宽的试纸条，最后将试剂条装入卡壳内，完成试剂卡的组装，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。
1）检测方法
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：将检测卡水平放置，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15分钟后，将检测卡***荧光层析检测仪器中，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，即可获得样本中幽门螺杆菌抗体的含量（U/ml），也即实现了对幽门螺杆菌抗体的定量检测。
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5倍梯度稀释100U/ml幽门螺杆菌抗体检测标准品，分别制备20U/ml、4U/ml、0.8U/ml的幽门螺杆菌抗体检测标准品。分别取100U/ml、20U/ml、4U/ml、0.8U/ml的幽门螺杆菌抗体检测标准品各50μL 加到幽门螺杆菌抗体试剂卡的加样孔中，室温反应15min，然后将卡条***荧光层析检测仪器中，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U值为纵坐标，得到幽门螺杆菌抗体检测标准曲线。
上述100U/ml的幽门螺杆菌抗体检测标准品的制备方法如下：收集20份幽门螺杆菌抗体阳性血清，混合后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 1mg/ml纯化幽门螺杆菌抗体，定为100U/ml。
2）特异性试验
以幽门螺杆菌IgG抗体阳性血清、乙型肝炎病毒IgG抗体阳性血清、巨细胞病毒IgG抗体阳性血清、单纯疱疹病毒IgG抗体阳性血清、腺病毒IgG抗体阳性血清、呼吸道合胞病毒IgG抗体阳性血清和幽门螺杆菌IgG抗体阴性血清采用本发明上述实施例的荧光层析定量检测幽门螺杆菌抗体试剂卡进行检测，只有幽门螺杆菌IgG抗体阳性血清检测结果为阳性，其它均为阴性。结论：本发明的试剂卡特异性良好。
3）重复性试验
随机抽取30份幽门螺杆菌IgG阳性血清、阴性血清分别于不同的时间用本发明上述实施例的荧光层析定量检测幽门螺杆菌抗体试剂卡进行重复检测，变异系数均<5%。结论：本发明的试剂卡具有良好的稳定性和重复性。
4）临床标本检测对比试验
以德国微润赛润公司的幽门螺杆菌IgG抗体ELISA检测试剂盒为对比试剂。对100份临床标本用本发明上述实施例的试剂卡和德国微润赛润公司的幽门螺杆菌IgG抗体ELISA检测试剂盒分别进行检测，比较阴性与阳结果，如表1所示。
表1 临床标本检测对比试验结果
Figure 343884dest_path_image001
对100份标本用两种试剂检测共有95例相符(95%)，只有5例不符（5%），说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。
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 (10)

  1. 荧光层析定量检测幽门螺杆菌抗体试剂卡，包括卡壳和装于卡壳内的试纸条；其特征在于：
    所述试纸条包括底板，以及粘贴于底板上的硝酸纤维素膜、结合垫和样品垫；所述样品垫、结合垫和硝酸纤维素膜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；所述结合垫上喷涂有包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球。
  2. 根据权利要求1所述的荧光层析定量检测幽门螺杆菌抗体试剂卡，其特征在于，所述包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球通过以下方法步骤制备：
    步骤Ⅰ．在20℃～25℃温度条件下，将幽门螺杆菌重组蛋白抗原包被镧系元素标记的乳胶微球4～6h，包被浓度为20～50μg幽门螺杆菌重组蛋白抗原/1mg镧系元素标记的乳胶微球；
    步骤Ⅱ．12000～15000r/m条件下离心30min，分离出包被幽门螺杆菌重组蛋白抗原的的乳胶微球并用0.01mol/L的PBS洗涤3次，然后加入到终浓度为1～2%BSA和1～2％酪蛋白的封闭液中封闭包被幽门螺杆菌重组蛋白抗原的乳胶微球30min；
    步骤Ⅲ．用0.01mol/L的PBS洗涤3次封闭后的包被幽门螺杆菌重组蛋白抗原的乳胶微球；
    步骤．用0.01mol/L的PBS重悬包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球，备用。
  3. 根据权利要求2所述的荧光层析定量检测幽门螺杆菌抗体试剂卡，其特征在于，所述步骤I中，用于包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球的粒径为100～150nm。
  4. 根据权利要求2或3所述的荧光层析定量检测幽门螺杆菌抗体试剂卡，其特征在于，所述包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球通过点膜仪喷涂于结合垫上，喷涂完成后在干燥间室温干燥。
  5. 根据权利要求1所述的荧光层析定量检测幽门螺杆菌抗体试剂卡，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。
  6. 根据权利要求1所述的荧光层析定量检测幽门螺杆菌抗体试剂卡，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。
  7. 根据权利要求1所述的荧光层析定量检测幽门螺杆菌抗体试剂卡，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。
  8. 根据权利要求1所述的荧光层析定量检测幽门螺杆菌抗体试剂卡，其特征在于，所述试剂卡的组装在干燥间内完成，具体步骤如下：
    步骤一、在底板中央贴上包被幽门螺杆菌天然抗原的硝酸纤维素膜；
    步骤二、在硝酸纤维素膜上缘粘贴喷涂有包被幽门螺杆菌重组蛋白抗原的镧系元素标记的乳胶微球的结合垫；
    步骤三、在结合垫上缘粘贴样品垫，获得试纸板；
    步骤四、用裁切机将贴好的试纸板切成宽度与卡壳适配的试纸条，然后将试剂条装入卡壳内，完成试剂卡的组装。
  9. 荧光层析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1-8任一权利要求的荧光层析定量检测幽门螺杆菌抗体试剂卡对幽门螺杆菌抗体进行定量检测；
    检测过程如下：将幽门螺杆菌抗体试剂卡水平放置，滴加1滴血清或血浆样本在试剂卡的加样孔中，室温反应15分钟后，将试剂卡***荧光层析检测仪器中，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（U/ml）。
  10. 根据权利要求9所述的荧光层析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步骤：
    步骤A、用幽门螺杆菌抗体阴性血清按5倍梯度稀释100U/ml的幽门螺杆菌抗体检测标准品，制备成20U/ml、4U/ml、0.8U/ml的幽门螺杆菌抗体检测标准品；
    步骤B、分别取100U/ml的幽门螺杆菌抗体检测标准品以及步骤A制备的20U/ml、4U/ml、0.8U/ml的幽门螺杆菌抗体检测标准品各50μL加到试剂卡的加样孔中，室温反应15min；
    步骤C、将试剂卡***荧光层析检测仪器中，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U值为纵坐标，得到幽门螺杆菌抗体检测标准曲线；
    所述100U/ml的幽门螺杆菌抗体检测标准品的制备方法如下：收集20份幽门螺杆菌抗体阳性血清，混合后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 1mg/ml纯化幽门螺杆菌抗体，定为100U/ml。
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