WO2022053072A1 - Reagent card for quantitatively detecting helicobacter pylori antibodies by fluorescence chromatography and detection method - Google Patents

Abstract

A reagent card for quantitatively detecting helicobacter pylori antibodies by fluorescence chromatography in the present invention, comprising a card case and a test strip provided in the card case. The test strip comprises a bottom plate, and a nitrocellulose membrane, a binding pad and a sample pad which are pasted to the bottom plate; the sample pad, the binding pad, and the nitrocellulose membrane are sequentially overlapped end to end for a preset length; the nitrocellulose membrane is coated with helicobacter pylori natural antigens by lineation; and latex microspheres which are coated with helicobacter pylori recombinant protein antigens and marked by lanthanides are sprayed to the binding pad. The reagent card in the present invention is a reagent card for quantitatively detecting the lanthanides of the helicobacter pylori antibodies by time-resolved fluorescence chromatography, which is prepared by a double-antigen sandwich method in combination with the helicobacter pylori recombinant protein antigens and natural antigens, and can achieve fast quantitative detection of the helicobacter pylori antibodies (IgG antibodies). Correspondingly, the present invention also provides a detection method for quantitatively detecting the helicobacter pylori antibodies by fluorescence chromatography.

Description

Fluorescence chromatography quantitative detection of Helicobacter pylori antibody reagent card and detection method technical field

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.

Background technique

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 μm long and 0.5-1.0 μm 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. In 1994, the World Health Organization/International Agency for Research on Cancer (WHO/IARC) designated Helicobacter pylori as a class I carcinogen. In Asia, 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%. , and 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.

At present, many methods have been developed for the diagnosis of Helicobacter pylori infection, including bacteriology, pathology, serology, isotope tracing, and molecular biology. But in general, from the perspective of specimen collection, it can be divided into two categories: invasive and non-invasive. The invasive method 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. Among them, 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. At present, there are indirect methods and double-antigen sandwich methods for the detection of antibodies by fluorescence chromatography. Among them, the indirect method only needs one antigen, and the development is relatively simple, but it is difficult to keep the sensitivity and specificity at a good level at the same time, and it is prone to some false positives and False negative results; the double-antigen sandwich method requires paired antigens, which is more difficult to develop, but the detection sensitivity and specificity are significantly higher than those of the indirect method. At present, there is no lanthanide time-resolved fluorescence chromatography double-antigen sandwich assay reagent that can quantitatively detect Helicobacter pylori antibodies on the market.

technical problem

In order to overcome the deficiencies in the prior art, 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). Correspondingly, the present invention also provides a detection method for quantitatively detecting Helicobacter pylori antibodies by fluorescence chromatography.

technical solutions

As for the reagent card, 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.

As an optimization, 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℃～25℃, the Helicobacter pylori recombinant protein antigen was coated with lanthanide-labeled latex microspheres for 4-6h, and the coating concentration was 20-50μg Helicobacter pylori recombinant protein antigen/1mg lanthanide labeling. The latex microspheres; step II. Centrifuge at 12000～15000r/m for 30min, separate the latex microspheres coated with Helicobacter pylori recombinant protein antigen and wash 3 times with 0.01mol/L PBS, then add to the final concentration of 1～2%BSA and 1～ The latex microspheres coated with the Helicobacter pylori recombinant protein antigen were sealed in the blocking solution of 2% casein for 30 min; step III. Washing the latex microspheres coated with Helicobacter pylori recombinant protein antigen after blocking for 3 times with 0.01mol/L PBS; Step IV. The lanthanide-labeled latex microspheres coated with the Helicobacter pylori recombinant protein antigen were resuspended with 0.01 mol/L PBS, and set aside.

As an optimization, in the step I, the particle size of the lanthanide-labeled latex microspheres used to coat the Helicobacter pylori recombinant protein antigen is 100-150 nm.

As an optimization, 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.

As an optimization, 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 1µl/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.

As an optimization, the nitrocellulose membrane was also coated with mouse anti-Helicobacter pylori monoclonal antibody by streaking for quality control. Further, 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 1µl/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.

As an optimization, the assembly of the reagent card is completed in the drying room, and the specific steps are as follows: 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.

Compared with the prior art, 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).

As for the detection method, 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.

As an optimization, 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. ml, 4U/ml, 0.8U/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 μL/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. Taking the T/C value as the abscissa and the U value of the Helicobacter pylori antibody detection standard as the ordinate, the standard curve for the detection of Helicobacter pylori antibodies is obtained. 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.

beneficial effect

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.

Description of drawings

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.

Embodiments of the present invention

The present invention will be further described below in conjunction with specific embodiments, but not as a basis for limiting the present invention.

Referring to FIG. 1, 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.

Wherein, the nitrocellulose membrane 4 is coated with the Helicobacter pylori natural antigen by scribing to form a detection line 401; ball.

Example:

The reagent card for quantitative detection of Helicobacter pylori antibody by fluorescence chromatography in this embodiment is prepared according to the following steps.

1. 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 μg 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. And washed 3 times with 0.01mol/L PBS, then added to the final concentration of 1-2% BSA and 1-2% casein, and blocked the latex microspheres coated with Helicobacter pylori recombinant protein antigen for 30min; Wash the latex microspheres with 0.01mol/L PBS for 3 times to block the coated Helicobacter pylori recombinant protein antigen; Latex microspheres, spare.

2. 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.

3. 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 µl/cm to form a detection line 401 .

4. Coating the nitrocellulose membrane with mouse anti-Helicobacter pylori monoclonal antibody: Dilute the mouse anti-Helicobacter pylori monoclonal antibody to 0.3 mg/ml with 0.01mol/L PBS, and then use the BIODOT dot film instrument to apply the mouse anti-Helicobacter pylori The bacterial monoclonal antibody was streaked on the nitrocellulose membrane 4 that had been coated with the natural antigen of Helicobacter pylori at 1 µl/cm; after the mouse anti-Helicobacter pylori monoclonal antibody was coated, the nitrocellulose membrane 4 was dried. Dry at room temperature for 8h and set aside. (Nitrocellulose membrane 4 is coated with mouse anti-Helicobacter pylori monoclonal antibody to form quality control line 402 for quality control. When testing clinical specimens, add quality control solution dropwise, if the mouse anti-Helicobacter pylori monoclonal antibody test is negative , indicating that the detection system is abnormal, and the IgG antibody test results cannot be trusted)

5. Assembly of reagent card: prepare 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 present invention will be further described below in conjunction with the detection method and some necessary tests.

1) Detection method

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. Take 100U/ml, 20U/ml, 4U/ml, and 0.8U/ml of Helicobacter pylori antibody detection standard 50μL, 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.

2) Specificity test

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.

3) Repeated test

30 Helicobacter pylori IgG positive sera and negative sera were randomly selected for repeated detection at different times using the fluorescent chromatography quantitative detection of Helicobacter pylori antibody reagent card of the above embodiment of the present invention, and the coefficient of variation was <5%. Conclusion: The reagent card of the present invention has good stability and repeatability.

4) Comparative test of clinical specimen detection

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.

Table 1 The results of the comparative test of clinical specimen detection

For 100 specimens detected by the two reagents, a total of 95 cases are consistent (95%), and only 5 cases are inconsistent (5%), indicating that the reagent of the present invention has high sensitivity and specificity.

The above description is based on a specific embodiment of the present invention. Within the scope of the technical solution of the present invention, other specific embodiments formed by adjusting the values of the relevant parameters in the above-mentioned embodiments can also achieve the purpose of the present invention. Not listed one by one.

The above general description of the invention involved in this application and the description of its specific embodiments should not be construed as limiting the technical solution of the invention. According to the disclosure of the present application, those skilled in the art can add, reduce or combine the technical features disclosed in the above general description or/and the embodiments without departing from the constituent elements of the invention involved, to form a structure belonging to the present invention. Apply for other technical solutions within the scope of protection.

Claims (10)

1. A reagent card for quantitative detection of Helicobacter pylori antibodies by fluorescence chromatography, comprising a cartridge and a test strip installed in the cartridge; it is characterized in that:

   The test strip includes a bottom plate, and a nitrocellulose membrane, a binding pad and a sample pad pasted on the bottom plate; the sample pad, the binding pad and the nitrocellulose membrane are sequentially overlapped end to end for a preset length;

   The nitrocellulose membrane is coated with the natural antigen of Helicobacter pylori by scribing; the binding pad is sprayed with lanthanide-labeled latex microspheres coated with the recombinant protein antigen of Helicobacter pylori.
2. The fluorescent chromatography quantitative detection Helicobacter pylori antibody reagent card according to claim 1, wherein 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℃～25℃, the Helicobacter pylori recombinant protein antigen was coated with lanthanide-labeled latex microspheres for 4-6h, and the coating concentration was 20-50μg Helicobacter pylori recombinant protein antigen/1mg lanthanide labeling. latex microspheres;

   Step II. Centrifuge at 12000～15000r/m for 30min, separate the latex microspheres coated with Helicobacter pylori recombinant protein antigen and wash 3 times with 0.01mol/L PBS, then add to the final concentration of 1～2%BSA and 1～ The latex microspheres coated with the recombinant protein antigen of Helicobacter pylori were sealed in the blocking solution of 2% casein for 30 minutes;

   Step III. Wash the latex microspheres coated with Helicobacter pylori recombinant protein antigen after blocking with 0.01mol/L PBS for 3 times;

   step. The lanthanide-labeled latex microspheres coated with the Helicobacter pylori recombinant protein antigen were resuspended with 0.01 mol/L PBS, and set aside.
3. Fluorescence chromatography quantitative detection Helicobacter pylori antibody reagent card according to claim 2, is characterized in that, in described step 1, is used to coat the particles of lanthanide-labeled latex microspheres of Helicobacter pylori recombinant protein antigen. The diameter is 100 to 150 nm.
4. The fluorescence chromatography quantitative detection Helicobacter pylori antibody reagent card according to claim 2 or 3, characterized in that, the lanthanide-labeled latex microspheres coated with the Helicobacter pylori recombinant protein antigen are sprayed on the spot by a dot film instrument. On the bonding pad, dry at room temperature in the drying room after spraying is completed.
5. Fluorescence chromatography quantitative detection Helicobacter pylori antibody reagent card according to claim 1, is characterized in that, on described nitrocellulose membrane, the method step of coating Helicobacter pylori natural antigen by scribing is as follows:

   Step i. Dilute the Helicobacter pylori natural antigen to 0.2-0.5mg/ml with 0.01mol/L PBS;

   Step ii. Use a dot membrane apparatus to coat the natural antigen of Helicobacter pylori diluted in step i on a nitrocellulose membrane at 1µl/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.
6. The reagent card for quantitative detection of Helicobacter pylori antibody by fluorescence chromatography according to claim 1, wherein the nitrocellulose membrane is also coated with mouse anti-Helicobacter pylori monoclonal antibody by scribing for quality control.
7. Fluorescence chromatography quantitative detection Helicobacter pylori antibody reagent card according to claim 1, is characterized in that, on described nitrocellulose membrane, the method steps of coating mouse anti-Helicobacter pylori monoclonal antibody by scribing 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 1µl/cm using a dot membrane 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.
8. The reagent card for quantitative detection of Helicobacter pylori antibodies by fluorescence chromatography according to claim 1, wherein the assembly of the reagent card is completed in a drying room, and the specific steps are as follows:

   Step 1, paste the nitrocellulose membrane coated with the natural antigen of Helicobacter pylori in the center of the bottom plate;

   Step 2, pasting and spraying the binding pad of latex microspheres labeled with lanthanide elements coated with Helicobacter pylori recombinant protein antigen on the upper edge of the nitrocellulose membrane;

   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 test board into test strips whose width is suitable for the card case, and then put the reagent strip into the card case to complete the assembly of the reagent card.
9. A detection method for quantitatively detecting Helicobacter pylori antibodies by fluorescence chromatography, characterized in that the method uses the fluorescent chromatography quantitative detection Helicobacter pylori antibody reagent card of any one of claims 1-8 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 The ratio of the peak area of the T line to the C line is the T/C value, which is substituted into the standard curve of Helicobacter pylori antibody detection to obtain the content of Helicobacter pylori antibody (U/ml) in the sample.
10. The method for quantitatively detecting Helicobacter pylori antibodies by fluorescence chromatography according to claim 9, wherein the method for preparing the standard curve for detecting Helicobacter pylori antibodies comprises the following steps:

    Step A, use the Helicobacter pylori antibody negative serum to dilute the 100U/ml Helicobacter pylori antibody detection standard by 5-fold gradient to prepare the Helicobacter pylori antibody detection standard of 20U/ml, 4U/ml and 0.8U/ml;

    Step B. Take 100U/ml of Helicobacter pylori antibody detection standard and 20U/ml, 4U/ml, 0.8U/ml of Helicobacter pylori antibody detection standard prepared in Step A. Add 50 μL to the sample addition of the reagent card In the hole, react at room temperature for 15 min;

    Step C, insert the reagent card into the fluorescence chromatography detection instrument, 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 U value as the The ordinate is to obtain the standard curve of Helicobacter pylori antibody detection;

    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.