Helicobacter pylori antibody typing detection test strip and kit

Abstract

The invention relates to the field of in-vitro diagnosis immunodetection, in particular to a helicobacter pylori antibody typing detection test strip and a kit. The helicobacter pylori antibody typing detection test strip comprises a bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, and the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially overlapped and pasted on the bottom plate in the chromatography direction; the nitrocellulose membrane is coated with three detection lines and one quality control line; the first detection line is coated with a Urease antigen, the second detection line is coated with a CagA antigen, the third detection line is coated with a VacA antigen, and the quality control line is coated with rabbit anti-DNP-BSA or goat anti-chicken IgY; the combination pad is coated with a biotinylation ProteinA / G-antibody marked by colored microspheres, and a biotinylation antibody or antigen marked by colored microspheres; the invention discloses a helicobacter pylori antibody typing detection test strip and a kit, which are good in stability and higher in sensitivity compared with similar products.

Description

Technical Field

[0001] The present application relates to the field of in vitro diagnostic immunoassays, and in particular to a Helicobacter pylori antibody typing test strip and a kit. Background Art

[0002] Helicobacter pylori (HP) is a spiral bacterium that parasitizes on the submucus epithelial cells on the surface of the gastric mucosa and continuously releases toxic substances such as urease, vacuolating toxin, phospholipase, biogenic amines, etc., which cause epithelial cell degeneration and necrosis, destroy the mucosal barrier, lead to the occurrence of gastritis, promote the formation of gastric and duodenal ulcers, and even induce gastric cancer.

[0003] Usually, patients with gastric ulcers are more likely to suffer from stomach pain and chronic gastritis after being infected with Helicobacter pylori. The main clinical manifestations of chronic gastritis caused by Helicobacter pylori infection include upper abdominal discomfort, dull pain, sometimes belching, acid reflux, nausea, and vomiting. The course of the disease is relatively slow, but it is easy to relapse.

[0004] At present, the complete gene sequence of Helicobacter pylori has been determined, among which the urease gene has four open reading frames, namely UreA, UreB, UreC and UreD. The polypeptides encoded by UreA and UreB are equivalent to the two subunit structures of the urease structure. The urease of Helicobacter pylori is extremely rich, containing about 15% of the bacterial protein, and the activity is equivalent to 400 times that of Proteus. Urease catalyzes the hydrolysis of urea to form ammonia, which can protect bacteria from surviving in a high acid environment. In addition, there are VacA gene and CagA gene, which encode vacuolating toxin and cytotoxin-related proteins respectively. According to the expression of these two genes, Helicobacter pylori strains are divided into two main types: Type I contains CagA and VacA genes and expresses two proteins, Type II does not contain CagA genes and does not express two proteins, and some are intermediate expression types, that is, they express one of the virulence factors. It is now generally believed that Type I is closely related to gastric diseases.

[0005] Currently, Helicobacter pylori antibody typing detection reagents include immunoblotting, immunochromatography, etc.; the immunoblotting method is cumbersome to operate and time-consuming, and the immunochromatography reagents have problems such as insufficient sensitivity.

[0006] Therefore, it is of great significance to develop a Helicobacter pylori antibody typing detection reagent with higher sensitivity and easier operation. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present application provides a Helicobacter pylori antibody typing test strip and a kit.

[0008] In the first aspect, the present application provides a Helicobacter pylori antibody typing test strip and a kit, which adopt the following technical scheme: A Helicobacter pylori antibody typing test strip, the test strip comprising a base plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water absorbent pad, wherein the sample pad, the conjugation pad, the nitrocellulose membrane and the water absorbent pad are sequentially overlapped and pasted on the base plate in a chromatography direction; The nitrocellulose membrane is coated with three detection lines and one quality control line; the first detection line is coated with Urease antigen, the second detection line is coated with CagA antigen, the third detection line is coated with VacA antigen, and the quality control line is coated with rabbit anti-DNP-BSA or sheep anti-chicken IgY; The conjugate pad is coated with biotinylated ProteinA / G-antibodies labeled with colored microspheres, and biotinylated antibodies or antigens labeled with colored microspheres.

[0009] In a specific embodiment, the conjugate pad is coated with biotinylated ProteinA / G-antibody labeled with colored microspheres and biotinylated antibody labeled with colored microspheres.

[0010] In a specific embodiment, the conjugate pad is coated with biotinylated ProteinA / G-antibody labeled with colored microspheres and biotinylated antigen labeled with colored microspheres.

[0011] Preferably, the biotinylated ProteinA / G-antibody labeled with colored microspheres is biotinylated ProteinA / G-mouse anti-human IgG labeled with colored microspheres.

[0012] Preferably, the preparation method of the biotinylated ProteinA / G-mouse anti-human IgG labeled with colored microspheres comprises the following steps: (1) adding Protein A / G to biotin and mixing them to obtain biotinylated Protein A / G; (2) adding the streptavidin-modified colored microspheres to the biotinylated Protein A / G and mixing them to obtain colored microsphere-SA-biotin-Protein A / G conjugates; (3) adding mouse anti-human IgG monoclonal antibody to the colored microsphere-SA-biotin-ProteinA / G conjugate and mixing them to obtain mouse anti-human IgG monoclonal antibody microsphere marker; (4) Add a blocking agent to the mouse anti-human IgG monoclonal antibody microsphere marker to block the marker, thereby obtaining a biotinylated ProteinA / G-mouse anti-human IgG labeled with a colored microsphere.

[0013] Preferably, the molar ratio of biotin to ProteinA / G in step (1) is (20-40):1.

[0014] Preferably, in step (2), the mass ratio of the streptavidin-modified colored microspheres to the biotinylated ProteinA / G is 1:(0.01-0.02).

[0015] Preferably, in the step (3), the mass ratio of mouse anti-human IgG monoclonal antibody to colored microsphere-SA-biotin-ProteinA / G conjugate is (0.03-0.05):1.

[0016] Preferably, in step (4), the mass ratio of the mouse anti-human IgG monoclonal antibody microsphere marker to the blocker is 1:(0.02-0.05).

[0017] Preferably, the biotin is NHS-PEG12-biotin.

[0018] By adopting the above technical solution, since biotin has a small molecular weight, the formed biotinylated label is easily affected by the steric hindrance effect of large molecular proteins. This kit adopts NHS-PEG12-biotin to make the labeled conjugate have a longer spacer arm to reduce the problem of low sensitivity due to limited binding caused by spatial steric hindrance when antigen-antibody and biotin and SA are combined. At the same time, the amplification of the biotin-avidin system and the extension of the distance between the biotin parent structure and the antibody are more conducive to the binding of biotin and avidin. Biotin and ProteinA / G are coupled and then the antibody and ProteinA / G are blocked by a blocking agent. Since ProteinA / G has the common characteristics of ProteinA and ProteinG, it has a larger specific surface area and more antibody binding sites, so it can provide a high number of antibody binding, which is conducive to improving the sensitivity of the reagent. The sensitivity of the reagent is improved by amplifying the biotin-avidin system and introducing ProteinA / G.

[0019] Preferably, the biotinylated antibody or antigen labeled with colored microspheres is biotinylated chicken IgY labeled with colored microspheres or biotinylated DNP-BSA labeled with colored microspheres.

[0020] Preferably, the method for preparing the biotinylated chicken IgY antibody labeled with colored microspheres comprises the following steps: (1) adding chicken IgY to biotin and mixing them to obtain biotin-chicken IgY; (2) Add the streptavidin-modified colored microspheres to biotin-chicken IgY and mix them to obtain colored microspheres-SA-biotin-chicken IgY.

[0021] Preferably, in step (1), the molar ratio of biotin to chicken IgY is (10-30):1; Preferably, in step (2), the mass ratio of the streptavidin-modified colored microspheres to biotin-chicken IgY is 1:(0.03-0.05).

[0022] Preferably, the method for preparing the biotinylated DNP-BSA antigen labeled with colored microspheres comprises the following steps: (1) adding DNP-BSA to biotin and mixing them to obtain biotin-DNP-BSA; (2) Add the streptavidin-modified colored microspheres to biotin-DNP-BSA and mix them to obtain colored microspheres-SA-biotin-DNP-BSA.

[0023] Preferably, in step (1), the molar ratio of biotin to DNP-BSA is (10-30):1; Preferably, in step (2), the mass ratio of the streptavidin-modified colored microspheres to biotin-DNP-BSA is 1:(0.03-0.05).

[0024] Preferably, the biotin is NHS-PEG12-biotin.

[0025] In a second aspect, the present application provides a kit for detecting interleukin-6, which adopts the following technical solution: A Helicobacter pylori antibody typing test paper kit comprises a test card, the test card comprises a card shell, and the test paper strip is arranged in the card shell.

[0026] Preferably, the kit further comprises a diluent.

[0027] In summary, the present application includes at least one of the following beneficial technical effects: The present application discloses a Helicobacter pylori antibody typing test strip and a test kit, which have good stability, higher sensitivity than similar products, wider detection range, simple operation, and are suitable for rapid detection without high requirements on personnel professionalism. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further illustrated by specific embodiments below. The specific embodiments do not limit the protection scope of the present invention; some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0029] The raw materials involved in this application are all commercially available products, among which: Blocker: The manufacturer is Roche MAK33.

[0030] NHS-PEG12-biotin: manufactured by Thermo Fisher Scientific.

[0031] Streptavidin colored microspheres, commercially available, commercialized streptavidin colored microspheres, particle size 300nm.

[0032] The present application is further described in detail below in conjunction with embodiments and comparative examples.

[0033] The solution used in this example is configured as follows: Example 1: A Helicobacter pylori antibody typing detection kit 1. Preparation of conjugate pad 1.1. Preparation of biotinylated ProteinA / G-mouse anti-human IgG labeled with colored microspheres (1) Add 1 mL of 2 mg / mL Protein A / G to 4.8 μL of 0.25 mol / L biotin (NHS-PEG12-biotin) and react for 30 minutes to obtain biotinylated Protein A / G; (2) Add 1 mg of streptavidin-modified colored microspheres to 10 μg of biotinylated Protein A / G to obtain colored microsphere-SA-biotin-Protein A / G conjugates; (3) adding 50 μg of mouse anti-human IgG monoclonal antibody to 1 mg of colored microsphere-SA-biotin-ProteinA / G conjugate and mixing them to obtain mouse anti-human IgG monoclonal antibody microsphere marker; (4) Add 50 μg of blocking agent (Roche MAK33) to 1 mg of mouse anti-human IgG monoclonal antibody microsphere marker for blocking to obtain colored microsphere-biotin-ProteinA / G-mouse anti-human IgG monoclonal antibody conjugate.

[0034] 1.2. Preparation of biotinylated chicken IgY labeled with colored microspheres: (1) 1 mL of 2 mg / mL chicken IgY antibody was mixed with 0.9 μL of 0.25 mol / L biotin (NHS-PEG12-biotin) for 30 min to obtain biotin-chicken IgY; (2) 1 mg of streptavidin-modified colored microspheres was mixed with 40 μg of biotin-chicken IgY antibody to obtain colored microsphere-SA-biotin-chicken IgY conjugate.

[0035] 1.3. Preparation of conjugate pad (1) Evenly apply the conjugate pad with the conjugate pad blocking solution to completely wet it, dry it, and cut it; (2) Mixing the colored microsphere-biotin-ProteinA / G-mouse anti-human IgG monoclonal antibody conjugate and the colored microsphere-SA-biotin-chicken IgY conjugate with a conjugate pad treatment solution to obtain a colored microsphere probe conjugate solution; wherein the colored microsphere-biotin-ProteinA / G-mouse anti-human IgG monoclonal antibody conjugate is diluted 40 times, and the colored microsphere-SA-biotin-chicken IgY conjugate is diluted 60 times; (3) Spray the colored microsphere probe coupling liquid at 5 μL / cm onto the dried and cut binding pad. After spraying, dry it for later use.

[0036] 2. Preparation of sample pad: The sample pad was evenly coated with the sample pad blocking solution to completely wet it, and then dried at 55°C and cut.

[0037] 3. Preparation of nitrocellulose membrane: (1) Before scratching, the nitrocellulose membrane needs to be equilibrated in a humidity environment of 45%-60% for 3-4 hours; (2) Preparation of test line: Urease antigen was prepared with coating buffer to a concentration of 1 mg / mL and streaked onto the T1 line area of ​​the NC membrane using a gold spray streaking instrument with a streaking volume of 1 μL / cm; The CagA antigen was prepared with coating buffer to a concentration of 1 mg / mL and was streaked onto the T2 line area of ​​the NC membrane using a gold spray streaking instrument with a streaking volume of 1 μL / cm; VacA antigen was prepared with coating buffer to a concentration of 1 mg / mL and streaked onto the T3 line area of ​​the NC membrane using a gold spray streaking instrument with a streaking volume of 1 μL / cm; The scored film was dried at 55°C for 2-3 days.

[0038] (3) Preparation of quality control line: The goat anti-chicken IgY antibody was prepared into a concentration of 1 mg / mL using a coating buffer, and the NC membrane was streaked with a gold spray streaker at a streaking volume of 1 μL / cm. The streaked membrane was dried at 55°C for 3 days for later use.

[0039] 4. Preparation of test strips: The sample pad, the conjugate pad, the nitrocellulose membrane and the absorbent pad are overlapped and pasted on the bottom plate in sequence according to the chromatography direction, and the test strips are cut.

[0040] 5. Preparation of the kit: (1) Test card: Put the prepared test strip into the card housing to obtain a test card; (2) Preparation of sample diluent.

[0041] Example 2: A Helicobacter pylori antibody typing detection kit 1. Preparation of conjugate pad 1.1. Preparation of biotinylated ProteinA / G-mouse anti-human IgG labeled with colored microspheres (1) Add 1 mL of 2 mg / mL Protein A / G to 4.8 μL of 0.25 mol / L biotin (NHS-PEG12-biotin) and react for 30 min to obtain biotinylated Protein A / G; (2) adding 1 mg of streptavidin-modified colored microspheres to 15 μg of biotinylated Protein A / G and mixing them to obtain colored microsphere-SA-biotin-Protein A / G conjugates; (3) adding 50 μg of mouse anti-human IgG monoclonal antibody to 1 mg of colored microsphere-SA-biotin-ProteinA / G conjugate and mixing them to obtain mouse anti-human IgG monoclonal antibody microsphere marker; (4) Add 50 μg of blocking agent (Roche MAK33) to 1 mg of mouse anti-human IgG monoclonal antibody microsphere marker for blocking to obtain colored microsphere-biotin-ProteinA / G-mouse anti-human IgG monoclonal antibody conjugate.

[0042] 1.2. Preparation of biotinylated IgY labeled with colored microspheres: (1) Mix 1 mL of 2 mg / mL chicken IgY with 0.9 μL of 0.25 mol / L biotin (NHS-PEG12-biotin) for 30 min to obtain biotin-chicken IgY; (2) 1 mg of streptavidin-modified colored microspheres was mixed with 40 μg of biotin-chicken IgY to obtain a colored microsphere-SA-biotin-chicken IgY conjugate.

[0043] 1.3. Preparation of conjugate pad (1) Evenly apply the conjugate pad with the conjugate pad blocking solution to completely wet it, dry it, and cut it; (2) Mixing the colored microsphere-biotin-ProteinA / G-mouse anti-human IgG monoclonal antibody conjugate and the colored microsphere-SA-biotin-chicken IgY conjugate with a conjugate pad treatment solution to obtain a colored microsphere probe conjugate solution; wherein the colored microsphere-biotin-ProteinA / G-mouse anti-human IgG monoclonal antibody conjugate is diluted 40 times, and the colored microsphere-SA-biotin-chicken IgY conjugate is diluted 60 times; (3) Spray the colored microsphere probe coupling liquid at 5 μL / cm onto the dried and cut binding pad. After spraying, dry it for later use.

[0044] 2. Preparation of sample pad: The sample pad was evenly coated with the sample pad blocking solution to completely wet it, and then dried at 55°C and cut.

[0045] 3. Preparation of nitrocellulose membrane: (1) Before scratching, the nitrocellulose membrane needs to be equilibrated in a humidity environment of 45%-60% for 3-4 hours; (2) Preparation of test line: Urease antigen was prepared with coating buffer to a concentration of 1 mg / mL and streaked onto the T1 line area of ​​the NC membrane using a gold spray streaking instrument with a streaking volume of 1 μL / cm; The CagA antigen was prepared with coating buffer to a concentration of 1 mg / mL and was streaked onto the T2 line area of ​​the NC membrane using a gold spray streaking instrument with a streaking volume of 1 μL / cm; VacA antigen was prepared with coating buffer to a concentration of 1 mg / mL and streaked onto the T3 line area of ​​the NC membrane using a gold spray streaking instrument with a streaking volume of 1 μL / cm; The scored film was dried at 55°C for 2-3 days.

[0046] (3) Preparation of quality control line: The goat anti-chicken IgY antibody was prepared into a concentration of 1 mg / mL using a coating buffer, and the NC membrane was streaked with a gold spray streaker at a streaking volume of 1 μL / cm. The streaked membrane was dried at 55°C for 3 days for later use.

[0047] 4. Preparation of test strips: The sample pad, the conjugate pad, the nitrocellulose membrane and the absorbent pad are overlapped and pasted on the bottom plate in sequence according to the chromatography direction, and the test strips are cut.

[0048] 5. Preparation of the kit: (1) Test card: Put the prepared test strip into the card housing to obtain a test card; (2) Preparation of sample diluent.

[0049] Example 3: A Helicobacter pylori antibody typing detection kit 1. Preparation of conjugate pad 1.1. Preparation of biotinylated ProteinA / G-mouse anti-human IgG labeled with colored microspheres (1) Add 1 mL of 2 mg / mL Protein A / G to 4.8 μL of 0.25 mol / L biotin (NHS-PEG12-biotin) and react for 30 min to obtain biotinylated Protein A / G; (2) adding 1 mg of streptavidin-modified colored microspheres to 20 μg of biotinylated Protein A / G and mixing them to obtain colored microsphere-SA-biotin-Protein A / G conjugates; (3) adding 50 μg of mouse anti-human IgG monoclonal antibody to 1 mg of colored microsphere-SA-biotin-ProteinA / G conjugate and mixing them to obtain mouse anti-human IgG monoclonal antibody microsphere marker; (4) Add 50 μg of blocking agent (Roche MAK33) to 1 mg of mouse anti-human IgG monoclonal antibody microsphere marker for blocking to obtain colored microsphere-biotin-ProteinA / G-mouse anti-human IgG monoclonal antibody conjugate.

[0050] 1.2. Preparation of biotinylated IgY labeled with colored microspheres: (1) Mix 1 mL of 2 mg / mL chicken IgY with 0.9 μL of 0.25 mol / L biotin (NHS-PEG12-biotin) for 30 min to obtain biotin-chicken IgY; (2) 1 mg of streptavidin-modified colored microspheres was mixed with 40 μg of biotin-chicken IgY to obtain a colored microsphere-SA-biotin-chicken IgY conjugate.

[0051] 1.3. Preparation of conjugate pad (1) Evenly apply the conjugate pad with the conjugate pad blocking solution to completely wet it, dry it, and cut it; (2) Mixing the colored microsphere-biotin-ProteinA / G-mouse anti-human IgG monoclonal antibody conjugate and the colored microsphere-SA-biotin-chicken IgY conjugate with a conjugate pad treatment solution to obtain a colored microsphere probe conjugate solution; wherein the colored microsphere-biotin-ProteinA / G-mouse anti-human IgG monoclonal antibody conjugate is diluted 40 times, and the colored microsphere-SA-biotin-chicken IgY conjugate is diluted 60 times; (3) Spray the colored microsphere probe coupling liquid at 5 μL / cm onto the dried and cut binding pad. After spraying, dry it for later use.

[0052] 2. Preparation of sample pad: The sample pad was evenly coated with the sample pad blocking solution to completely wet it, and then dried at 55°C and cut.

[0053] 3. Preparation of nitrocellulose membrane: (1) Before scratching, the nitrocellulose membrane needs to be equilibrated in a humidity environment of 45%-60% for 3-4 hours; (2) Preparation of test line: Urease antigen was prepared with coating buffer to a concentration of 1 mg / mL and streaked onto the T1 line area of ​​the NC membrane using a gold spray streaking instrument with a streaking volume of 1 μL / cm; The CagA antigen was prepared with coating buffer to a concentration of 1 mg / mL and was streaked onto the T2 line area of ​​the NC membrane using a gold spray streaking instrument with a streaking volume of 1 μL / cm; VacA antigen was prepared with coating buffer to a concentration of 1 mg / mL and streaked onto the T3 line area of ​​the NC membrane using a gold spray streaking instrument with a streaking volume of 1 μL / cm; The scored film was dried at 55°C for 2-3 days.

[0054] (3) Preparation of quality control line: The goat anti-chicken IgY antibody was prepared into a concentration of 1 mg / mL using a coating buffer, and the NC membrane was streaked with a gold spray streaker at a streaking volume of 1 μL / cm. The streaked membrane was dried at 55°C for 3 days for later use.

[0055] 4. Preparation of test strips: The sample pad, the conjugate pad, the nitrocellulose membrane and the absorbent pad are overlapped and pasted on the bottom plate in sequence according to the chromatography direction, and the test strips are cut.

[0056] 5. Preparation of the kit: (1) Test card: Put the prepared test strip into the card housing to obtain a test card; (2) Preparation of sample diluent.

[0057] Performance test 1: 1. Detection method A comparison test was conducted on 110 serum samples collected from a medical institution (determined by immunoblotting), including 27 negative samples (Ure:-, VacA:-, CagA:-); 34 type II Ure positive samples (Ure:+, VacA:-, CagA:-); and 49 type I samples (Ure:+, VacA:+, CagA:+, 19 cases; VacA:-, 14 cases were positive for the other two items; CagA:-, 16 cases were positive for the other two items): 2. Test results 2.1. The above samples were tested using the kit prepared in Example 1. The test results are as follows: Table 1 Test results of Example 1 Table 2 Example 1 Test results evaluation four-grid table The test results of Example 1: Combining Table 1 and Table 2, we can know that the positive coincidence rate and negative coincidence rate of the test kit are calculated, and the results are as follows: Ure positive coincidence rate = 81 / (81+2) × 100% = 97.59%; Ure negative coincidence rate = 27 / (0+27)×100% = 100%; CagA positive coincidence rate = 31 / (31+2)×100% = 93.93%; CagA negative coincidence rate = 77 / (0+77)×100% = 100%; VacA positive coincidence rate = 34 / (34+1)×100% = 97.14%; VacA negative coincidence rate = 75 / (0+75)×100% = 100%.

[0058] 2.2. The above samples were tested using the kit prepared in Example 2. The test results are as follows: Table 3 Example 2 test results Table 4 Example 2 Test results evaluation four-grid table Combining Table 3 and Table 4, it can be seen that the test results of Example 2 are as follows: Ure positive coincidence rate = 83 / (83+0)×100% = 100%; Ure negative coincidence rate = 27 / (0+27)×100% = 100%; CagA positive coincidence rate = 33 / (33+0)×100% = 100%; CagA negative coincidence rate = 77 / (0+77)×100% = 100%; VacA positive coincidence rate = 35 / (35+0)×100% = 100%; VacA negative coincidence rate = 75 / (0+75)×100% = 100%.

[0059] 2.3. The above samples were tested using the kit prepared in Example 3. The test results are as follows: Table 5 Example 3 test results Table 6 Example 3 Test results evaluation four-grid table Combining Table 5 and Table 6, it can be seen that the test results of Example 3 are as follows: Ure positive coincidence rate = 83 / (83+0)×100% = 100%; Ure negative coincidence rate = 25 / (2+25)×100% = 92.59%; CagA positive coincidence rate = 33 / (33+0)×100% = 100%; CagA negative coincidence rate = 75 / / (2+75)×100% = 97.40%; VacA positive coincidence rate = 35 / (35+0)×100% = 100%; VacA negative compliance rate = 74 / (1+74)×100% = 98.67%; In summary, the test kits prepared in Examples 1-3 were used to test the above samples, and the test results all met the requirements, and Example 2 had the highest compliance rate.

[0060] The compliance rate of Example 1 is lower than that of Example 2 because the amount of biotinylated antibody coated in Example 1 is less than that in Example 2, resulting in missed detection in Example 1.

[0061] The compliance rate of Example 3 is lower than that of Example 2 because the amount of antibody coated in Example 3 is greater than that in Example 2, resulting in false positives in Example 3.

[0062] Performance test 2: The kit prepared in Example 2 was divided into 4 parts, and placed at 2-8°C and 37°C for accelerated aging, respectively. Negative control product C1, low value control product C2, medium value control product C3, and high value control product C4 were used for testing. The test results are shown in the following table; Table 7 Stability of the kit of Example 2 It can be seen from the test results that after the kit was placed in high temperature accelerated aging, the negative low, medium and high value quality control products were tested respectively and the results of the 2-8°C control were consistent with the color development, indicating that the kit of Example 2 has good stability.

Claims

1. A Helicobacter pylori antibody typing test strip, characterized in that: The test strip comprises a bottom plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad, wherein the sample pad, the conjugation pad, the nitrocellulose membrane and the water-absorbing pad are overlapped and pasted on the bottom plate in sequence according to the chromatography direction; The nitrocellulose membrane is coated with three detection lines and one quality control line; the first detection line is coated with Urease antigen, the second detection line is coated with CagA antigen, the third detection line is coated with VacA antigen, and the quality control line is coated with rabbit anti-DNP-BSA or sheep anti-chicken IgY; The conjugate pad is coated with biotinylated ProteinA / G-antibodies labeled with colored microspheres, and biotinylated antibodies or antigens labeled with colored microspheres.

2. A Helicobacter pylori antibody typing test strip according to claim 1, characterized in that: The biotinylated ProteinA / G-antibody labeled with colored microspheres is biotinylated ProteinA / G-mouse anti-human IgG labeled with colored microspheres.

3. A Helicobacter pylori antibody typing test strip according to claim 2, characterized in that: The preparation method of the biotinylated ProteinA / G-mouse anti-human IgG labeled with colored microspheres comprises the following steps: (1) Adding Protein A / G to biotin and mixing them to obtain biotinylated Protein A / G; (2) Adding the streptavidin-modified colored microspheres to the biotinylated Protein A / G and mixing them to obtain colored microsphere-SA-biotin-Protein A / G conjugates; (3) adding mouse anti-human IgG monoclonal antibody to the colored microsphere-SA-biotin-ProteinA / G conjugate and mixing them to obtain mouse anti-human IgG monoclonal antibody microsphere marker; (4) Add a blocking agent to the mouse anti-human IgG monoclonal antibody microsphere marker to block it and obtain biotinylated ProteinA / G-mouse anti-human IgG labeled with colored microspheres.

4. A Helicobacter pylori antibody typing test strip according to claim 3, characterized in that: In the step (1), the molar ratio of biotin to ProteinA / G is (20-40):1; In step (2), the mass ratio of the streptavidin-modified colored microspheres to the biotinylated ProteinA / G is 1:(0.01-0.02); In the step (3), the mass ratio of mouse anti-human IgG monoclonal antibody to colored microsphere-SA-biotin-ProteinA / G conjugate is (0.03-0.05): 1; In the step (4), the mass ratio of the mouse anti-human IgG monoclonal antibody microsphere marker to the blocking agent is 1:(0.02-0.05).

5. A Helicobacter pylori antibody typing test strip according to claim 1, characterized in that: The biotinylated antibody or antigen labeled with the colored microspheres is biotinylated chicken IgY labeled with the colored microspheres or biotinylated DNP-BSA labeled with the colored microspheres.

6. A Helicobacter pylori antibody typing test strip according to claim 5, characterized in that: The preparation method of the biotinylated antibody or antigen labeled with the colored microspheres comprises the following steps: (1) adding the antibody or antigen to biotin and mixing them to obtain biotin-antibody or antigen; (2) adding the streptavidin-modified colored microspheres to the biotin-antibody or antigen and mixing them to obtain the colored microsphere-labeled biotinylated antibody or antigen; The antibody is chicken IgY; the antigen is DNP-BSA.

7. A Helicobacter pylori antibody typing test strip according to claim 6, characterized in that: In step (1), the molar ratio of biotin to antibody or antigen is (10-30):1; In step (2), the mass ratio of the streptavidin-modified colored microspheres to the biotin-antibody or antigen is 1:(0.03-0.05).

8. A Helicobacter pylori antibody typing test strip according to claim 3 or 6, characterized in that: The biotin is NHS-PEG12-biotin.

9. A Helicobacter pylori antibody typing test paper kit, characterized in that: The test kit comprises a test card, which comprises a card housing, in which the test strip according to any one of claims 1 to 8 is arranged.

10. A Helicobacter pylori antibody typing test kit according to claim 9, characterized in that: The kit also includes a diluent.

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