Helicobacter pylori detection reagent strip, reagent pen and application thereof

Abstract

The present application relates to the field of veterinary in vitro diagnostic technology, in particular to a Helicobacter pylori detection reagent strip, a reagent pen and application thereof. The present application takes Helicobacter pylori antibody II as a coating antibody of a detection zone, and is used for preparing a product for detecting pet Helicobacter pylori. The product for detecting pet Helicobacter pylori detects pet Helicobacter pylori through an immunochromatography method, and the chromatography direction of a sample to be detected is vertical from bottom to top in the detection process. Therefore, the original pen appearance is improved, the present application not only provides a novel detection method for dog / cat Helicobacter pylori antigen in the animal industry, but also shortens the sample processing time, and the main advantage is that the urease content of the Helicobacter pylori positive fecal sample can be detected by one-step method when the content exceeds 5 ng / mL.

Description

Technical Field

[0001] This invention relates to the field of veterinary in vitro diagnostic technology, and in particular to Helicobacter pylori detection reagent strips, reagent pens, and their applications. Background Technology

[0002] Helicobacter pylori (HP) is a spiral-shaped, Gram-negative bacterium that is one of the most common pathogens of the digestive tract, affecting about 50% of the global population. Its infection is a major cause of diseases such as gastritis, gastric ulcers, adenocarcinoma, and lymphoid hyperplasia, and it is classified as a Group I carcinogen.

[0003] Humans and pet cats and dogs harboring *Helicobacter pylori* (HP) urease-containing strains include *H. pylori*, *H. canis*, and *H. bizzozeronii*. Therefore, research on urease is indispensable in the preparation of ideal HP antibodies. Urease is one of the most common enzymes found in bacterial infections of the gastrointestinal and urinary tracts of humans and animals. *H. pylori* urease has a molecular weight of 550 kDa, and each monomer consists of two subunits, A and B, which combine to form a hexamer structure. Urease B is the main outer membrane antigenic component of *H. pylori*, encoded by amino acid residues 569. It is a major protective protein, characterized by non-toxicity, conservation, and strong antigenicity, making it the preferred antigenic epitope for vaccines against this bacterium.

[0004] Helicobacter pylori (H. pylori) has nearly 100% homology with humans in dogs and cats. It is primarily transmitted through the fecal-oral and oral-oral routes. Infection occurs in various races and regions worldwide, making it arguably the most widespread chronic bacterial infection between pets and humans. Humans infected with H. pylori may directly or indirectly infect their pets, and conversely, pets infected with H. pylori may transmit the infection to humans. Clinical symptoms in dogs and cats with H. pylori infection mainly include vomiting, diarrhea, lethargy, loss of appetite, severe halitosis, and pica. Severe cases may develop psychotic symptoms. Therefore, daily prevention is crucial, including regular pet checkups and disinfection of the home environment.

[0005] Currently, the main methods for detecting H. pylori infection include: direct examination, urease activity assay, and immunological testing.

[0006] (1) Direct examination of bacteria: HP is detected by preparing tissue sections by forceps, staining and culturing bacteria. This method has the advantages of high sensitivity and strong specificity and can be used as the "gold standard" for verifying diagnostic tests. However, it has great limitations and requires professional personnel to operate.

[0007] (2) Urease test: Since HP can produce a large amount of urease in the body, HP infection can be diagnosed by detecting urease. Urease decomposes urea in the stomach to produce ammonia and carbon dioxide, which reduces the urea concentration and increases the ammonia concentration. Therefore, gastric biopsy tissue urease test; gastric juice urea or urea nitrogen determination; 15N-urea test; breath test can be performed.

[0008] (3) Immunological detection: Currently, the main immunological methods are to diagnose HP infection by measuring HP antibodies in serum, including complement fixation test, agglutination test, passive hemagglutination assay, immunoblotting and enzyme-linked immunosorbent assay (ELISA).

[0009] Unlike testing for Helicobacter pylori in humans, the types of samples that can be easily collected when testing pets are limited. Furthermore, testing samples from the excretory system, which are readily available and quick, is significantly more challenging than testing natural liquid samples such as oral permeate, thus placing higher demands on the sensitivity and specificity of antibodies. Additionally, the bottom-up chromatography method of pen-type tests requires a collection section; during the chromatography process, the retention effect of the collection section and the influence of gravity both reduce the chromatographic effect, further increasing the difficulty of the test. Currently, there are few reagents available in China for detecting Helicobacter pylori in dogs and cats, and a pen-type test kit that combines sampling and detection is not yet available on the market.

[0010] Therefore, developing an accurate, rapid, one-step pen-type reagent for detecting Helicobacter pylori (HP) would facilitate the widespread application of HP testing in veterinary hospitals or at home, and would have significant clinical implications for the prevention and treatment of related diseases caused by HP in pets. Summary of the Invention

[0011] The purpose of this invention is to provide a test strip and / or test pen for Helicobacter pylori in pets, to develop a clinical testing product with good specificity, high sensitivity, rapid detection and simple sampling for pet diseases, and at the same time to provide strong protection for the health of both humans and pets.

[0012] It should be noted that the "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domain of the heavy chain can be referred to as "VH," and the variable domain of the light chain as "VL." These domains are typically the most variable parts of the antibody and contain antigen-binding sites. The variable region of the light or heavy chain consists of a framework region interrupted by three hypervariable regions called "complementarity-determining regions" or "CDRs." The framework region of the antibody, that is, the framework region of the combination of the light and heavy chains, plays a role in locating and aligning the CDRs, which are primarily responsible for binding to the antigen.

[0013] The “framework” or “FR” region refers to the region outside of those defined as CDRs of the antibody variable domain. Each antibody variable domain framework can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3, and FR4).

[0014] Typically, the variable regions VL / VH of heavy and light chains can be obtained by connecting the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0015] In this invention, CDR1-VH, CDR2-VH and CDR3-VH refer to the three highly variable regions of the heavy chain variable region, and correspondingly, CDR1-VL, CDR2-VL and CDR3-VL refer to the three highly variable regions of the light chain variable region.

[0016] To solve the above-mentioned technical problems and achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0017] In a first aspect, the present invention provides the application of murine Helicobacter pylori antibody II in the preparation of a product for detecting Helicobacter pylori in pets; the product for detecting Helicobacter pylori in pets is prepared by immunochromatography, wherein the chromatography direction of the sample to be tested is vertical from bottom to top during the detection process, and the murine Helicobacter pylori antibody is coated in the detection area of ​​the immunochromatography.

[0018] The variable region of the murine Helicobacter pylori antibody II includes: a complementarity-determining region CDR1-VH having the amino acid sequence shown in SEQ ID NO.1, a complementarity-determining region CDR2-VH having the amino acid sequence shown in SEQ ID NO.2, a complementarity-determining region CDR3-VH having the amino acid sequence shown in SEQ ID NO.3, a complementarity-determining region CDR1-VL having the amino acid sequence shown in SEQ ID NO.4, a complementarity-determining region CDR2-VL having the amino acid sequence shown in SEQ ID NO.19, and a complementarity-determining region CDR3-VL having the amino acid sequence shown in SEQ ID NO.5.

[0019] CDR1-VH:DYTFTNSW (SEQ ID NO.1);

[0020] CDR2-VH: INPSTGST (SEQ ID NO. 2);

[0021] CDR3-VH:ASEEYDGFDY (SEQ ID NO.3);

[0022] CDR1-VL: SSIIYM (SEQ ID NO.4);

[0023] CDR2-VL:DTS (SEQ ID NO.19);

[0024] CDR3-VL: QQWSSSPYT (SEQ ID NO. 5).

[0025] As a further technical solution, the variable region amino acid sequence of the heavy chain of the murine Helicobacter pylori antibody II is shown in SEQ ID NO.6:

[0026] PGASVKMSCRASDYTFTNSWMHWVKQRPGQGLEWVGYINPSTGSTDYNQKFRDKATLTADKSSSTAYMQLSSLTSEDSAVYYCASEEYDGFDYWGQGTTLTVSS (SEQ ID NO. 6);

[0027] The amino acid sequence of the variable region of the light chain of the murine Helicobacter pylori antibody II is shown in SEQ ID NO.7:

[0028] DIVITQSPAIMSASPGEKVTMTCSANSSIIYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSSPYTFGGGTKLEIK (SEQ ID NO. 7).

[0029] As a further technical solution, the variable region nucleotide sequence of the heavy chain of the murine Helicobacter pylori antibody II is shown in SEQ ID NO.8:

[0030] CCTGGGGCCTCAGTGAAGATGTCCTGCAGGGCTTCTGACTACACCTTTACTAACTCCTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGGTTGGATACATTAATCCTAGCACTGGTTCTACTGACTACAATCAGAAGTTCAGGGA CAAGGCCACTTTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGCGAGGAGTACGACGGCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO.8);

[0031] The variable region nucleotide sequence of the light chain of the murine Helicobacter pylori antibody II is shown in SEQ ID NO. 9:

[0032] GATATTGTGATAACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAACTCAAGTATAATTTACATGCATTGGTACCAGCAGAAGTCAGGCACGTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGC TTCTGGAGTCCCTGCTCGTTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAGCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO.9).

[0033] Secondly, the present invention provides a Helicobacter pylori detection strip, wherein the strip comprises, from top to bottom, a quality control area, a detection area, a polyester film containing latex marker I, a sample pad, and absorbent paper; the detection area is coated with murine Helicobacter pylori antibody II.

[0034] The variable region of the murine Helicobacter pylori antibody II includes: a complementarity-determining region CDR1-VH having the amino acid sequence shown in SEQ ID NO.1, a complementarity-determining region CDR2-VH having the amino acid sequence shown in SEQ ID NO.2, a complementarity-determining region CDR3-VH having the amino acid sequence shown in SEQ ID NO.3, a complementarity-determining region CDR1-VL having the amino acid sequence shown in SEQ ID NO.4, a complementarity-determining region CDR2-VL having the amino acid sequence shown in SEQ ID NO.19, and a complementarity-determining region CDR3-VL having the amino acid sequence shown in SEQ ID NO.5;

[0035] Preferably, the base material of the reagent strip is a nitrocellulose membrane.

[0036] As a further technical solution, the latex marker I is a latex microsphere conjugated with Helicobacter pylori antibody I;

[0037] The variable region of the Helicobacter pylori antibody I includes: a complementarity-determining region CDR1-VH having the amino acid sequence shown in SEQ ID NO.10, a complementarity-determining region CDR2-VH having the amino acid sequence shown in SEQ ID NO.11, a complementarity-determining region CDR3-VH having the amino acid sequence shown in SEQ ID NO.12, a complementarity-determining region CDR1-VL having the amino acid sequence shown in SEQ ID NO.13, a complementarity-determining region CDR2-VL having the amino acid sequence shown in SEQ ID NO.20, and a complementarity-determining region CDR3-VL having the amino acid sequence shown in SEQ ID NO.14;

[0038] CDR1-VH: GYTFTKYG (SEQ ID NO. 10);

[0039] CDR2-VH: INTYTGEP (SEQ ID NO. 11);

[0040] CDR3-VH:ASPFGY (SEQ ID NO.12);

[0041] CDR1-VL: QTIVYSNGKTY (SEQ ID NO.13);

[0042] CDR2-VL: KVS (SEQ ID NO. 20);

[0043] CDR3-VL: FQGSHVPWT (SEQ ID NO. 14).

[0044] As a further technical solution, the variable region amino acid sequence of the heavy chain of the Helicobacter pylori antibody I is shown in SEQ ID NO.15:

[0045] VQVQESGPELKKPGETVKISCKASGYTFTKYGMNWVKQAPGKDLKWMGWINTYTGEPTYADDFKGRFAFSLETFASTAYLQINNLKNEDTATYFCASPFGYWGQGTLVTVS (SEQ ID NO. 15);

[0046] The variable region amino acid sequence of the light chain of the Helicobacter pylori antibody I is shown in SEQ ID NO.16:

[0047] DVLMTQTPPSLPVSLGDQASISCRSSQTIVYSNGKTYLDWYLQKPGQSPNLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLKSK (SEQ ID NO. 16).

[0048] As a further technical solution, the variable region nucleotide sequence of the heavy chain of the Helicobacter pylori antibody I is shown in SEQ ID NO.17:

[0049] GTGCAGGTGCAGGAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCAAAAATATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGATTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAG CCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTTTTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGCCCGTTTGGTTACTGGGGCCAAGGGACTCTGGTCACAGTCTCT (SEQ ID NO17);

[0050] The variable region nucleotide sequence of the light chain of the Helicobacter pylori antibody I is shown in SEQ ID NO.18:

[0051] GATGTTTTGATGACCCAAACTCCACCCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGACCATTGTATATAGTAATGGAAAAACCTATTTAGACTGGTACCTGCAGAAACCAGGCCAGTCTCCAAATCTCCTGATCTACAAAGTTTC CAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGATGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGAAATCAAAA (SEQ ID NO.18).

[0052] Preferably, the latex is a C-latex, selected from C-Latex1, C-Latex2 or C-Latex3; more preferably, it is C-Latex3.

[0053] As a further technical solution, the preparation method of the latex marker I includes: carboxyl-activated latex microspheres being coupled with Helicobacter pylori antibody I via a coupling agent under the action of a catalyst;

[0054] Preferably, the catalyst is a combination catalyst of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide salt;

[0055] Preferably, the particle size of the latex microspheres is in the range of 50 nm to 250 nm;

[0056] As a further technical solution, the method for preparing the polyester film containing latex marker I includes spraying the diluted latex marker I onto the polyester film;

[0057] Preferably, the components of the diluent used for dilution include bovine serum albumin, tris(hydroxymethyl)aminomethane, and sodium azide;

[0058] Preferably, a gold spraying machine is used for spraying, and the spraying speed is further preferably 0.5 to 1.5 μL / cm, more preferably 1.0 μL / cm;

[0059] Preferably, before spraying, the polyester film is treated with a treatment solution, the components of which include disodium hydrogen phosphate, bovine serum albumin, polyvinyl alcohol, and Triton X-100.

[0060] As a further technical solution, the quality control area is coated with a quality control line, and the preparation method of the quality control line includes streaking antibody III diluted with buffer onto the quality control area, wherein the antibody III is a goat anti-mouse polyclonal antibody;

[0061] The detection area is coated with a detection line, and the method for preparing the detection line includes streaking a line of mouse Helicobacter pylori antibody II diluted with buffer onto the detection area.

[0062] Preferably, the buffer solution comprises PBS buffer;

[0063] Preferably, the PBS buffer comprises sodium chloride, anhydrous disodium hydrogen phosphate, anhydrous sodium dihydrogen phosphate, and sodium azide, and the pH of the PBS buffer is 7.4 ± 0.1.

[0064] Preferably, the ratio of the coating mass of antibody III and murine Helicobacter pylori antibody II coated on the nitrocellulose membrane to the length of the reagent strip is 0.5-1 μg / cm and 0.1-0.5 μg / cm, respectively, and more preferably 1 μg / cm and 0.5 μg / cm.

[0065] Preferably, after dilution with buffer, the final concentration of antibody III or murine Helicobacter pylori antibody II is 0.5–1 g / L, preferably 1 g / L.

[0066] As a further technical solution, the sample pad is coated with a sample pad treatment solution, the components of which include buffer salts, polyvinylpyrrolidone, casein and surfactants;

[0067] Preferably, the sample pad treatment solution comprises 0.1 mol / L buffer salt, 0.8% (w / v) polyvinylpyrrolidone, 0.5% (w / v) casein and 1% (w / v) surfactant;

[0068] Preferably, the pH of the sample pad treatment solution is 7.4 ± 0.1.

[0069] As a further technical solution, the distance between the detection line in the detection area and the quality control line in the quality control area is 5 to 10 mm, preferably 7 mm.

[0070] Thirdly, the present invention provides a Helicobacter pylori detection reagent pen, the reagent pen including the reagent strip and a reagent pen shell sleeved outside the reagent strip, one end of the reagent pen shell having a collection part.

[0071] As a further technical solution, an absorbent material is provided between the collection part and the reagent strip, and the absorbent material is in contact with and connected to the collection part and the reagent strip respectively;

[0072] Preferably, the absorbent material includes an absorbent sponge.

[0073] The technical advantages of this invention are as follows: This invention uses Helicobacter pylori antibody II as the coating antibody for the detection area to prepare a product for detecting Helicobacter pylori in pets; the product detects Helicobacter pylori in pets using immunochromatography, and the chromatography direction of the sample is vertical from bottom to top during the detection process. Therefore, the original pen-shaped appearance of this invention was improved, and the composition of the reagent strip was optimized to select the reagent formula with the best performance. By comparing the test reagent strip with the test reagent pen, similar effects were achieved, meeting the requirements of sensitivity and other indicators. This invention not only provides a novel detection method for canine / feline Helicobacter pylori antigen detection reagents in the animal industry, but also shortens the sample processing time during the detection process. Its main advantage is that it can detect approximately 5 ng / mL of urease in Helicobacter pylori-positive fecal samples. Attached Figure Description

[0074] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0075] Figure 1 This is a schematic diagram of the structure of the Helicobacter pylori detection reagent pen for dogs / cats provided by the present invention. Detailed Implementation

[0076] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0077] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0078] It should be noted that, in the following embodiments, the variable region amino acid sequence of the heavy chain of mouse Helicobacter pylori antibody II is shown in SEQ ID NO. 6, and the variable region amino acid sequence of the light chain is shown in SEQ ID NO. 7; the variable region amino acid sequence of the heavy chain of Helicobacter pylori antibody I is shown in SEQ ID NO. 15, and the variable region amino acid sequence of the light chain is shown in SEQ ID NO. 16.

[0079] In one specific embodiment, the present invention provides a method for preparing a canine / feline Helicobacter pylori detection reagent pen, which specifically includes the following steps:

[0080] (1) Preparation of latex marker I: Latex microspheres were purchased from Magsphere. Electron microscopy was used to detect the latex particles, which had an average size of 200 nm. The microspheres were washed with carboxyl activation buffer, centrifuged and the supernatant was discarded. The washing was repeated 2-3 times. After adding carboxyl activation buffer again, the microspheres were sonicated. N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide salt solution were added and incubated for 20 min. After centrifugation and discarding the supernatant, the microspheres were coupled with a coupling agent. The prepared Helicobacter pylori antibody I (Hangzhou Xuke Biotechnology Co., Ltd., R0176) was added to the microspheres to form HP antibody-latex marker I. After shaking for 2 h, the microspheres were washed 2-3 times with PBS / BSA and then stored in preservation solution.

[0081] (2) Preparation of polyester film: The HP antibody-latex marker I obtained in step (1) is diluted with latex dilution buffer (the main components of the dilution buffer include: bovine serum albumin, tris(hydroxymethyl)aminomethane, sodium azide), and a certain amount of spraying is selected by the gold spraying machine and evenly sprayed onto the treated polyester film (the polyester film is first treated with polyester film treatment solution, the main components of which include: disodium hydrogen phosphate, bovine serum albumin, polyvinyl alcohol, Triton X-100).

[0082] (3) Antibody coating for feline / canine Helicobacter pylori detection reagent: The raw materials for the control line (C line) and the test line (T line) were diluted to 1.0 mg / mL with PBS buffer (main components include sodium chloride, anhydrous disodium hydrogen phosphate, anhydrous sodium dihydrogen phosphate, sodium azide, pH adjusted to 7.4±0.1). Anti-canine / feline mouse Helicobacter pylori antibody II (Hangzhou Xuke Biotechnology Co., Ltd., RM0023) and goat anti-mouse polyclonal antibody were respectively streaked on the PVC board of the prepared nitrocellulose membrane (purchased from Sartorius CN140) and dried for 24 h to prepare the immunonitrocellulose membrane.

[0083] (4) Sample pad preparation: Purchase (8964#) glass fiber membrane from Austron Corporation, prepare a solution with the following main chemical components: (0.1 mol / L buffer salt, 0.8% polyvinylpyrrolidone (PVP), 0.5% casein and 1% surfactant), adjust the pH to a suitable level, coat the prepared solution onto the fiber membrane, and then dry it overnight in a 37°C oven for later use.

[0084] (5) Assemble the prepared polyester membrane, immunonitrocellulose membrane, sample pad, and absorbent paper in sequence, then assemble the reagent strip and reagent pen, and cover them. The structural diagram of the resulting reagent pen is shown below. Figure 1 As shown, 1 is absorbent paper, 2 is the quality control area, 3 is the detection area, 4 is a polyester film containing latex marker I, 5 is the sample pad, and 6 is the collection area.

[0085] 1. Sample collection and processing:

[0086] Remove the reagent pen and its protective cap. Insert the pen's sampling tip into the collection site or / and the fecal sample, rotating it 3-5 times. After collection, slowly and vertically insert the pen's sampling tip into the sample diluent.

[0087] 2. Sample detection and result interpretation:

[0088] Negative result: Only one band appeared in the control area (C line), and no band appeared in the test area (T line). The negative result indicates that Helicobacter pylori was not detected in the sample.

[0089] Positive result: Both bands appeared. One was located in the detection zone (T line), and the other was located in the control zone (C line). The positive result indicates that the sample contains Helicobacter pylori.

[0090] Invalid: No purple-red band appears in the control area (C line), indicating incorrect operation or that the reagent has deteriorated or been damaged. In this case, the instructions should be read carefully again and the test should be repeated with new reagent.

[0091] Example 1: Screening of different antibody concentration ratios for canine / feline Helicobacter pylori detection strips

[0092] Referring to the specific implementation method described above, in the preparation of antibody coating for the feline / canine Helicobacter pylori detection reagent, three different dilution concentrations were selected to dilute the detection line (T line) raw material to 0.5 mg / mL, 1.0 mg / mL, and 1.5 mg / mL, respectively. The rest of the preparation process remained the same, and the reagent pen was finally assembled for detection.

[0093] Test method:

[0094] 1. Prepare reagents: Take the canine / feline Helicobacter pylori test reagent pen and place it on a horizontal table to allow it to return to room temperature (15℃~25℃). Take the canine / feline Helicobacter pylori test reagent plate and place it on the table to allow it to return to room temperature (15℃~25℃).

[0095] 2. Preparation of test samples: Prepare Helicobacter pylori urease concentrations of 2.5 ng / mL, 5 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL as standard samples.

[0096] 3. Sample testing and result observation: Take 2-3 drops of sample diluent with a pipette and drop it onto the sponge tip. Then, insert the pen tip vertically into the lysis tube containing the sample diluent and let it stand on the table for 10-15 minutes before interpreting the results.

[0097] Table 1 Comparison of T-line color development at different antibody concentrations in canine / feline Helicobacter pylori detection reagent pens

[0098]

[0099] A comparison of different antibody concentration ratios reveals that using Helicobacter pylori antibody ratios of 1.0 mg / mL and 1.5 mg / mL (T-line concentration) provides better sensitivity, while the 0.5 mg / mL antibody concentration exhibits poorer sensitivity and weaker gradient color development. To save costs, the 1.0 mg / mL antibody concentration is chosen to achieve the best effect and highest titer with a lower concentration.

[0100] Example 2: Screening of latex microspheres for canine / feline Helicobacter pylori detection reagent pens

[0101] 1. Test method:

[0102] Referring to the method for preparing reagent strips described above, three different C-Latex materials were selected during the preparation of the latex microsphere labeling: C-Latex1, C-Latex2, and C-Latex3. C-Latex1 was black, C-Latex2 was light red, and C-Latex3 was red. After labeling the latex microspheres, the remaining preparation process remained consistent, and the final assembly was used to create a reagent pen for detection. Comparative verification was performed to assess the color intensity of the three different latex microspheres and the sensitivity of the reagent.

[0103] 2. Preparation of test samples: Prepare Helicobacter pylori urease concentrations of 5 ng / mL, 20 ng / mL, and 50 ng / mL, using clinically negative stool samples and blank diluents as controls.

[0104] Table 2 Comparison of C / T line color development of different latex particles in canine / feline Helicobacter pylori detection reagent pens

[0105]

[0106] Note: Color rendering gradually increases from L2 to L10.

[0107] By comparing the types of latex particles used in the test strip formulations, it can be found that the C-Latex3 latex particles have a stronger color development intensity than C-Latex1 and C-Latex2, and also have the best performance.

[0108] Example 3: Sample pad treatment and pH screening for canine / feline Helicobacter pylori detection reagent pens.

[0109] 1. Test method:

[0110] Referring to the preparation method of the reagent strips provided in the above specific embodiments, the sample pad preparation process was optimized. 8964# glass fiber membrane was selected, and solutions were prepared according to the following proportions: the main chemical components were (0.1 mol / L buffer salt, 0.8% polyvinylpyrrolidone (PVP), 0.5% casein, and 1% surfactant). Three solutions were prepared, and the pH values ​​were adjusted to pH=6.0, pH=7.4, and pH=9.0, respectively. The prepared solutions were then coated onto the fiber membrane and dried overnight in a 37°C oven. The remaining preparation process remained consistent.

[0111] 2. Preparation of test samples: Prepare Helicobacter pylori urease concentrations of 2.5 ng / mL, 5 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL, respectively, using clinically negative stool samples and blank diluents as controls.

[0112] 3. Sample testing and result observation: Take 2-3 drops of sample diluent with a pipette and drop it onto the sponge tip. Then, insert the pen tip vertically into the lysis tube containing the sample diluent and let it stand on the table for 10-15 minutes before interpreting the results.

[0113] Table 3 Comparison of sample pads with different pH values ​​for canine / feline Helicobacter pylori detection reagent pens

[0114]

[0115] The results showed that comparing the sample pads with three different pH values ​​simulated corresponding buffer reaction environments. The sample pads treated with a slightly acidic pH exhibited reduced sensitivity, weak colorimetric gradient, and poor colorimetric results. Meanwhile, the sample pads treated with a slightly alkaline pH resulted in false positives in the negative control during the colorimetric process, and the reagent strips showed a reddish tint and slower overall migration speed, significantly impacting the colorimetric results. Therefore, the sample pad with pH=7.4 was selected for comparative analysis.

[0116] Example 4: Comparison of running speed between canine / feline Helicobacter pylori detection reagent pen and detection plate.

[0117] Detection method:

[0118] 1. Prepare reagents: Take three canine / feline Helicobacter pylori test reagent pens and place them on a horizontal table to allow them to return to room temperature (15℃~25℃). Take three canine / feline Helicobacter pylori test reagent plates and place them on a table to allow them to return to room temperature (15℃~25℃).

[0119] 2. Prepare the test sample: Prepare 50 ng / mL of the company's quality control reference standard.

[0120] 3. Sample testing and result observation:

[0121] Migration rate = L / t (L is the distance from the C line to the bottom of the sample pad, and t is the time it takes for the C line to appear). Slowly and vertically insert the reagent pen sampling tip into the sample diluent. Start timing with a stopwatch after adding the sample and stop timing when the liquid reaches the C line. Record the time taken as (t). Measure the length from the C line to the bottom of the sample pad with a vernier caliper and record it as (L).

[0122] Table 4. Comparison of running speed between the canine / feline Helicobacter pylori detection reagent pen and the reagent plate.

[0123]

[0124] The results showed that, by comparing the running speed of the canine / feline Helicobacter pylori detection pen and the detection plate, it could be seen that the sample migration rate of the detection pen was 11.4 mm / min and the detection migration rate of the detection plate was 12.3 mm / min. The sample running speed of the two products was basically the same, with no significant difference.

[0125] Example 5: Sensitivity Comparison Experiment of Canine / Cat Helicobacter pylori Detection Reagent Pen and Reagent Plate

[0126] Detection method:

[0127] 1. Prepare reagents: Take the canine / feline Helicobacter pylori test reagent pen and place it on a horizontal table to allow it to return to room temperature (15℃~25℃). Take the canine / feline Helicobacter pylori test reagent plate and place it on the table to allow it to return to room temperature (15℃~25℃).

[0128] 2. Prepare test samples: Prepare Helicobacter pylori urease concentrations of 2.5 ng / mL, 5 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL for the company's quality control reference.

[0129] 3. Sample testing and result observation: Take 2-3 drops of sample diluent with a pipette and drop it onto the sponge tip. Then, insert the pen tip vertically into the lysis tube containing the sample diluent and let it stand on the table for 10-15 minutes before interpreting the results.

[0130] Table 5. Comparison of sensitivity between canine / feline Helicobacter pylori detection reagent pens and reagent plates.

[0131]

[0132] The results demonstrate that, through comparison of the detection results of the canine / feline Helicobacter pylori detection pen and the reagent plate, the sensitivity of the canine / feline Helicobacter pylori detection pen is close to that of the reagent plate. Furthermore, the sensitivity of the canine / feline Helicobacter pylori detection pen prepared in this invention is 5 ng / mL, which meets the detection requirements.

[0133] Example 6: Repeatability and stability test of canine / feline Helicobacter pylori detection reagent pen

[0134] 1. Test method:

[0135] Three batches of canine / feline Helicobacter pylori detection pens were prepared and labeled A1, B1, and C1, respectively. Each pen was used to test for Helicobacter pylori urease at concentrations of 5 ng / mL, 20 ng / mL, and 50 ng / mL (complicated 5 times per concentration). The pens were packaged in aluminum foil bags containing desiccant and placed in an oven at 55°C. Five pens were removed at 0, 3, 7, 14, and 30 days, and tested for Helicobacter pylori urease at concentrations of 5 ng / mL, 20 ng / mL, and 50 ng / mL using the Helicobacter pylori urease control reference. This was done to study the accelerated stability and to assess the repeatability and sensitivity of the pens.

[0136] 2. Experimental Results:

[0137] Table 6. Stability results of the canine / feline Helicobacter pylori detection reagent pen

[0138]

[0139] The results demonstrate that the intra- and inter-batch differences in the canine / feline Helicobacter pylori detection reagent pen are minimal, with 100% repeatability, and no false positives or false negatives were observed. The three batches of samples developed remained stable at 55℃ for at least 30 days. According to the Arrhenius equation, the reagent remains stable for 2 years at normal room temperature, with stable sensitivity and repeatability, showing no significant changes.

[0140] Example 7: Clinical trial of a canine / feline Helicobacter pylori detection reagent pen

[0141] Eight pet hospitals in Zhejiang Province were randomly selected. Based on preliminary clinical diagnoses of possible Helicobacter pylori infection, a total of 160 clinical fecal samples were collected, including 79 cat samples and 81 dog samples. The canine / feline Helicobacter pylori detection reagent pen of this invention was used to test these clinical samples. The plastic cap was removed, and the pen tip (sponge tip) was gently rotated 3-5 times around the sample. The pen tip was then vertically inserted into a lysis tube containing sample diluent, and the samples were left to stand for 10-15 minutes before interpretation. The results showed that 63 fecal samples from cats and 27 fecal samples from dogs were positive, with the remainder being negative. No test pens failed to develop either of the two lines.

[0142] Based on the clinical test results of eight pet hospitals, the results of the above 160 clinical fecal tests were statistically analyzed, as shown in Table 7:

[0143] Table 7 Results of Clinical Testing and Reagent Pen Testing

[0144]

[0145] Table 8 Summary of Results

[0146]

[0147] Based on the results of clinical trials, the sensitivity of the canine / feline Helicobacter pylori detection reagent pen of this invention is 90 / (90+1)×100%=98.9%. The specificity (SPE) is 69 / 69×100%=100%, the true positive rate (PPV) is 90 / 90×100%=100%, and the concurrent concordance rate is (90+68) / 160*100%=98.7%.

[0148] As can be seen from the above-mentioned experimental examples, the canine / feline Helicobacter pylori detection reagent pen prepared by this invention is consistent with the existing test results of pet hospitals. The product has good sensitivity and specificity, and is convenient for sampling, simple to operate, novel in design, and provides intuitive and rapid results. It is suitable for pet hospitals, pet rescue stations, and pet owners to diagnose whether their pets are infected with Helicobacter pylori.

[0149] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of mouse-derived Helicobacter pylori antibody II in the preparation of products for detecting Helicobacter pylori in pets; the products for detecting Helicobacter pylori in pets are prepared by immunochromatography, wherein the chromatography direction of the sample to be tested is vertical from bottom to top, and the mouse-derived Helicobacter pylori antibody II is coated in the detection area of ​​the immunochromatography. The variable region of the murine Helicobacter pylori antibody II includes: The complementarity-determining regions (CDR1-VH) of the amino acid sequence shown in SEQ ID NO.1, the CDR2-VH of the amino acid sequence shown in SEQ ID NO.2, the CDR3-VH of the amino acid sequence shown in SEQ ID NO.3, the CDR1-VL of the amino acid sequence shown in SEQ ID NO.4, the CDR2-VL of the amino acid sequence shown in SEQ ID NO.19, and the CDR3-VL of the amino acid sequence shown in SEQ ID NO.

5.

2. A Helicobacter pylori detection strip, characterized in that, The reagent strip comprises, from top to bottom, a quality control area, a detection area, a polyester film containing latex marker I, a sample pad, and absorbent paper; the detection area is coated with murine Helicobacter pylori antibody II. The variable region of the murine Helicobacter pylori antibody II includes: the complementarity-determining region CDR1-VH of the amino acid sequence shown in SEQ ID NO.1, the complementarity-determining region CDR2-VH of the amino acid sequence shown in SEQ ID NO.2, the complementarity-determining region CDR3-VH of the amino acid sequence shown in SEQ ID NO.3, the complementarity-determining region CDR1-VL of the amino acid sequence shown in SEQ ID NO.4, the complementarity-determining region CDR2-VL of the amino acid sequence shown in SEQ ID NO.19, and the complementarity-determining region CDR3-VL of the amino acid sequence shown in SEQ ID NO.

5.

3. The reagent strip according to claim 2, characterized in that, The substrate material of the reagent strip is a nitrocellulose membrane.

4. The reagent strip according to claim 2, characterized in that, The latex marker I is a latex microsphere conjugated with Helicobacter pylori antibody I; The variable region of the Helicobacter pylori antibody I includes: the complementarity-determining region CDR1-VH of the amino acid sequence shown in SEQ ID NO.10, the complementarity-determining region CDR2-VH of the amino acid sequence shown in SEQ ID NO.11, the complementarity-determining region CDR3-VH of the amino acid sequence shown in SEQ ID NO.12, the complementarity-determining region CDR1-VL of the amino acid sequence shown in SEQ ID NO.13, the complementarity-determining region CDR2-VL of the amino acid sequence shown in SEQ ID NO.20, and the complementarity-determining region CDR3-VL of the amino acid sequence shown in SEQ ID NO.

14.

5. The reagent strip according to claim 4, characterized in that, The latex is a C-latex, selected from C-Latex1, C-Latex2 or C-Latex3.

6. The reagent strip according to claim 5, characterized in that, The latex is C-Latex3.

7. The reagent strip according to claim 4, characterized in that, The preparation method of the latex marker I includes coupling latex microspheres with Helicobacter pylori antibody I via a coupling agent under the action of a catalyst.

8. The reagent strip according to claim 7, characterized in that, The catalyst is a combination catalyst of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide salt.

9. The reagent strip according to claim 7, characterized in that, The particle size of the latex microspheres ranges from 50 nm to 250 nm.

10. The reagent strip according to claim 2, characterized in that, The method for preparing the polyester film containing latex marker I includes spraying diluted latex marker I as described in claim 4 onto the polyester film.

11. The reagent strip according to claim 10, characterized in that, The components of the diluent used for dilution include bovine serum albumin, tris(hydroxymethyl)aminomethane, and sodium azide; The gold spraying machine is used for spraying, and the spraying speed is 0.5 to 1.5 μL / cm; Before spraying, the polyester film is treated with a treatment solution, the components of which include disodium hydrogen phosphate, bovine serum albumin, polyvinyl alcohol, and Triton X-100.

12. The reagent strip according to claim 2, characterized in that, The quality control area is coated with a quality control line, and the preparation method of the quality control line includes streaking antibody III diluted with buffer onto the quality control area, wherein antibody III is a goat anti-mouse polyclonal antibody. The detection area is coated with a detection line, and the method for preparing the detection line includes drawing a line on the detection area with mouse Helicobacter pylori antibody II diluted with buffer solution.

13. The reagent strip according to claim 12, characterized in that, The buffer solution includes PBS buffer; The PBS buffer comprises sodium chloride, anhydrous disodium hydrogen phosphate, anhydrous sodium dihydrogen phosphate, and sodium azide, and the pH of the PBS buffer is 7.4 ± 0.

1. The ratios of antibody III and murine Helicobacter pylori antibody II coated on nitrocellulose membranes to the length of the test strip were 0.5–1 μg / cm and 0.1–0.5 μg / cm, respectively. After dilution with buffer, the final concentration of antibody III or murine Helicobacter pylori antibody II is 0.5–1 g / L.

14. The reagent strip according to claim 13, characterized in that, The ratios of antibody III and murine Helicobacter pylori antibody II coated on nitrocellulose membranes to the length of the test strip were 1 μg / cm and 0.5 μg / cm, respectively. After dilution with buffer, the final concentration of antibody III or murine Helicobacter pylori antibody II is 1 g / L.

15. The reagent strip according to claim 2, characterized in that, The sample pad is coated with a sample pad treatment solution, the components of which include buffer salts, polyvinylpyrrolidone, casein, and surfactants.

16. The reagent strip according to claim 15, characterized in that, The sample pad treatment solution comprises 0.1 mol / L buffer salt, 0.8% polyvinylpyrrolidone, 0.5% casein and 1% surfactant; The pH of the sample pad treatment solution is 7.4 ± 0.

1.

17. The reagent strip according to any one of claims 2 to 16, characterized in that, The distance between the test line in the test area and the quality control line in the quality control area is 5 to 10 mm.

18. The reagent strip according to any one of claims 2 to 16, characterized in that, The distance between the test line in the test area and the quality control line in the quality control area is 7 mm.

19. A Helicobacter pylori detection pen, characterized in that, The reagent pen includes a reagent strip as described in any one of claims 2 to 18, and a reagent pen shell sleeved over the reagent strip, wherein one end of the reagent pen shell is provided with a collection part.

20. The reagent pen according to claim 19, characterized in that, A water-absorbing material is provided between the collection part and the reagent strip, and the water-absorbing material is in contact with and connected to the collection part and the reagent strip respectively.

21. The reagent pen according to claim 20, characterized in that, The absorbent material includes an absorbent sponge.

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