Antigenic epitope peptide of helicobacter pylori vacuolating toxin in feces and application thereof

Abstract

The present application relates to the technical field of in vitro diagnosis, and particularly relates to an antigen epitope peptide of Helicobacter pylori vacuolating cytotoxin in feces and application thereof. The amino acid sequence of the antigen epitope peptide comprises one or more of DTV, DMK and WRI. By using the antigen epitope peptide provided in the present application, more specific antibodies against different antigen epitopes of VacA can be obtained, so that the accuracy of immunological methods for detecting the degradation product of VacA in feces is improved, the detection time of Helicobacter pylori infection is greatly saved, and the human body is not damaged during sampling.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, and in particular to an antigenic epitope peptide of Helicobacter pylori vacuolating toxin in feces and its application. Background Technology

[0002] Helicobacter pylori (HP) is a spiral-shaped, microaerophilic, Gram-negative bacterium with extremely demanding growth requirements. First successfully isolated from gastric mucosal biopsy tissue of patients with chronic active gastritis in 1983, it is currently the only known microorganism capable of surviving in the human stomach. More than 50% of the world's population has been infected with Helicobacter pylori, and in some countries, nearly 90% of the population has been infected with this bacterium. Studies have shown that infection with this bacterium can cause chronic gastritis, gastric ulcers, and gastric atrophy, and is closely related to the development of gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer. The International Agency for Research on Cancer (IARC) of the World Health Organization classifies Helicobacter pylori (infection) as a Group 1 carcinogen.

[0003] Currently, there are two main categories of methods for detecting Helicobacter pylori (HP): invasive and non-invasive. Invasive methods mainly rely on gastroscopy for sample collection, which is more traumatic and expensive. Non-invasive methods include PCR testing, serum antibody testing, and breath tests, but they also have certain limitations. For example, PCR testing requires a high level of professional knowledge and operational skills from the personnel, and the testing equipment and consumables used are relatively expensive. Serum antibody testing can only prove that a patient has been infected or is currently infected, and cannot accurately determine whether a patient has eradicated HP after treatment. Breath tests currently mainly use 13C and 14C isotopes for tracing, but the accuracy of the results can be affected by the use of antibiotics or proton pump inhibitors (PPIs).

[0004] As research into the virulence of *Helicobacter pylori* (HP) has deepened, its identified pathogenic factors include flagellin, urease, heat shock protein, vacuolating cytotoxic agent (VacA), and cytotoxic-associated factor (CagA). VacA is a key pathogenic gene for HP, encoding a secreted protein that induces vacuolating degeneration in eukaryotic cells, leading to apoptosis, cytoskeleton rearrangement, and other morphological changes. Studies have shown that VacA is detected in 63% of gastric cancer patients. Therefore, detecting VacA protein levels in feces is of great significance for the prevention and control of gastric cancer and can overcome the limitations of PCR testing, serum antibody testing, and breath tests for detecting HP infection.

[0005] The gene encoding VacA is present in all *Helicobacter pylori* strains, with a full length of 3846 bp. After translation and modification, it forms a mature toxin with a relative molecular mass of 95 kDa (including a 37 kDa activating subunit and a 58 kDa binding subunit). The complete VacA molecule is petal-shaped or snowflake-shaped, composed of 6–7 radially symmetrical monomers forming an oligomeric complex, which depolymerizes into monomers under acidic conditions. By the time VacA reaches feces, it has undergone a series of degradations into polypeptides. Some antigenic epitopes present in the full-length VacA are randomly degraded, making it impossible for antibodies targeting these epitopes to detect VacA degradation products in feces. Because the degradation sites of VacA before reaching feces are random, the degraded antigenic epitopes are not fixed. Therefore, exploring more antigenic epitopes in VacA can help develop specific antibodies against different VacA epitopes, thereby improving the accuracy of detecting VacA degradation products in feces. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an antigenic epitope peptide of Helicobacter pylori vacuolating toxin in feces and its application. Utilizing the antigenic epitope peptide provided in this invention can promote the development of specific antibodies against different antigenic epitopes of VacA, thereby improving the accuracy of immunological methods for detecting VacA degradation products in feces. This has broad application prospects and practical value in the detection of Helicobacter pylori.

[0007] The specific technical solution of this invention is as follows:

[0008] In a first aspect, the present invention provides an antigenic epitope peptide of Helicobacter pylori vacuolating toxin in feces, wherein the amino acid sequence of the antigenic epitope peptide includes one or more of DTV, DMK and WRI.

[0009] The inventors discovered three antigenic epitopes in the degradation products (peptides) of vacuole toxin (VacA) in human feces. Each epitope consists of three amino acids in tandem: DTV, DMK, and WRI. When any one of the three antigenic epitopes is present in the peptide, it can bind to VacA-specific antibodies.

[0010] The discovery of these antigenic epitopes facilitates the development of more specific antibodies against different antigenic epitopes in VacA, thereby improving the accuracy of immunological methods for detecting VacA degradation products in feces. This significantly reduces the detection time for Helicobacter pylori infection, and the sampling process is non-invasive. Furthermore, compared to traditionally recombinantly expressed proteins, antigens designed and synthesized targeting these specific epitopes are less expensive, easier to handle, easier to store, and more stable. Specific antibodies prepared using these antigens exhibit a higher positive rate for detecting degradation products in feces.

[0011] Secondly, the present invention provides a fecal Helicobacter pylori vacuole toxin antigen, wherein the antigenic epitopes include one or more of DTV, DMK and WRI.

[0012] DTV, DMK, and WRI serve as antigenic epitopes for VacA degradation products in feces, allowing for the direct design and synthesis of artificial VacA antigenic peptides, which is beneficial for the production of specific antibodies against VacA degradation products in feces.

[0013] Preferably, the amino acid sequence of the antigen includes one or more of the sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0014] Furthermore, the antigen is a conjugate of a hapten and a carrier protein; the amino acid sequence of the hapten includes the sequence shown in SEQ ID NO:1, the sequence shown in SEQ ID NO:2 and the sequence shown in SEQ ID NO:3.

[0015] Preferably, the amino acid sequence of the antigen is amino acids 34 to 420 from the N-terminus of the vacuole toxin. This antigen can be obtained through recombinant expression.

[0016] Thirdly, the present invention provides the use of the said epitope peptide or the said antigen in the preparation of specific antibodies against Helicobacter pylori vacuolating toxin in feces.

[0017] Antigens containing DTV, DMK, or WRI peptides can trigger an immune response in animals, producing specific antibodies against VacA degradation products in feces.

[0018] Fourthly, the present invention provides a specific antibody against the said antigenic epitope peptide or the said antigen.

[0019] Fifthly, the present invention provides the use of the antigenic epitope peptide or the antigen or the specific antibody in the preparation of a diagnostic reagent for the diagnosis of Helicobacter pylori infection.

[0020] Preferably, the diagnostic reagent is used to detect feces.

[0021] Preferably, the diagnostic reagent is an ELISA test kit or a colloidal gold test strip.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] This invention provides three antigenic epitopes of vacuole toxin. By utilizing these epitopes, more specific antibodies against different antigenic epitopes of VacA can be obtained, thereby improving the accuracy of immunological methods in detecting VacA degradation products in feces and helping to achieve non-invasive and rapid detection of Helicobacter pylori. Attached Figure Description

[0024] Figure 1 It is a PCR product of amino acid fragment 34-420 of VacA protein.

[0025] Figure 2 It is the purified product of the full-length VacA protein.

[0026] Figure 3 It is a purified product of amino acid fragments from position 34 to 420 of the VacA protein.

[0027] Figure 4 This is a schematic diagram and an analysis of the test results for the colloidal gold reagent strip. Among them, Figure 4 (A) is a schematic diagram of a colloidal gold reagent strip. Figure 4 (B) is a graph showing the analysis of the test results. Detailed Implementation

[0028] The present invention will be further described below with reference to embodiments.

[0029] General Implementation Examples

[0030] An antigenic epitope peptide of Helicobacter pylori vacuolating toxin found in feces, wherein the amino acid sequence of the antigenic epitope peptide includes one or more of DTV, DMK, and WRI.

[0031] A fecal Helicobacter pylori vacuolating toxin antigen, wherein the antigenic epitopes include one or more of DTV, DMK, and WRI.

[0032] In one specific embodiment, the amino acid sequence of the antigen includes one or more of the sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0033] In one specific embodiment, the antigen is a conjugate of a hapten and a carrier protein; the amino acid sequence of the hapten includes one or more of the sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0034] In one specific embodiment, the amino acid sequence of the antigen is amino acid 34 to 420 from the N-terminus of the vacuole toxin.

[0035] The use of the antigenic epitope peptide or the antigen in the preparation of specific antibodies against Helicobacter pylori vacuolating toxin in feces.

[0036] A specific antibody against the said antigenic epitope peptide or the said antigen.

[0037] The application of the antigenic epitope peptide, the antigen, or the specific antibody in the preparation of a diagnostic reagent for the diagnosis of Helicobacter pylori infection.

[0038] In one specific implementation, the diagnostic reagent is used to detect feces.

[0039] In one specific embodiment, the diagnostic reagent is an ELISA test kit or a colloidal gold test strip.

[0040] Example 1: Recombinant expression of VacA protein in prokaryotic cells (including full-length protein and amino acid fragments from position 34 to 420) (1) Synthesis of full-length plasmid containing VacA:

[0041] Based on the human VacA DNA sequence provided by GenBank, a commercial company was commissioned to chemically synthesize the full-length VacA DNA sequence and clone it into a suitable cloning vector (such as pUC19, pUC57, etc.; pUC57 was used in this example).

[0042] The VacA-pUC57 and PET-28a expression vectors were digested with (BamHI / XhoHI), then recovered by 1% agarose gel electrophoresis and gel excision. The VacA fragment and PET-42a fragment were mixed at a ratio of 9:1 and ligated with T4 ligase. The ligation product was then transformed into freshly prepared competent cells and screened on Kana medium. Single colonies were selected for small-scale plasmid extraction, followed by double enzyme digestion and 1% agarose gel electrophoresis for preliminary identification. Finally, plasmids with the correct band positions were selected for sequencing verification, obtaining full-length plasmids containing VacA.

[0043] (2) Preparation of full-length VacA protein:

[0044] The obtained full-length VacA plasmid was transformed into DE3 and Rosetta cells, respectively. Colonies were picked and cultured at 37°C with shaking until the OD value reached 0.8. After adding glycerol, the bacterial strain was stored at -80°C. Another bacterial strain was cultured again with shaking. Different concentrations of IPTG and different temperature conditions were used for induction. After bacterial lysis, the supernatant and precipitate were collected and detected by SDS electrophoresis to screen for the optimal induction conditions.

[0045] The bacterial cells were collected after being cultured under the selected induction conditions (DE3, 37℃, 0.25mM IPTG, high expression of inclusion bodies).

[0046] Inclusion body refolding was performed using Novagen's inclusion body refolding kit. The specific procedure is as follows:

[0047] 1) Resuspend the bacterial cells on ice with 1× IB Wash Buffer;

[0048] 2) Add lysozyme to a final concentration of 100 μg / mL and incubate at 30°C for 15 minutes;

[0049] 3) Sonicate until the solution is no longer viscous;

[0050] 4) Centrifuge at 10000g for 10 minutes, then discard the supernatant.

[0051] 5) Resuspend in 1× IB Wash Buffer, centrifuge at 10000g for 10 minutes, and discard the supernatant;

[0052] 6) Repeat step 5) once;

[0053] 7) Weigh the contents and calculate the volume of the solution to bring the inclusion body concentration to 10–20 mg / mL;

[0054] 8) At room temperature, mix 5 mL of 10×IB Solubilization Buffer, 0.5 mL of 30 wt% N-lauroylsarcosine, 50 μL of 1 M DTT and 44.45 mL of deionized water to prepare a 50 mL solution;

[0055] 9) Add the solution to the inclusion body precipitate, mix by blowing and stirring, incubate at room temperature for 15 minutes, centrifuge at 10000g for 10 minutes, and collect the supernatant;

[0056] 10) Dialyze twice at 4°C for 3 hours each time using 1× Dialysis Buffer containing 0.01 wt% DTT solution;

[0057] 11) Dialyze twice with 1× Dialysis Buffer at 4°C, 3 hours each time;

[0058] 12) Dialyze overnight at 4°C using a solution of 1× Dialysis Buffer containing 1 mM reduced glutathione and 0.2 mM oxidized glutathione.

[0059] After refolding, the protein was purified using a nickel column, dialyzed against 30% glycerol, and then frozen. Protein quantification was performed using the BCA method (see purified product). Figure 2 ).

[0060] (3) Preparation of the amino acid fragment from position 34 to 420 of VacA:

[0061] Based on the mass spectrometry results, upstream and downstream primers were designed and synthesized. The DNA sequence of the VacA protein, specifically amino acid fragments 34–420 from the N-terminus (i.e., segments 100–1260 bp encoding the VacA protein), was amplified by PCR. The PCR product is shown below. Figure 1 Then, following steps (1) and (2) above, the recombinant expressed and purified amino acid fragment of VacA from position 34 to 420 was obtained (see purification product). Figure 3 The amino acid sequence from position 34 to 420 of VacA, starting from the N-terminus, is shown in SEQ ID NO:4, namely: AFFTTVIIPAIVGGIATGAAVGTVSGLLGWGLKQAEEANKTPDKPDKV WRI QAGKGFNEFPNKEYDLYRSLLSSKIDGGWDWGNAATHYWVKGGQWNKLEV DMK DAVGTYNLSGLRNFTGGDLDVNMQKATLRLGQFNGNSFTSYKDSADRTTRVDFNAKNILIDNFLEINNRVGSGAGRKASSTVLTLQASEGITSSKNAEISLYDGATLNLASNSVKLMGNVWMGRLQ YVGAYLAPSYSTINTSKVTGEVNFNHLTVGDHNAAQAGIIASNKTHIGTLDLWQSAGLNIIAPPEGGYKDKPKDKPSNTTQNNANNNQQNSAQNNSNTQVINPPNSAQKTEIQPTQVIDGPFAGGK DTV VNIDRINTNADGTIKVGGYKASLTTNAA.

[0062] Example 2: Preparation and screening of monoclonal antibodies against VacA degradation products in feces using full-length VacA (1) Preparation of monoclonal antibodies:

[0063] Using the purified full-length VacA protein from Example 1 as an immunogen, female Balb / c mice aged 6-8 weeks were immunized multiple times, and the best mouse spleens were selected for cell fusion to obtain hybridoma cells.

[0064] Screening for hybridoma cell lines secreting specific antibodies: Positive clones were first selected using recombinant full-length VacA protein expressed in prokaryotic cells. Then, sequential saturation mode ELISA blocking reactions were performed using either 0.9 wt% sodium chloride saline extract or 0.1 wt% Triton X-100 pH 7.4, 100 mM phosphate buffer extract from HP-positive patients. Antibodies that reacted positively with the full-length protein but negatively with the full-length protein after mixing with the fecal extract were identified as specifically recognizing human fecal VacA protein degradation products. Nine antibodies were ultimately screened and named 1D11, 1H12, 6A11, 8A1, 9C3, 13A2, 13A8, 13G7, and 19F10.

[0065] (2) Screening for antibodies against different epitopes:

[0066] The nine antibodies from step (1) were labeled with biotin and their titers were detected.

[0067] Nine antibodies were coated onto plates at a concentration of 10 μg / mL. After blocking, 100 μg / mL of full-length VacA protein was added as the primary antibody. After incubation and washing, eight other biotinylated antibodies were added. Finally, avidin-HRP antibody was added for detection. The results showed that antibodies 8A1, 1H12, and 6A11 were homologous; antibodies 9C3 and 13A2 were homologous; and antibodies 1D11, 13A8, 13G7, and 19F10 were homologous. Antibodies with different homologous homologs could pair with each other. This demonstrates that antibodies specifically recognizing human fecal VacA protein degradation products have three different binding sites.

[0068] Example 3: Epitope Identification of VacA Antigen

[0069] (1) Using the three monoclonal antibodies 8A1, 13A2, and 13G7 obtained in Example 2, affinity chromatography columns were prepared:

[0070] 1) Take 20 mg of the mixture of the above 3 monoclonal antibodies and dialyze overnight with 0.1 M sodium bicarbonate solution;

[0071] 2) Weigh 1.3g of CNBr-Activated agarose (SIGMA, catalog number C9210), soak it in purified water at pH 1.0 for 30 minutes, pack it into a column, wash for 5 column volumes, and then mix it with the dialyzed monoclonal antibody at room temperature for 3 hours.

[0072] 3) Wash with 100mM sodium bicarbonate + 300mM sodium chloride mixed solution (pH 8.3), 100mM sodium acetate + 300mM sodium chloride mixed solution (pH 4.0), and 100mM Tris + 300mM sodium chloride mixed solution (pH 8.0) as washing solutions for 3 cycles, washing 5 column volumes with each washing solution, and then sealing overnight.

[0073] (2) Mass spectrometry identification of polypeptide sequences:

[0074] 1) The full-length VacA protein obtained in Example 1 was enzymatically digested (mass spectrometry grade trypsin) for 4 hours, and an inhibitor (trypsin inhibitor, TI) was added to obtain artificially simulated VacA protein degradation products from human feces.

[0075] 2) Load the artificially simulated VacA protein peptide (obtained in step (1)) onto a column, wash with 100mM PBS at pH 7.4 for 5 column volumes, and then elute the bound peptide with 100mM glycine at pH 2.8. Keep the pH of the collection solution neutral during collection.

[0076] 3) Mass spectrometry analysis was performed on the collected peptides (Suzhou Putai Biotechnology Co., Ltd.).

[0077] (3) Epitope identification:

[0078] Based on the mass spectrometry detection results, the polypeptide sequences in Table 1 were designed and synthesized.

[0079] Table 1. Peptide sequences

[0080]

[0081]

[0082] Using full-length VacA protein to coat ELISA plates, a mixture of the three monoclonal antibodies 8A1, 13A2, and 13G7 obtained in Example 2 was reacted with the peptides in Table 1 and then added to a blocked ELISA plate. The results showed that peptides numbered 4, 5, 8, 9, 13, 14, 17, 21, 22, 23, and 26 could not be blocked, while all other peptides could be blocked. Therefore, it was inferred that the epitope of the VacA antigen in human feces is the three tandem amino acids DTV, DMK, or WRI. Furthermore, it was inferred that the amino acids of the poorly soluble Helicobacter pylori vacuole toxin VacA degradation products in feces are from amino acids 34 to 420 (i.e., the corresponding nucleotide sequences from 100 to 1260 bp).

[0083] Example 4: Preparation and specificity testing of specific monoclonal antibodies targeting the three epitopes DTV, DMK, and WRI

[0084] (1) Synthesize polypeptide-carrier protein conjugates:

[0085] Three peptides, DGPFAGGKDTVVNIDRINTKAD (SEQ ID NO:1), HYWIKGGQWNKLEVDMKDAVGTYKL (SEQ ID NO:2), and ANKTPDKPDKVWRIQAGKGKGFNID (SEQ ID NO:3), were synthesized using peptide synthesis technology, with HPLC purity exceeding 95%. The synthesized peptides were then directly conjugated to bovine serum albumin (BSA) and hemocyanin (KLH) using the bifunctional conjugate EDC, respectively, yielding three peptide-BSA and three peptide-KLH conjugates.

[0086] (2) Immunization of mice with peptide-BSA conjugate:

[0087] Five × three inbred female Balb / c mice aged 6–8 weeks were selected. Three different polypeptide-BSA conjugates were used as antigens. The initial immunization concentration was 1 mg / mL, and the immunization dose was 0.1 mL / mouse. The conjugates were emulsified with Freund's complete adjuvant and injected subcutaneously at multiple sites. For the second to fourth immunizations, the antigen concentration was adjusted to 0.5 mg / mL, and the immunization dose was 0.1 mL / mouse. The conjugates were emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple sites. Subsequent immunizations were spaced 14 days apart. 7–10 days after the fourth immunization, blood was collected from the submandibular vein of the immunized mice. Plasma was collected by centrifugation, and antibody titers were detected using ELISA with full-length VacA protein coated on a plate.

[0088] (3) Hybridoma cell fusion:

[0089] Mice with the highest serum titers were selected and intraperitoneally injected with the peptide-BSA conjugate antigen for stimulation. Three days later, spleen cells were harvested for cell fusion. Mice were euthanized by cervical dislocation under aseptic conditions, and the spleens were removed. After mechanical disruption, a spleen cell suspension was prepared and mixed with myeloma cells of the same lineage at a 1:1 ratio. A preheated fusion promoter was added, and the cells were transferred to xanthine-aminopterin-thymidine (HAT) selective medium for selection culture.

[0090] (4) ELISA clone screening:

[0091] The cells were coated overnight at 4°C with 20 μg / mL, 100 μL / well of the corresponding peptide-KLH conjugate. After washing 5 times with TBST, the cells were blocked with 10% fetal bovine serum for 1 hour. 50 μL of hybridoma cell culture supernatant was added and incubated at 37°C for 1 hour, followed by 5 washes. Goat anti-mouse-HRP was then added and incubated at 37°C for 30 minutes, followed by washing and drying. Finally, TMB chromogenic solution was added, and stop solution was added after 5 minutes of development. The OD value was detected at 450 nm, and positive clones were selected. 50 μL of the selected positive clone cell supernatant + 50 μL of 10% fetal bovine serum were used as controls, and 50 μL of the positive clone cell supernatant + 50 μL of HP-positive fecal lysis buffer were incubated together at 37°C for 1 hour. Then, both were added separately to the blocked ELISA plates and incubated at 37°C for 1 hour, followed by 5 washes. Goat anti-mouse-HRP was then added and incubated at 37°C for 30 minutes, followed by washing and drying. Finally, add TMB chromogenic solution, and after 5 minutes of chromogenic development, add stop solution. Detect OD value using a wavelength of 450nm. Cell lines that show significantly lower or no reaction after blocking the reaction with HP-positive fecal lysate compared to the control are the initially screened monoclonal antibodies that can secrete specific antibodies to recognize empty toxin degradation products in fecal samples.

[0092] By subcloning hybridoma cells of monoclonal antibodies (using limiting dilution), a cell line capable of producing highly efficient monoclonal antibodies against vacuole toxin degradation products in human feces can be obtained; after large-scale culture, the cells are cryopreserved. Three monoclonal antibodies targeting epitopes DTV, DMK, and WRI can be produced from these cell lines; these are named mAb-DTV, mAb-DMK, and mAb-WRI, respectively.

[0093] (5) Specificity test of monoclonal antibody:

[0094] The plates were coated with full-length VacA protein at a final concentration of 5 μg / mL, 100 μL / well, and incubated overnight at 4°C. After washing 5 times with TBST, the plates were blocked with 10% fetal bovine serum for 1 hour. At the same time, the mixture of the three monoclonal antibodies in step (4) was serially diluted from 1:50000 and mixed 1:1 with 0.5 mg / mL CagA protein, urease α and β subunits, heat shock protein and E. coli lysate, respectively. The mixture was incubated at 37°C for 1 hour. The mixture was then added to the blocked ELISA plates and incubated at 37°C for 1 hour. After washing 5 times, goat anti-mouse-HRP was added and the plates were incubated at 37°C for 30 minutes. The plates were then washed and patted dry. Finally, TMB chromogenic solution was added, and after 5 minutes of chromogenic development, stop solution was added. The OD value was detected at a wavelength of 450 nm. The results are shown in Table 2, which demonstrates that the monoclonal antibody has a cross-reactivity of <5% with other proteins of HP bacteria, such as CagA protein, α and β subunits of urease, and heat shock proteins, and no cross-reactivity with E. coli.

[0095] Table 2 Cross-reactivity between monoclonal antibodies and other proteins in HP

[0096]

[0097]

[0098] The monoclonal antibody mixture was diluted and incubated with HP-positive fecal lysate, and then reacted with a blocked ELISA plate. The experiment was repeated twice, and the OD values ​​were measured as shown in Table 3, which proved that this monoclonal antibody specifically binds to VacA protein in human feces.

[0099] Table 3. Monoclonal antibody-specific binding to VacA protein in human feces.

[0100]

[0101] Example 5: Preparation of rabbit polyclonal antibodies targeting amino acids 34-420 of VacA

[0102] (1) Preparation of polyclonal antibodies:

[0103] Two healthy, approximately 6-week-old White rabbits (about 2 kg each) were selected. After stabilization, blood was collected from their ears to serve as negative control serum. 0.5 mg of fully adjuvanted emulsified antigen (the antigen used was the purified amino acid fragment 34-420 from Example 1) was injected into the palmar and plantar surfaces of both rabbits. 7-10 days later, 0.5 mg of partially adjuvanted emulsified antigen was injected at multiple sites along both sides of the spine. This step was repeated twice, with an interval of 7-10 days between each injection. Seven days after the last injection, blood was collected for ELISA titer testing. Both rabbits showed titers above 100,000. Blood was collected from both rabbits, and antibodies were purified. The resulting rabbit polyclonal antibodies were labeled R1 and R2, respectively.

[0104] (2) HRP Coupling:

[0105] Take 5 mg of each of the two rabbit polyantibodies R1 and R2 obtained in step (1) and dialyze overnight with 0.01 M CBS at pH 9.6; weigh 5 mg of HRP powder, dissolve it in 1 mL of purified water, add 200 μL of 0.1 M sodium periodate, stir for 30 minutes in the dark, and dialyze overnight with 1 mM sodium acetate solution at pH 4.4; the next day, take the R1 and R2 antibodies after the ambush and mix them with HRP solution respectively, stir at room temperature for 2 hours, add 0.1 mL of freshly prepared 4 mg / mL sodium borohydride solution, stir at 4℃ for another 2 hours, precipitate with saturated ammonium sulfate, and dialyze with PBS to obtain R1-HRP conjugate and R2-HRP conjugate.

[0106] (3) Detection of rabbit polyclonal antibody-HRP titer:

[0107] The VacA amino acid fragments 34-420 purified in Example 1 were plated at a concentration of 10 μg / mL and incubated overnight at 4°C. The R1-HRP conjugate and R2-HRP conjugate obtained in step (2) were diluted 1:1000, and the rabbit polyclonal antibody R1-HRP titer was 1:2000 and R2-HRP titer was 1:4000.

[0108] Example 6: ELISA kit for detecting VacA degradation products in feces

[0109] The ELISA kit for detecting VacA degradation products in feces includes the following reagents:

[0110] 1) Microplates coated with a mixture of monoclonal antibodies targeting three different epitopes: The monoclonal antibodies were prepared in Example 4, and the microplates were transparent polystyrene microplates with flat bottoms at the bottom of the wells.

[0111] 2) Enzyme-labeled secondary antibody: Rabbit polyclonal antibody R2-HRP 1:4000 obtained in Example 5 was used;

[0112] 3) Standard: Full-length VacA protein obtained in Example 1, 4000 ng / vial;

[0113] 4) Colorimetric solution: 1 wt% 3,3',5,5'-tetramethylbenzidine solution;

[0114] 5) Termination solution: hydrochloric acid;

[0115] 6) Detergent: TBST;

[0116] 7) Sample diluent: 0.9 wt% sodium chloride solution containing 0.1 wt% Triton X-100.

[0117] The above-mentioned ELISA kit was used to detect VacA degradation products in feces. The specific steps are as follows:

[0118] A 0.1 wt% Triton X-100 solution with 0.9 wt% sodium chloride was used as the lysis buffer. The lysis buffer was mixed with the fecal sample at a mass-to-volume ratio of 3 mL: 1 g. The supernatant was added to the microplates (microplates coated with monoclonal antibodies) and incubated. Proteins that may be present in the sample bind to the monoclonal antibodies in the microplates. After washing the plate, enzyme-labeled secondary antibody was added, and the plate was incubated again and washed. Colorimetric solution was added, and stop solution was added after the reaction was completed. The sample was then detected using an ELISA reader. Finally, the content of vacuole toxin VacA degradation products in the sample was calculated using a standard curve.

[0119] Fifteen fecal samples were randomly selected for testing, and the content of VacA degradation products was determined according to the method described above. When plotting the standard curve, each standard concentration was measured three times. Each fecal sample was tested 2–3 times. The OD values ​​measured by ELSA at different standard concentrations when plotting the standard curve, the OD results of the samples measured by ELSA, and the VacA content in the samples calculated based on these results are shown in Tables 4–6 below.

[0120] Table 4. OD results of standards at different concentrations detected by ELSA method

[0121]

[0122] Table 5 ELSA method for detecting sample OD

[0123]

[0124] Table 6. VacA content in samples

[0125]

[0126] Accuracy verification:

[0127] 1) Blocking experiment:

[0128] Using the full-length VacA protein prepared in Example 1 to coat the plate, the three monoclonal antibodies obtained in Example 4 and the rabbit polyclonal antibody R2-HRP obtained in Example 5 were reacted with the lysate of human HP-positive fecal samples for 1 hour. Then, they were added as primary antibodies to the blocked plate, and after adding enzyme-labeled antibody, the plate was developed. The result was completely white, which proved that the prepared monoclonal antibodies and polyclonal antibodies could specifically recognize VacA degradation antigens in feces.

[0129] 2) Recovery rate test:

[0130] Recovery rates are used to determine whether analyte detection is affected by differences in the sample matrix. The matrix (such as fecal lysate) can be an interfering factor, affecting the ability of ELISA to accurately quantify analytes. Recovery rates are calculated by spiked known amounts of analyte into fecal lysate and then performing the detection. Generally, if the average recovery rate is 80%–120%, the sample matrix is ​​considered to have minimal impact on the ability to accurately quantify analytes. The results, shown in Table 7, indicate that the recovery rates of this ELISA kit all fall within the range of 80%–120%, suggesting that the fecal lysate has a relatively small impact on accurate analyte quantification.

[0131] Table 7. Recovery rate test results of the ELISA kit.

[0132]

[0133] Example 7: Colloidal gold reagent strip for detecting VacA degradation products in feces

[0134] The specific steps for preparing a colloidal gold reagent strip for detecting VacA degradation products in feces are as follows:

[0135] One end of the PVC backing was sequentially glued with absorbent paper and a glass fiber membrane, with a nitrocellulose membrane attached in the middle, and absorbent fiber attached to the other end. The binding pad contained a mixture of three monoclonal antibodies targeting different epitopes obtained in Example 4, labeled with colloidal gold. The T-line of the NC membrane contained the purified rabbit polyclonal antibody R2-HRP obtained in Example 5. The C-line of the NC membrane was coated with a secondary antibody that specifically binds to the gold-labeled antibody, located 5 mm from the T-line (see...). Figure 4 (A)). Then cut the PVC material into strips about 5mm wide, assemble them into the base groove of the plastic card, dry them, and seal them for storage.

[0136] The above-mentioned colloidal gold reagent strip was used to detect VacA degradation products in feces. The specific steps are as follows:

[0137] Fifteen fecal samples were selected, and a 0.1 wt% Triton X-100 solution with 0.9 wt% sodium chloride was used as the lysis buffer. The lysis buffer was mixed with the fecal sample at a mass-to-volume ratio of 3 mL: 1 g. The supernatant was then added dropwise to the sample pad, and the reaction was allowed to proceed for 15 minutes. The results were observed. A schematic diagram of negative, positive, and invalid samples is shown below. Figure 4 (B). The test results are shown in Table 8 below, where “–” indicates a negative result and “+” indicates a positive result, and the more “+” signs there are, the stronger the color development.

[0138] Table 8. Results of Colloidal Gold Detection in Fecal Samples

[0139] First experiment Second experiment Sample 1 + + Sample 2 ++ ++ Sample 3 + + Sample 4 +++ +++ Sample 5 + + Sample 6 + + Sample 7 ++ ++ Sample 8 + + Sample 9 ++ +++ Sample 10 +++ +++ Sample 11 – – Sample 12 – – Sample 13 – – Sample 14 – – Sample 15 – –

[0140] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0141] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A fecal Helicobacter pylori vacuolating toxin antigen, characterized in that, It includes antigen A, antigen B and antigen C; antigen A, antigen B and antigen C are all conjugates of haptens and carrier proteins; the amino acid sequence of the hapten of antigen A is shown in SEQ ID NO:1, the amino acid sequence of the hapten of antigen B is shown in SEQ ID NO:2 and the amino acid sequence of the hapten of antigen C is shown in SEQ ID NO:

3.

2. The antigen as described in claim 1, characterized in that, The carrier protein in antigen A is bovine serum albumin.

3. The antigen as described in claim 1, characterized in that, The carrier protein in antigen B is bovine serum albumin.

4. The antigen as described in claim 1, characterized in that, The carrier protein in antigen C is bovine serum albumin.

5. The antigen as described in claim 1, characterized in that, The carrier protein in antigen A is hemocyanin.

6. The antigen as described in claim 1, characterized in that, The carrier protein in antigen B is hemocyanin.

7. The antigen as described in claim 1, characterized in that, The carrier protein in antigen C is hemocyanin.

8. The use of the antigen as described in any one of claims 1 to 7 in the preparation of specific antibodies against Helicobacter pylori vacuolating toxin in feces.

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