Kit for detecting helicobacter pylori CagA subtype and application thereof

Abstract

According to the kit for detecting the helicobacter pylori CagA subtype and the application of the kit, 3E67 and 5T68 monoclonal antibodies are prepared by using CagA dominant immune epitopes, and the monoclonal antibodies are used in a homogeneous chemiluminescence system for the first time to prepare an antigen for detecting the helicobacter pylori CagA subtype. A murine antibody is prepared through immunization of dominant epitopes of helicobacter pylori CagA protein, an antibody pair with good specificity and strong binding force is obtained through an in-vitro screening pairing strategy, and amino acid sequences of a heavy chain variable region and a light chain variable region of the antibody pair are obtained through sequencing. Products for detecting the helicobacter pylori-related CagA antigen are developed on the basis of a chemiluminescence platform, an enzyme-linked immunosorbent assay platform and an immune layer system platform, and the kit has the advantages of high sensitivity, good specificity and rapid detection.

Description

Technical Field

[0001] The invention belongs to the field of medical detection and relates to a kit for detecting Helicobacter pylori CagA subtype in gastric mucosal tissue and application thereof. Background Art

[0002] Helicobacter pylori (HP), first discovered by Barry J. Marshall and J. Robin Warren, is the only known microorganism capable of surviving in the human stomach and is classified as a Group 1 carcinogen by the World Health Organization's International Agency for Research on Cancer. It is a bacterium that can grow and multiply in the human body and is transmitted through oral-oral-fecal-oral routes. Globally, HP infection rates are nearly 60%. In China, the average infection rate is as high as 53%, representing nearly 700 million people. The main symptoms of H. pylori infection include acid reflux, heartburn, stomach pain, and bad breath. This is because H. pylori infection triggers excessive gastrin secretion, resulting in acidity and heartburn. For patients with gastric ulcers, the primary symptom is stomach pain. H. pylori can also cause chronic gastritis, the main clinical manifestations of which include upper abdominal discomfort and dull pain, sometimes accompanied by belching, acid reflux, nausea, and vomiting. The disease progresses slowly but is prone to recurrence. When patients are infected with Helicobacter pylori, they will induce the production of multiple pathogenic factors, thereby causing damage to the gastric mucosa. The occurrence of clinical diseases is diverse, and patients often experience acid reflux, belching, bloating, etc.

[0003] There are many methods for detecting Helicobacter pylori infection, including direct bacterial examination, urease activity measurement, immunological testing, and polymerase chain reaction. However, these detection methods have their limitations. The most obvious one is that even if the test result is positive, it cannot confirm whether there is a virulent strain infection, which makes it difficult to accurately use clinical medication: 1. C-14 Breathalyzer + Breathalyzer Bag: It requires only five minutes of blowing and is pain-free. It is currently one of the ideal testing methods and the gold standard in the medical field for HP testing. A positive test indicates HP infection, but it does not confirm whether the patient has the pathogenic HP. Approximately one in six patients infected with Helicobacter pylori may develop peptic ulcer disease, so prescribing medication based on a positive test is irrational. The complete genome sequence of Helicobacter pylori has been determined. Type I contains the CagA and VacA genes and expresses two proteins. Type I is currently considered to be closely associated with gastric diseases. A negative result for CagA and VacA antigens indicates infection with a non-virulent strain, while a positive result indicates infection with a virulent strain. Therefore, a positive result for CagA and VacA antigens is crucial in determining whether a patient needs clinical medication.

[0004] 2. Immunological testing: Currently, there are multiple immunological testing methods that detect Helicobacter pylori infection by measuring Helicobacter pylori antibodies in serum, including complement fixation tests, agglutination tests, passive hemagglutination assays, immunoblotting techniques, and enzyme-linked immunosorbent assays (ELISAs). Currently, the main clinical test is for HP antibodies. A positive HP antibody test only indicates that the patient has been infected with HP and does not confirm whether the infection is a virulent strain. Therefore, HP antibody testing has limited clinical significance.

[0005] 3. Polymerase chain reaction (PCR): Detecting HP nucleic acid in gastric mucosal tissue is highly sensitive and can confirm HP infection, but it cannot confirm whether the infection is a virulent strain. PCR testing also requires advanced technology and a demanding laboratory environment, making it challenging.

[0006] Therefore, there is an urgent need for new technologies and methods to detect new markers of Helicobacter pylori for rapid clinical diagnosis and treatment guidance. To address the above-mentioned difficulty in diagnosing Helicobacter pylori infection, the present invention has developed a rapid detection kit for Helicobacter pylori-related CagA subtype antigens in gastric mucosal tissue. Summary of the Invention

[0007] This invention utilizes the dominant antigenic epitope of the Helicobacter pylori CagA protein to immunize and generate murine antibodies. Through an in vitro screening and pairing strategy, a pair of antibodies with excellent specificity and strong binding affinity is obtained, and the amino acid sequences of their heavy and light chain variable regions are sequenced. This invention is the first to develop a product for detecting Helicobacter pylori-associated CagA antigens based on a chemiluminescence platform, offering the advantages of high sensitivity, good specificity, and rapid detection.

[0008] Furthermore, the first aspect of the present invention provides a monoclonal antibody pair (antibody 3E67 and antibody 5T68) or antigen-binding fragment involved in the kit, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region: the heavy chain variable region VH comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region VL comprises LCDR1, LCDR2 and LCDR3; Specifically, the amino acid sequence of the dominant antigenic epitope of the selected Helicobacter pylori CagA protein is shown in SEQ ID NO: 21: MTNETIDQQPQTEAAFNPQQFINNLQVAFLKVDNAVASYDPDQKPIVDKNDRDNRQAFDGISQLREEYSNKAIKNPAKKNQYFSDFINKSNDLINKDALIDVESSTKSFQKFGDQRYRIFTSWVSHQNDPSKINTRSIRDFMEHTIQPPIPDDK EKAEFLKSAKQSFAGIIIGNQIRTDQKFMGVFDESLKERQEAEKNGGPTGGDWLDIFLSFIFDKKQSSDVKEAINQEPVPHVQPDIATSTTHIQGLPPESRDLLDERGNFSKFTLGDMEMLDVEGVADMDPNYKFNQLLIHNNTLSSVLMGSHNG The two antibodies obtained are antibody 3E67 and antibody 5T68.

[0009] The amino acid sequence of the heavy chain variable region of antibody 3E67 is shown in SEQ ID NO: 1: DMSVESGGGLVKPGGSLKLSCAASGFTFN SYAMS WVRQTPEKRLEWVA SSGGNTYYPDSVKG RFTISRDNARNIYLQMSSLRSEDTAMYYCAC GFESPYDTAMYYFDY WGQGTTARASWL; The amino acid sequence of HCDR1 is shown in SEQ ID NO:9: SYAMS , the amino acid sequence of HCDR2 is shown in SEQ ID NO: 10: SSGGNTYYPDSVKG , the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11: GFESPYDTAMYYFDY ; The amino acid sequence of the light chain variable region of antibody 3E67 is shown in SEQ ID NO: 2: VIEMTQSPASLAVSLGQRTTISY RASKSVSTSGYSYMH WNQQKPGQPPRLLY LVSNLES GVPARFGGSGSGTDFTLNIHPVEEEDTAT YYCQHIREL TRSEGGPSWLT; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 12: RASKSVSTSGYSYMH , the amino acid sequence of LCDR2 is shown in SEQ ID NO: 13: LVSNLES , the amino acid sequence of LCDR3 is shown in SEQ ID NO: 14: YYCQHIREL ; The amino acid sequence of the heavy chain variable region of the antibody 5T68 is shown in SEQ ID NO: 3: QIMESGPDLVKPSQSLTCIVIGYSIT SGYNWHN WIRQFPGYGTEWMG YIHYRGTSSYNISLKS RISITRDSTISSPLSVTTTAYYCAC ADDFYSRDY MGQGTIVIVSSED; The amino acid sequence of HCDR1 is shown in SEQ ID NO: 15: SGYNWHN , the amino acid sequence of HCDR2 is shown in SEQ ID NO: 16: YIHYRGTSSYNISLKS , the amino acid sequence of HCDR3 is shown in SEQ ID NO: 17: ADDFYSRDY ; The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4: ELVMTMSASPGEKVTMTC SASSSGVSYMNH WYQQKSSPKRWIY DTSRKLPSSER GVPGRFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPTER FGAGEVKQIPITKLE; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 18: SASSSGVSYMNH , the amino acid sequence of LCDR2 is shown in SEQ ID NO: 19: DTSRKLPSSER , the amino acid sequence of LCDR3 is shown in SEQ ID NO: 20: QQWSSNPPTER。

[0010] Furthermore, the nucleotide sequence of the heavy chain variable region of antibody 3E67 is shown in SEQ ID NO: 5: Gacatgagcgtggagagcggcggcggcctggtgaagcccggcggcagcctgaagctgagctgcgccgccagcggcttcaccttcaacagctacgccatgagctgggtgagacagacccccgagaagagactggagtgggtggccagcagcggcggcaacacctactaccccgacagcgtgaagggcagattcaccatcagcagagacaacgccagaaacatctacctgcagatgagcagcctgagaagcgaggacaccgccatgtactactgcgcctgcggcttcgagagcccctacgacaccgccatgtactacttcgactactggggccagggcaccaccgccagagccagctggctg; And the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO: 6: gtgatcgagatgacccagagccccgccagcctggccgtgagcctgggccagagaaccaccatcagctacagagccagcaagagcgtgagcaccagcggctacagctacatgcactggaaccagcagaagcccggccagccccccagactgctgtacctggtgagcaacctggagagcggcgtgcccgccagattcggcggcagcggcagcggcaccgacttcaccctgaacatccaccccgtggaggaggaggacaccgccacctactactgccagcacatcagagagctgaccagaagcgagggcggccccagctgg; The nucleotide sequence of the heavy chain variable region of antibody 5T68 is as shown in SEQ ID NO: 7: Cagatcatggagagcggccccgacctggtgaagcccagccagagcctgacctgcatcgtgatcggctacagcatcaccagcggctacaactggcacaactggatcagacagttccccggctacggcaccgagtggatgggctacatccactacagaggcaccagc agctacaacatcagcctgaagagcagaatcagcatcaccagagacagcaccatcagcagccccctgagcgtgaccaccaccgcctactactgcgcctgcgccgacgacttctacagcagagactacatgggccagggcaccatcgtgatcgtgagcagcgaggac The nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 8: gagctggtgatgaccatgagcgccagccccggcgagaaggtgaccatgacctgcagcgccagcagcagcggcgtgagctacatgaaccactggtaccagcagaagagcagccccaagagatggatctacgacaccagcagaaagctgcccagcagcgagagaggcgtgccc ggcagattcagcggcagcggcagcggcaccagctacagcctgaccatcagcagcatggaggccgaggacgccgccacctactactgccagcagtggagcagcaacccccccaccgagagattcggcgccggcgaggtgaagcagatccccatcaccaagctggagctgacc.

[0011] The present invention first screens the dominant antigenic epitopes of CagA, intercepts and constructs recombinant peptide segments, and uses them as immunogens to prepare mouse-specific IgG antibodies. The ELISA platform is used to screen antibody pairs with high titer and good pairing affinity in vitro for kit development.

[0012] Furthermore, the kit is a chemiluminescence kit, an enzyme-linked immunosorbent assay kit, or an immunochromatography kit.

[0013] Among them, the chemiluminescence kit is a chemiluminescence kit for the NMHL reaction system, including an enzyme marker, a biotin marker, an auxiliary agent, an acridinium ester derivative biomarker and a calibrator.

[0014] The calibrator contains CagA and 0.1 M phosphate buffer; The enzyme marker is peroxidase-labeled streptavidin and 0.05M phosphate buffer; The biotin marker is biotin-labeled anti-CagA monoclonal antibody 3E67 and 0.05M phosphate buffer; The acridinium ester derivative label is an anti-CagA monoclonal antibody 5T68 labeled with an acridinium ester derivative and 0.05M Tris buffer; The auxiliary agent includes a luminescence auxiliary agent and a citrate buffer; The substrate solution includes H2O2 and 0.05M Tris buffer.

[0015] The ELISA kit includes an ELISA plate, antibody 3E67, mouse anti-human HRP, a colorimetric solution, a stop solution, a diluent, a washing solution, and a standard. The plate is coated with antibody 5T68. The concentration of antibody 3E67 is 1 μg / ml to 5 μg / mL, and the concentration of antibody 5T68 is 1 μg / ml to 5 μg / mL.

[0016] The immunochromatographic kit includes a test card, which includes: a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper; the sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper are sequentially overlapped and adhered to the base plate; the conjugate pad is sprayed with a tracer marker labeled with antibody 3E67, a C line, and a T line, wherein the C line is fixed with mouse anti-human IgG antibody, and the T line is fixed with antibody 5T68.

[0017] Another object of the present invention is to provide the use of the above-mentioned kit for detecting Helicobacter pylori CagA subtypes in the preparation of products for detecting Helicobacter pylori CagA subtypes.

[0018] It should be noted that the antibody pair of the present invention can also be used in various kits known in the art for the detection of Helicobacter pylori CagA antigen.

[0019] The samples applicable to the present invention include human fecal samples, or digestive tract secretions and tissue samples.

[0020] The beneficial technical effects achieved by the present invention include a kit for detecting Helicobacter pylori CagA subtypes and its application. Mouse antibodies are prepared by immunization using dominant antigenic epitopes of the Helicobacter pylori CagA protein. Through an in vitro screening and pairing strategy, a pair of antibodies with high specificity and strong binding affinity is obtained, and the amino acid sequences of the heavy and light chain variable regions are sequenced. The present invention develops a product for detecting Helicobacter pylori-associated CagA antigens based on chemiluminescence, enzyme-linked immunosorbent assay, and immunohistochemistry platforms, offering the advantages of high sensitivity, good specificity, and rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The purification results of the CagA recombinant antigen in Example 1 of the present invention; wherein, B: bacterial solution before induction; A: bacterial solution after induction; E1-5: eluted target protein; M: protein maker; Figure 2 The purification results of the mouse anti-CagA IgG monoclonal antibody 3E67 in Example 1 of the present invention, wherein B: bacterial solution before induction; A: bacterial solution after induction; E1-4: eluted target protein; M: protein maker; Figure 3 Purification results of mouse anti-CagA IgG monoclonal antibody 5T68 in Example 1 of the present invention, B: bacterial solution before induction; A: bacterial solution after induction; E1-4: eluted target protein; M: protein maker; Figure 4 The standard curve (linear range) in specific embodiment 2 of the present invention. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0023] The present invention is further described below with reference to the embodiments. Example 1

[0024] (I) Plasmid construction First, (a) plasmid construction First, the dominant antigen epitope segment 1-309AA (Uniprot: T2SZC2) of CagA was screened and intercepted, and its amino acid sequence is shown in SEQ ID NO:21. The dominant antigen epitope segment was optimized into a nucleotide sequence according to the Escherichia coli codon principle, and then gene synthesis was completed (completed by GenScript Biotech).

[0025] The double enzyme digestion method was used to connect BamHI and EcoRI to the pGEX expression vector to construct an expression plasmid. 80-100 ng of the expression plasmid was extracted and added to DH5α competent cells. The competent cells after the addition of the plasmid were then heat-shocked at 42°C for 90 seconds. The transformed competent cells were added to LB liquid culture medium and cultured on a shaker at 37°C and 200 rpm for about 30 minutes. The suspension was then aspirated and spread on a plate containing ampicillin resistance and cultured in a 37°C incubator overnight. A single clone was then picked from the culture plate and added to 2-5 mL of LB culture medium, cultured at 37°C and 200 rpm for 8 hours, the bacterial solution was collected, centrifuged, the supernatant was removed, and the expression plasmid was extracted according to the instructions of the plasmid extraction kit produced by Tiangen Company.

[0026] (2) Antigen expression Transform the verified recombinant expression plasmid pGEX-CagA-GST into the BL21 strain. Spread the plate onto a pre-prepared LB plate containing 50 μg / ml kanamycin and invert the plate at 37°C. After incubation for 12–16 hours, isolate a single colony and expand it into liquid culture at 37°C and 230 rpm overnight. Inoculate the plate with a larger volume of culture and shake until the OD600 reaches 0.6–0.8. Induce expression by adding IPTG to a final concentration of 0.3 mM. Shake at 37°C and 230 rpm for 4 hours. Collect the cells by centrifugation at 4000 rpm for 40 minutes.

[0027] (3) Antigen purification Resuspend the cells in buffer 1, sonicate on ice for 20 minutes, and centrifuge at 12,000 rpm for 20 minutes at 4°C to collect the supernatant. The molecular weight of pGEX-CagA-GST is approximately 32.8 kDa. The buffer contains 150 mM Tris, 150 mM NaCl, 1 mM PMSF, and 0.1 M DTT at pH 7.5. Figure 1 shown.

[0028] The expressed antigens were bound to a GST column. After washing away impurities with buffer 2 (50mM Tris, 150mM NaCl, pH 8.0), the GST tag was removed on the column using PreScission enzyme. The target protein was eluted in batches and collected, and the peptides were verified by SDS-PAGE.

[0029] (IV) Immunogen preparation Prepare an EDC reagent with a final concentration of 1 M and dissolve it in coupling buffer (0.1 M MES pH 4.7) with a final concentration of carrier protein KLH of 10 mg / mL. Dissolve the purified antigen collected above in coupling buffer at a concentration of 4 mg / mL, add carrier protein, and the molar ratio of antigen protein to carrier protein is 10:1. Under magnetic stirring, slowly add EDC solution dropwise. The molar amount of EDC solution added is basically the same as that of the polypeptide. Dialysis and filtration are used to remove uncoupled antigen and EDC solution, and 1× PBS solution is added.

[0030] (5) Animal immunization The antigen was emulsified with Freund's complete adjuvant and five SPF BALB / c mice were selected and injected subcutaneously at 50µg / mouse. In the second and third weeks, the antigen emulsified with Freund's incomplete adjuvant was injected subcutaneously at 50µg / mouse. In the fourth week, the antigen protein solution was injected for boosting immunization.

[0031] Before each immunization, blood was collected from the tail vein to detect changes in serum antibody levels; on the fifth day after the last immunization, blood was collected from the mouse eyeballs, the blood was collected and allowed to stand until the serum was completely separated, and the serum was centrifuged at 3000 rpm for 5 minutes and packaged into aliquots and frozen at -70°C for later use.

[0032] (VI) Monoclonal antibody screening and preparation After immunization, BALB / c mice were eye-removed and blood was collected to serve as a positive control serum. Mice were then sacrificed by cervical dislocation and disinfected with 75% alcohol. The spleens were removed and a splenocyte suspension (counted under a microscope) was prepared. The suspension was mixed with SP2 / 0 myeloma cells (counted under a microscope) in serum-free DMEM medium at a ratio of 5:1. The cells were centrifuged at 2000 rpm for 5 minutes, the supernatant removed, and the suspension was resuspended. Preheated 50% PEG4000 was immediately added to fuse the cells. After 1 minute of fusion, serum-free DMEM was added to terminate fusion. The suspension was allowed to stand at 37°C for 10 minutes, centrifuged, the supernatant removed, and the cells resuspended in HAT medium. The cells were aliquoted into 96-well plates and cultured in a cell culture incubator for approximately 10 days until the fused cells covered 20%-50% of the well bottom. Positive clones were screened by indirect ELISA.

[0033] The antigen collected in step (3) of the coating was used, and HRP-labeled goat anti-mouse IgG was used as the secondary antibody. Serum collected after enucleation of immunized mice served as a positive control, and supernatant from cultured myeloma SP2 / 0 cells served as a negative control. A total of eight anti-CagA antibodies were obtained.

[0034] (VII) Antibody pairing The eight antibodies obtained were screened using the ELISA double antibody sandwich method, and a pair of hybridoma cells was finally identified. The selected hybridoma cell pair was subcloned continuously using the limiting dilution method until the positive rate of the subcloned cells reached 100%, and then the cells were expanded and cultured.

[0035] (8) Antibody purification The screened hybridoma cells were cultured on a large scale in DMEM medium supplemented with 20% calf serum using the cell roller bottle culture method. The culture conditions were: 37°C, 220rpm, 5% CO2 to prepare antibody IgG. When the cell survival rate was less than 50%, the culture medium was removed and collected into a centrifuge bottle. The supernatant was collected by centrifugation at 12000rpm for 10 minutes. Impurities were removed by filtration through a 0.45μm filter membrane. After purification by Protein A affinity chromatography column, the protein properties were determined by SDS-PAGE electrophoresis. After confirmation, it was dialyzed into 1×PBS pH7.4 buffer, sampled, and sequenced (sent to GenScript). Two antibodies were obtained: monoclonal antibody 3E67, such as Figure 2 As shown, and monoclonal antibody 5T68, as Figure 3 shown.

[0036] The amino acid sequences of the two antibodies are as follows: Antibody 3E67: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; Antibody 5T68: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 3; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; The sequence was reverse translated and codon optimized for recombinant expression, and the nucleotide sequence obtained was as follows: Antibody 3E67: The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 5; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 6; Antibody 5T68: The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 7; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 8. Example 2

[0037] The two antibodies obtained are used to develop and prepare a detection kit for Helicobacter pylori CagA antigen. The kit comprises a calibrator, an enzyme marker, a biotin marker, an acridinium ester derivative marker, an auxiliary agent, and a substrate solution.

[0038] (I) Reagent components are shown in Table 1 Table 1

[0039] (2) Preparation process 1. Preparation of Calibrators 1.1 Preparation of calibrator diluent: The calibrator diluent can be prepared using conventional techniques without special requirements.

[0040] 1.2 Preparation of calibrators and negative and positive quality controls Dilute the purified CagA calibrator with calibrator diluent to concentrations of 0, 5.0 ng / mL, 10.0 ng / mL, 50.0 ng / mL, 100.0 ng / mL, 200.0 ng / mL, and 500.0 ng / mL, respectively. Store at 2-8°C until ready for use.

[0041] Negative control: Add 3% calf serum to the calibrator diluent.

[0042] Positive quality control: Weak positive: Dilute the CagA calibrator with calibrator diluent to a concentration of 0.5 ng / mL.

[0043] Strong positive: Dilute the CagA calibrator with calibrator diluent to a concentration of 5.0 ng / mL.

[0044] 2. R1: Preparation of enzyme markers 2.1 1 mg of HRP was reacted with 0.1 ml of 60 mmol / L NaIO4 at 4°C for 30 minutes. 0.1 ml of 0.16 mol / L ethylene glycol was then added. 30 minutes later, 1 mg of SA was added. The solution was dialyzed at 4°C for 24 hours to obtain an HRP-SA solution.

[0045] 2.2 Add an equal volume of saturated ammonium sulfate solution and centrifuge at 4000 rpm for 15 min. Remove the supernatant and dissolve the precipitate in PBS (pH 7.4). Measure the absorbance at 280 nm.

[0046] 2.3 Pack a Sephadex G-75 column and load 100 μL of HRP-SA. Elute with 0.025 mol / L KCl-0.2 mol / L acetate buffer at 0.5 ml / 2 min, then collect the sample. Measure the OD280 value of the collected sample using a DU800 UV spectrophotometer, plot the chromatogram (elution curve), and collect the single white peak based on this value. Concentrate the dialysate with PEG-2000.

[0047] 2.4 Take 10 μl of HRP-SA and add 10 times the volume of deionized double-distilled water. Scan the UV spectrophotometer in the wavelength range of 200-280 nm. HRP-SA has an absorption peak at 280 nm.

[0048] 3. Reagent 2: Biotinylated Antibody 3E67 Dilute the antibody 3E67 solution with 0.1 mol / L sodium bicarbonate buffer (pH 8.0) or 0.5 mol / L borate buffer (pH 8.6) to a concentration of 1 mg / mL. Dialyze the antibody 3E67 solution alternately with 0.1 mol / L sodium bicarbonate buffer (pH 8.0) or 0.5 mol / L borate buffer (pH 8.6). Dissolve 1 mg of NHSB in 1 mL of DMSO to obtain an NHSB solution. Add 120 μL of NHSB solution to 1 mL of the 3E67 solution. Stir continuously at room temperature for 2-4 hours. Add 9.6 μL of 1 mol / L NH₄⁻¹ and stir at room temperature for 10 minutes. Dialyze the solution thoroughly against PBS at 4°C to remove free biotin. Apply the solution to a 1 mL molecular sieve column and slowly elute with PBS. Collect the solution (1 mL / tube); the protein elutes when the concentration is between 1 and 3 mL. Add 50% redistilled glycerol to the eluate and store at 20°C.

[0049] 4. R3: Preparation of acridinium ester derivatives: Conventional techniques may be used for preparation, which is not limited in this application. The preparation may be carried out using the method disclosed in the applicant's published patent document (201811115054.7); 5. R4: Preparation of auxiliary agents: Conventional techniques may be used for preparation, which is not limited in this application. The preparation may be carried out using the method disclosed in the applicant's published patent document (201811115054.7); 6. R5: Preparation of substrate solution: Conventional techniques may be used for preparation, which is not limited in this application. The method disclosed in the applicant's published patent document (201811115054.7) may be used for preparation; 7. Preparation of gastric mucosal tissue treatment solution: 0.5M EDTA buffer containing 0.9% NaCl.

[0050] (3) Testing steps 1. Endoscopically obtain approximately 0.5 g of gastric mucosal tissue and place it in a centrifuge tube pre-filled with 1.0 mL of tissue processing solution. Mix thoroughly and store at 2-8°C. Detect within 2 weeks.

[0051] 2. Sample addition: Add 20uL of calibrator, positive and negative quality control, tissue extraction sample, R1: 40uL, R2: 20uL, R3: 20uL, R4: 5uL into the reaction tube and shake to mix; 3. Reaction: Incubate at 37°C for 5 minutes; 4. Detection: Add 75uL of R5, shake and mix, detect immediately, and read the signal value.

[0052] 5. Calculation: Perform a logistic four-parameter fit on the concentration and luminescence value of the calibrator, and calculate the sample concentration using the sample RLU.

[0053] 6. The results are judged as follows Figure 4 As shown: 6.1 Linear range: 0~500ng / mL.

[0054] 6.2 Detection sensitivity: 0.1ng / m3.

[0055] 6.3 Coefficient of variation: <5%.

[0056] 6.4 The quality control of yin and yang meets the requirements.

[0057] It should be noted that: Negative and positive judgment criteria: expressed by negative and positive results of CagA antigen.

[0058] Positive: the ratio of sample OD value to cut-off OD value is greater than 1; Negative: the ratio of sample OD value to cut-off OD value is less than 0.9; Gray area: The ratio of the sample OD value to the cut-off OD value is between 0.9 and 1.0.

[0059] 7. Clinical interpretation of the results: CagA antigen positive: infection with CagA subtype strain.

[0060] CagA antigen negative: no infection with CagA subtype strains.

[0061] CagA antigen gray zone: combined with clinical diagnosis, or regular review. Example 3

[0062] The enzyme-linked immunosorbent assay kit included an ELISA plate, antibody 3E67 (concentration ranged from 1 μg / ml to 5 μg / mL), mouse anti-human HRP, a colorimetric solution, a stop solution, a diluent, a washing solution, and a standard. The ELISA plate was coated with antibody 5T68 (concentration ranged from 1 μg / ml to 5 μg / mL). A double-antibody sandwich ELISA kit was prepared using conventional methods. The concentrations of antibody 3E67 were 2 μg / mL and antibody 5T68 were 2.4 μg / mL. The kit was used to test stool samples and digestive tract tissue samples. The results are shown in Table 2.

[0063] An immunochromatographic test kit includes a test card comprising a PVC base, a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper. The sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper are sequentially overlapped and affixed to the base. The conjugate pad is sprayed with a tracer marker labeled with the antibody 3E67, a control line (C line), and a test line (T line). The C line is immobilized with mouse anti-human IgG antibody, and the T line is immobilized with the antibody 5T68. Preferably, the tracer marker can be a nanoparticle, such as colloidal gold, latex microspheres, or fluorescent microspheres. A double-antibody sandwich immunochromatographic test kit was prepared using conventional methods. The concentrations of the antibody 3E67 and the antibody 5T68 were 1.2 μg / mL and 1.5 μg / mL, respectively. The kit was used to test stool samples and digestive tract tissue samples. The results are shown in Table 1.

[0064] Table 2 Test results of stool samples

[0065] Note: "+++" indicates strongly positive, "++" indicates moderately positive, "+" indicates weakly positive, and "-" indicates negative.

[0066] The present invention has been disclosed above with preferred embodiments, but it is not intended to limit the present invention. Or the technical solutions obtained by equivalent transformation solutions all fall within the protection scope of the present invention.

Claims

1. A kit for detecting Helicobacter pylori CagA subtype, characterized in that: The kit comprises: An antibody or antigen-binding fragment that binds to the CagA subtype antigen characteristics; the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region: the heavy chain variable region VH comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region VL comprises LCDR1, LCDR2, and LCDR3; the antibody or antigen-binding fragment is: Antibody 3E67: the amino acid sequence of HCDR1 is shown in SEQ ID NO:9, the amino acid sequence of HCDR2 is shown in SEQ ID NO:10, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:11; the amino acid sequence of LCDR1 is shown in SEQ ID NO:12, the amino acid sequence of LCDR2 is shown in SEQ ID NO:13, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:14; and Antibody 5T68: The amino acid sequence of HCDR1 is shown in SEQ ID NO:15, the amino acid sequence of HCDR2 is shown in SEQ ID NO:16, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:17; the amino acid sequence of LCDR1 is shown in SEQ ID NO:18, the amino acid sequence of LCDR2 is shown in SEQ ID NO:19, and the three primary color sequences of LCDR3 are shown in SEQ ID NO:

20.

2. The kit for detecting Helicobacter pylori CagA subtype according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of antibody 3E67 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region of antibody 5T68 is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4.

3. The kit for detecting Helicobacter pylori CagA subtype according to claim 2, characterized in that: The nucleotide sequence of the heavy chain variable region of the antibody 3E67 is shown in SEQ ID NO: 5, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 6; The nucleotide sequence of the heavy chain variable region of the antibody 5T68 is shown in SEQ ID NO: 7, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO:

8.

4. The kit for detecting Helicobacter pylori CagA subtype according to claim 1, characterized in that: The kits are chemiluminescence kits, enzyme-linked immunosorbent assay kits, and immunochromatography kits.

5. The kit for detecting Helicobacter pylori CagA subtype according to claim 4, characterized in that: The chemiluminescence kit is a chemiluminescence kit for the NMHL reaction system, including an enzyme marker, a biotin marker, an auxiliary agent, an acridinium ester derivative biomarker and a calibrator.

6. The kit for detecting Helicobacter pylori CagA subtype according to claim 5, characterized in that: The calibrator contains CagA and 0.1 M phosphate buffer; The enzyme marker is peroxidase-labeled streptavidin and 0.05M phosphate buffer; The biotin marker is biotin-labeled anti-CagA monoclonal antibody 3E67 and 0.05M phosphate buffer; The acridinium ester derivative label is an anti-CagA monoclonal antibody 5T68 labeled with an acridinium ester derivative and 0.05 M Tris buffer; The auxiliary agent includes a luminescence auxiliary agent and a citrate buffer; The substrate solution includes H2O2 and 0.05M Tris buffer.

7. The kit for detecting Helicobacter pylori CagA subtype according to claim 4, characterized in that: The enzyme-linked immunosorbent assay kit includes an enzyme-linked immunosorbent assay plate, antibody 3E67, mouse anti-human-HRP, a color developing solution, a stop solution, a diluent, a washing solution, and a standard; the enzyme-linked immunosorbent assay plate is coated with antibody 5T68.

8. The kit for detecting Helicobacter pylori CagA subtype according to claim 7, characterized in that: The concentration of antibody 3E67 is 1 μg / ml-5 μg / mL, and the concentration of antibody 5T68 is 1 μg / ml-5 μg / mL.

9. The kit for detecting Helicobacter pylori CagA subtype according to claim 4, characterized in that: The immunochromatographic kit includes a test card, which includes: a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper; the sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper are sequentially overlapped and adhered to the base plate; the conjugate pad is sprayed with a tracer marker labeled with antibody 3E67, a C line, and a T line, wherein the C line is fixed with mouse anti-human IgG antibody, and the T line is fixed with antibody 5T68.

10. Use of the kit for detecting Helicobacter pylori CagA subtype according to any one of claims 1 to 2 in preparing a product for detecting Helicobacter pylori CagA subtype.

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