A kit for detecting helicobacter pylori cag a subtype and application thereof

Abstract

The application provides a kit for detecting a CagA subtype of Helicobacter pylori and application thereof, 3E67 and 5T68 monoclonal antibodies are prepared by using a CagA dominant immunological epitope, and the monoclonal antibodies are first used in a homogeneous chemiluminescence system to prepare a detection Helicobacter pylori CagA subtype antigen. Mouse antibodies are prepared by immunization of a dominant antigen epitope of a Helicobacter pylori CagA protein, a pair of antibodies with good specificity and strong binding force are obtained by in-vitro screening and pairing strategies, and the amino acid sequences of the heavy chain variable region and the light chain variable region are sequenced. The application is based on a chemiluminescence platform, an enzyme-linked immunization platform and an immunization layer system to develop a product for detecting a Helicobacter pylori related CagA antigen, and the product has the advantages of high sensitivity, good specificity and rapid detection.

Description

Technical Field

[0001] This invention belongs to the field of medical testing and relates to a kit for detecting Helicobacter pylori CagA subtype in gastric mucosal tissue and its application. Background Technology

[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 International Agency for Research on Cancer (IARC) of the World Health Organization. It is a bacterium that can grow and multiply in the human body and is transmitted through the oral-fecal-oral route. Globally, nearly 60% of the population is infected with HP. In China, the average HP infection rate is as high as 53%, affecting nearly 700 million people. The main symptoms of Helicobacter pylori infection include acid reflux, heartburn, stomach pain, and bad breath. This is because infection with Helicobacter pylori induces excessive secretion of gastrin, leading to acid reflux and heartburn. Patients with peptic ulcers primarily experience stomach pain. Helicobacter pylori can also cause chronic gastritis, with main clinical manifestations including upper abdominal discomfort and dull pain, sometimes accompanied by belching, acid reflux, nausea, and vomiting. The disease progresses slowly but is prone to recurrence. After a patient is infected with Helicobacter pylori, it will induce the production of a variety of pathogenic factors, which will cause damage to the gastric mucosa. The occurrence of clinical diseases is diverse, and patients often experience symptoms such as acid reflux, belching, and bloating.

[0003] There are many methods for detecting Helicobacter pylori infection, mainly including direct bacterial examination, urease activity assay, immunological detection, and polymerase chain reaction (PCR). However, these methods all have their limitations. The most obvious one is that although the test result is positive, it cannot confirm whether there is an infection with the strain, which makes it difficult to determine the accuracy of clinical medication.

[0004] 1. C-14 Breath Tester + Breath Test Bag: Only requires 5 minutes of blowing, with no other discomfort. It is one of the ideal testing methods currently available. It is the gold standard in the medical field for detecting *Helicobacter pylori* (H. pylori). A positive test indicates H. pylori infection, but does not confirm infection with pathogenic H. pylori. Approximately 1 / 6 of *Helicobacter pylori* infections may develop peptic ulcer disease, so prescribing medication based solely on a positive test is unreasonable. The complete genome sequence of *Helicobacter pylori* has been determined. Type I contains CagA and VacA genes and expresses both proteins. Type I is now widely considered to be closely related to gastric diseases. A negative CagA and VacA antigen test indicates no viral infection, while a positive test indicates viral infection. Therefore, the positivity of CagA and VacA antigen tests is crucial in determining whether a patient requires clinical medication.

[0005] 2. Immunological testing: Various immunological testing methods are available to detect Helicobacter pylori infection by measuring serum antibodies. These include complement fixation tests, agglutination tests, passive hemagglutination assays, immunoblotting, and enzyme-linked immunosorbent assay (ELISA). Currently, clinical testing primarily focuses on detecting H. pylori antibodies. A positive H. pylori antibody test only indicates that the patient has been infected with H. pylori in the past; it does not confirm whether a specific strain is present. Therefore, the clinical significance of H. pylori antibody testing is limited.

[0006] 3. Polymerase Chain Reaction (PCR) technology: Detecting *Helicobacter pylori* (HP) nucleic acid in gastric mucosal tissue has high sensitivity and can confirm HP infection, but it cannot determine whether it is a specific strain. Furthermore, PCR testing requires advanced technology and a demanding laboratory environment, making the experiment quite challenging.

[0007] Therefore, there is an urgent need for a new technology and method to detect novel biomarkers for rapid clinical diagnosis and treatment guidance. To address the aforementioned challenge in diagnosing Helicobacter pylori infection, this invention develops a rapid detection kit for Helicobacter pylori-associated CagA subtype antigens in gastric mucosal tissue. Summary of the Invention

[0008] This invention utilizes the dominant antigenic epitopes of Helicobacter pylori CagA protein to prepare mouse antibodies via immunization. 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 their heavy chain variable regions and light chain variable regions are obtained through sequencing. This invention is the first product development based on a chemiluminescence platform for the detection of Helicobacter pylori-related CagA antigens, offering advantages such as high sensitivity, high specificity, and rapid detection.

[0009] 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, said antibody or antigen-binding fragment comprising 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.

[0010] Specifically, the amino acid sequence of the dominant antigenic epitope of the selected Helicobacter pylori CagA protein is shown in SEQ ID NO:21:

[0011] MTNETIDQQPQTEAAFNPQQFINNLQVAFLKVDNAVASYDPDQKPIVDKNDRDNRQAFDGISQLREEYSNKAIKNPAKKNQYFSDFINKSNDLINKDALIDVESSTKSFQKFGDQRYRIFTSWVSHQNDPSKINTRSIRDFMEHTIQPPIPDDK EKAEFLKSAKQSFAGIIIGNQIRTDQKFMGVFDESLKERQEAEKNGGPTGGDWLDIFLSFIFDKKQSSDVKEAINQEPVPHVQPDIATSTTHIQGLPPESRDLLDERGNFSKFTLGDMEMLDVEGVADMDPNYKFNQLLIHNNTLSSVLMGSHNG

[0012] The two antibodies obtained were antibody 3E67 and antibody 5T68.

[0013] The amino acid sequence of the variable region of the antibody 3E67 heavy chain is shown in SEQ ID NO:1:

[0014] DMSVESGGGLVKPGGSLKLSCAASGFTFN SYAMS WVRQTPEKRLEWVA SSGGNTYYPDSVKG RFTISRDNARNIYLQMSSLRSEDTAMYYCAC GFESPYDTAMYYFDY WGQGTTARASWL;

[0015] 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 ;

[0016] The amino acid sequence of the variable region of the light chain of antibody 3E67 is shown in SEQ ID NO:2:

[0017] VIEMTQSPASLAVSLGQRTTISY RASKSVSTSGYSYMH WNQQKPGQPPRLLY LVSNLES GVPARFGGSGSGTDFTLNIHPVEEEDTAT YYCQHIREL TRSEGGPSWLT;

[0018] The amino acid sequence of LCDR1 is shown in SEQ ID NO:12: RASKSVSTSGYSYMHThe 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 ;

[0019] The amino acid sequence of the heavy chain variable region of antibody 5T68 is shown in SEQ ID NO:3.

[0020] QIMESGPDLVKPSQSLTCIVIGYSIT SGYNWHN WIRQFPGYGTEWMG YIHYRGTSSYNISLKS RISITRDSTISSPLSVTTTAYYCAC ADDFYSRDY MGQGTIVIVSSED;

[0021] 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 ;

[0022] The amino acid sequence of the light chain variable region is shown in SEQ ID NO:4:

[0023] ELVMTMSASPGEKVTMTC SASSSGVSYMNH WYQQKSSPKRWIY DTSRKLPSSER GVPGRFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPPTER FGAGEVKQIPITKLE;

[0024] 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.

[0025] Furthermore, the nucleotide sequence of the heavy chain variable region of antibody 3E67 is shown in SEQ ID NO: 5:

[0026] Gacatgagcgtggagagcggcggcggcctggtgaagcccggcggcagcctgaagctgagctgcgccgccagcggcttcaccttcaacagctacgccatgagctgggtgagacagacccccgagaagagactggagtgggtggccagcagcggcggcaacacctactaccccgacagcgtgaagggcagattcaccatcagcagagacaacgccagaaacatctacctgcagatgagcagcctgagaagcgaggacaccgccatgtactactgcgcctgcggcttcgagagcccctacgacaccgccatgtactacttcgactactggggccagggcaccaccgccagagccagctggctg;

[0027] and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 6:

[0028] gtgatcgagatgacccagagccccgccagcctggccgtgagcctgggccagagaaccaccatcagctacagagccagcaagagcgtgagcaccagcggctacagctacatgcactggaaccagcagaagcccggccagccccccagactgctgtacctggtgagcaacctggagagcggcgtgcccgccagattcggcggcagcggcagcggcaccgacttcaccctgaacatccaccccgtggaggaggaggacaccgccacctactactgccagcacatcagagagctgaccagaagcgagggcggccccagctgg;

[0029] The nucleotide sequence of the heavy chain variable region of antibody 5T68 is shown in SEQ ID NO: 7:

[0030] Cagatcatggagagcggccccgacctggtgaagcccagccagagcctgacctgcatcgtgatcggctacagcatcaccagcggctacaactggcacaactggatcagacagttccccggctacggcaccgagtggatgggctacatccactacagaggcaccagc agctacaacatcagcctgaagagcagaatcagcatcaccagagacagcaccatcagcagccccctgagcgtgaccaccaccgcctactactgcgcctgcgccgacgacttctacagcagagactacatgggccagggcaccatcgtgatcgtgagcagcgaggac

[0031] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 8:

[0032] gagctggtgatgaccatgagcgccagccccggcgagaaggtgaccatgacctgcagcgccagcagcagcggcgtgagctacatgaaccactggtaccagcagaagagcagccccaagagatggatctacgacaccagcagaaagctgcccagcagcgagagaggcgtgccc ggcagattcagcggcagcggcagcggcaccagctacagcctgaccatcagcagcatggaggccgaggacgccgccacctactactgccagcagtggagcagcaacccccccaccgagagattcggcgccggcgaggtgaagcagatccccatcaccaagctggagctgacc.

[0033] This invention first screens for dominant antigenic epitopes of CagA, extracts and constructs recombinant peptides, and uses them as immunogens to prepare mouse-specific IgG antibodies. The ELISA platform is then used to screen antibody pairs with high titers and good pairing affinity in vitro for reagent kit development.

[0034] Furthermore, the kit is a chemiluminescence reagent kit, an enzyme-linked immunosorbent assay (ELISA) kit, or an immunochromatographic assay kit.

[0035] The chemiluminescence reagent kit is a chemiluminescence reagent kit based on the NMHL reaction system, which includes enzyme markers, biotin markers, adjuvants, acridinium ester derivative biomarkers, and calibrators.

[0036] The calibrator contains CagA and 0.1M phosphate buffer;

[0037] The enzyme label is streptavidin labeled with peroxidase and 0.05M phosphate buffer;

[0038] The biotin label is a biotin-labeled anti-CagA monoclonal antibody 3E67 and 0.05M phosphate buffer.

[0039] The acridinium ester derivative label is an acridinium ester derivative-labeled anti-CagA monoclonal antibody 5T68 in 0.05M Tris buffer;

[0040] The auxiliary agents include a luminescent auxiliary agent and a citrate buffer solution;

[0041] The substrate solution includes H2O2 and 0.05M Tris buffer.

[0042] The enzyme-linked immunosorbent assay (ELISA) kit includes an ELISA plate, antibody 3E67, mouse anti-human HRP, chromogenic solution, stop solution, diluent, washing solution, and standards; the ELISA plate is coated with antibody 5T68. 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.

[0043] The immunochromatographic kit includes a test card comprising: 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 coated 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.

[0044] Another object of the present invention is to provide the application of the above-described kit for detecting Helicobacter pylori CagA subtype in the preparation of products for detecting Helicobacter pylori CagA subtype.

[0045] It should be noted that the antibody pairs of the present invention can also be used in various kits known in the art for the detection of Helicobacter pylori CagA antigen.

[0046] The samples applicable to this invention include human fecal samples, or digestive tract secretions and tissue samples, etc.

[0047] The beneficial technical effects achieved by this invention are as follows: This invention provides a kit for detecting Helicobacter pylori CagA subtypes and its application. Mouse antibodies are prepared by immunization using the 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 their heavy chain variable regions and light chain variable regions are obtained by sequencing. This invention, based on chemiluminescence, enzyme-linked immunosorbent assay (ELISA), and immunochromatography platforms for the detection of Helicobacter pylori-related CagA antigens, has the advantages of high sensitivity, high specificity, and rapid detection. Attached Figure Description

[0048] Figure 1 The purification results of the CagA recombinant antigen in Specific Example 1 of this invention; wherein, B: bacterial culture before induction; A: bacterial culture after induction; E1-5: eluted target protein; M: protein marker;

[0049] Figure 2 The purification results of mouse anti-CagA IgG monoclonal antibody 3E67 in Specific Example 1 of this invention, wherein, B: bacterial culture before induction; A: bacterial culture after induction; E1-4: eluted target protein; M: protein marker;

[0050] Figure 3 Purification results of mouse anti-CagA IgG monoclonal antibody 5T68 in Specific Example 1 of this invention: B: bacterial culture before induction; A: bacterial culture after induction; E1-4: eluted target protein; M: protein marker;

[0051] Figure 4 The standard curve (linear range) in specific embodiment 2 of the present invention. Detailed Implementation

[0052] The present invention will be further described below with reference to specific embodiments. These embodiments are only used to more clearly illustrate the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention.

[0053] The present invention will be further described below with reference to the embodiments.

[0054] Example 1

[0055] (I) Plasmid Construction

[0056] First, (a) plasmid construction

[0057] First, the dominant antigenic epitope segment 1-309AA (Uniprot: T2SZC2) of CagA was screened and extracted. Its amino acid sequence is shown in SEQ ID NO:21. According to the codon principle of E. coli, the above dominant antigenic epitope segment was optimized into a nucleotide sequence, and then the gene was synthesized (completed by Genscript Biotech).

[0058] The above-mentioned double enzyme digestion method was used to ligate BamHI and EcoRI into the pGEX expression vector to construct the expression plasmid. 80-100 ng of the expression plasmid was extracted and added to DH5α competent cells. The competent cells were then heat-shocked at 42℃ for 90 s. The transformed competent cells were added to LB liquid medium and cultured on a shaker at 37℃ and 200 rpm for approximately 30 min. The cells were then removed, and the suspension was plated onto ampicillin-containing plates and incubated overnight at 37℃. Single colonies were then picked from the culture plates and cultured in 2-5 mL of LB medium at 37℃ and 200 rpm for 8 h. The bacterial culture was collected, centrifuged, and the supernatant was discarded. The expression plasmid was extracted according to the instructions of the plasmid mini-prep kit produced by Tiangen Pharmaceuticals.

[0059] (ii) Antigen expression

[0060] The validated recombinant expression plasmid pGEX-CagA-GST was transformed into strain BL21. The plasmid was spread onto pre-prepared LB agar plates containing 50 μg / ml kanamycin, inverted at 37°C, and incubated for 12–16 h. Single colonies were then transferred to liquid culture medium for further expansion, incubated overnight at 37°C and 230 rpm. The culture volume was then increased further, and the culture was shaken until the OD600 reached 0.6–0.8. Expression was induced by adding IPTG to a final concentration of 0.3 mM, shaking at 37°C and 230 rpm for 4 h, and then centrifuged at 4000 rpm for 40 min to collect the bacterial cells.

[0061] (III) Antigen purification

[0062] The bacterial cells were resuspended in buffer 1 and sonicated on ice for 20 min; the supernatant was collected by centrifugation at 12000 rpm for 20 min at 4℃. The molecular weight of pGEX-CagA-GST expression was approximately 32.8 kDa. The buffer solution consisted of 150 mM Tris, 150 mM NaCl, 1 mM PMSF, and 0.1 M DTT at pH 7.5. The results are as follows. Figure 1 As shown.

[0063] The expressed antigens were bound to a GST column, and after washing away impurities with buffer 2 (50 mM Tris, 150 mM NaCl, pH 8.0), the GST tags were cleaved using a PreScission enzyme column. The target proteins were eluted in batches, collected, and then validated by SDS-PAGE.

[0064] (iv) Immunogen preparation

[0065] Prepare a 1M EDC reagent solution by dissolving it in a coupling buffer and a KLH carrier protein solution with a final concentration of 10 mg / mL in a coupling buffer (0.1M MES pH 4.7). Dissolve the collected purified antigen in the coupling buffer to a concentration of 4 mg / mL. Add the carrier protein at a molar ratio of 10:1. Under magnetic stirring, slowly add the EDC solution dropwise. The molar amount of EDC solution added should be approximately the same as that of the peptide. Dialyze and filter to remove uncoupled antigen and EDC solution. Add 1×PBS solution.

[0066] (v) Animal immunization

[0067] The antigen was emulsified with Freund's complete adjuvant. Five SPF-grade BALB / c mice were selected and injected subcutaneously with 50 µg / mouse. In the second and third weeks, the antigen emulsified with Freund's incomplete adjuvant was injected subcutaneously with 50 µg / mouse. In the fourth week, the antigen protein solution was injected to boost the immunization.

[0068] 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 eyeballs of mice. The blood was collected and allowed to stand until the serum was completely separated. The serum was centrifuged at 3000 rpm for 5 minutes, aliquoted, and stored at -70°C for later use.

[0069] (vi) Monoclonal antibody screening and preparation

[0070] BALB / c mice that had been immunized had their eyes removed and blood collected as positive control serum. The mice were then euthanized by cervical dislocation, disinfected with 75% alcohol, and the spleen was harvested. A spleen cell suspension (microscopically counted) was prepared and mixed with myeloma cells SP2 / 0 (microscopically counted) in serum-free DMEM medium at a ratio of 5:1. The mixture was centrifuged at 2000 rpm for 5 minutes, the supernatant was removed, and the cells were resuspended. Preheated 50% PEG4000 fusion cells were immediately added, and after 1 minute of incubation, serum-free DMEM medium was added to terminate the fusion. The cells were incubated at 37°C for 10 minutes, centrifuged, the supernatant was removed, and the cells were 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 fusion cells covered 20%-50% of the bottom of the wells. Positive clones were screened using an indirect ELISA method.

[0071] The antigen collected in step (III) was coated with HRP-labeled goat anti-mouse IgG as a secondary antibody. Serum collected after enucleation of immunized mice served as a positive control, and the supernatant of cultured SP2 / 0 myeloma cells served as a negative control. A total of 8 anti-CagA antibodies were obtained.

[0072] (vii) Antibody pairing

[0073] Eight antibodies obtained were paired and screened using the double-antibody sandwich ELISA method to ultimately identify a pair of hybridoma cells. The selected pair of hybridoma cells was then subjected to serial subcloning using a limiting dilution method until the positive rate of the subcloned cells reached 100%, after which the cells were expanded into larger cultures.

[0074] (viii) Antibody purification

[0075] Hybridoma cells were cultured on a large scale in DMEM medium supplemented with 20% fetal bovine serum using a rolling flask method. The culture conditions were: 37℃, 220 rpm, 5% CO2. Antibody IgG was prepared. When cell viability was less than 50%, the culture medium was collected in centrifuge flasks and centrifuged at 12000 rpm for 10 min to collect the supernatant. Impurities were removed by filtration through a 0.45 μm filter. The supernatant was purified by Protein A affinity chromatography, and protein properties were determined by SDS-PAGE electrophoresis. After confirmation, the protein was dialyzed into 1×PBS pH 7.4 buffer, sampled, and sequenced (sent to GenScript Biotech). Two antibodies were obtained: monoclonal antibody 3E67, as shown in the image. Figure 2 As shown, and monoclonal antibody 5T68, as Figure 3 As shown.

[0076] The amino acid sequences of the two antibodies are as follows:

[0077] 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;

[0078] 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;

[0079] The sequence was reverse translated and then codon-optimized for use in recombinant expression, resulting in the following nucleotide sequence:

[0080] 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;

[0081] 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.

[0082] Example 2

[0083] A detection kit for Helicobacter pylori CagA antigen was developed using the two antibodies obtained. The kit includes calibrators, enzyme markers, biotin markers, acridinium ester derivative markers, auxiliary agents, and substrate solutions.

[0084] (a) The reagent components are shown in Table 1.

[0085] Table 1

[0086]

[0087] (II) Preparation process

[0088] 1. Preparation of calibrators

[0089] 1.1 Preparation of calibrator diluent: The calibrator diluent can be prepared using conventional techniques, with no special requirements.

[0090] 1.2 Preparation of calibrators and negative and positive quality control samples

[0091] Dilute the purified CagA calibrator 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 using calibrator diluent, and store at 2-8℃ for later use.

[0092] Negative control: Add 3% fetal bovine serum to the calibrator diluent.

[0093] Positive quality control:

[0094] Weak positive: Dilute the CagA calibrator with calibrator diluent to a concentration of 0.5 ng / mL.

[0095] Strong positive: Dilute the CagA calibrator with calibrator diluent to a concentration of 5.0 ng / mL.

[0096] 2. R1: Preparation of enzyme-labeled products

[0097] 2.1 1 mg of HRP and 0.1 ml of 60 mmol / L NaIO4 were reacted at 4 °C for 30 minutes. Then 0.1 ml of 0.16 mol / L ethylene glycol was added. After 30 minutes, 1 mg of SA was added. The mixture was allowed to stand at 4 °C for 24 hours for dialyzing to obtain an HRP-SA solution.

[0098] 2.2 Add an equal volume of saturated ammonium sulfate solution, centrifuge at 4000 r / min for 15 min, remove the supernatant, and dissolve the precipitate in PBS (pH 7.4). Measure the absorbance at 280 nm.

[0099] 2.3 The Sephadex G-75 column was packed, and 100 μL of HRP-SA was loaded. The sample was eluted with 0.025 mol / L KCl-0.2 mol / L acetate buffer at a rate of 0.5 mL / 2 min, and collected separately. The OD280 value of the collected sample was measured using a DU800 UV spectrophotometer, and a chromatography chromatogram (elution curve) was plotted. Single white peaks were collected based on this chromatogram, and the dialysate was concentrated with PEG-2000.

[0100] 2.4 Take 10 μL of HRP-SA, add 10 times the volume of deionized double-distilled water, and scan using a UV spectrophotometer in the wavelength range of 200-280 nm. HRP-SA has an absorption peak at 280 nm.

[0101] 3. Reagent 2: Biotin-labeled antibody 3E67

[0102] Antibody 3E67 was diluted 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. Antibody 3E67 was dialyzed thoroughly with alternating 0.1 mol / L sodium bicarbonate buffer (pH 8.0) or 0.5 mol / L borate buffer (pH 8.6). 1 mg of NHSB was dissolved in 1 mL of DMSO to obtain an NHSB solution. 120 μL of NHSB solution was added to 1 mL of antibody 3E67 solution. The solution was incubated at room temperature with continuous stirring for 2–4 hours. 9.6 μL of 1 mol / L NH4+ was added, and the solution was stirred at room temperature for 10 minutes. The solution was dialyzed thoroughly with PBS at 4°C to remove free biotin. The solution was then loaded onto a 1 mL molecular sieve column and slowly eluted with PBS, collecting 1 mL per tube; protein was eluted when the concentration was between 1–3 mL. Add 50% redistilled glycerol to the eluent and store at 20°C.

[0103] 4. R3: Preparation of acridinium ester derivatives: Conventional techniques can be used for preparation, and no limitation is made in this application. The method described in one of the applicant's published patent documents (201811115054.7) can be used for preparation.

[0104] 5. R4: Preparation of the excipient: It can be prepared using conventional techniques. This application does not impose any restrictions. The method described in one of the applicant's published patent documents (201811115054.7) can be used for preparation.

[0105] 6. R5: Preparation of substrate solution: It can be prepared using conventional techniques and is not limited in this application. The method described in one of the applicant's published patent documents (201811115054.7) can be used for preparation.

[0106] 7. Preparation of gastric mucosal tissue processing solution: 0.5M EDTA buffer containing 0.9% NaCl.

[0107] (III) Testing Procedures

[0108] 1. Take about 0.5 grams of gastric mucosal tissue under endoscopy, add it to a centrifuge tube, pre-fill the centrifuge tube with 1.0 mL of tissue processing solution, mix well, store at 2-8℃, and test within 2 weeks.

[0109] 2. Sample addition: Add calibrator, positive and negative quality controls, 20 μL of tissue extract sample, R1: 40 μL, R2: 20 μL, R3: 20 μL, R4: 5 μL to the reaction tube and vortex to mix.

[0110] 3. Reaction: Incubate at 37℃ for 5 min;

[0111] 4. Detection: Add R5: 75uL, oscillate to mix, detect immediately, and read the signal value.

[0112] 5. Calculation: Logistic four-parameter fitting is performed on the concentration and luminescence value of the calibrator, and the sample concentration is calculated through sample RLU.

[0113] 6. Result judgment as follows Figure 4 As shown:

[0114] 6.1 Linear range: 0~500ng / mL.

[0115] 6.2 Detection sensitivity: 0.1 ng / m.

[0116] 6.3 Coefficient of variation: <5%.

[0117] 6.4 The yin and yang properties meet the requirements.

[0118] It should be noted that:

[0119] Criteria for judging negative and positive results: expressed using CagA antigen negative and positive results.

[0120] Positive: The ratio of the sample OD value to the cut-off OD value is >1;

[0121] Negative: The ratio of the sample OD value to the cut-off OD value is <0.9;

[0122] Gray area: The ratio of the sample OD value to the cut-off OD value is between 0.9 and 1.0.

[0123] 7. Clinical interpretation of results:

[0124] CagA antigen positive: CagA subtype strain infection.

[0125] CagA antigen negative: No infection with CagA subtype strains.

[0126] CagA antigen gray zone: Combine with clinical diagnosis, or regular follow-up examination.

[0127] Example 3

[0128] The enzyme-linked immunosorbent assay (ELISA) kit includes an ELISA plate, antibody 3E67 (concentration of 1 μg / ml-5 μg / mL), mouse anti-human HRP, chromogenic solution, stop solution, diluent, washing solution, and standards; the ELISA plate is coated with antibody 5T68 (concentration of 1 μg / ml-5 μg / mL); a double-antibody sandwich ELISA kit was prepared using conventional methods, with antibody 3E67 concentration of 2 μg / mL and antibody 5T68 concentration of 2.4 μg / mL. The kit was used to detect fecal samples and digestive tract tissue samples, and the results are shown in Table 2.

[0129] Immunochromatographic kit: Includes a test card, which comprises 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 coated with a tracer marker labeled with 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 antibody 5T68. Preferably, the tracer marker can be nanoparticles, such as colloidal gold, latex microspheres, or fluorescent microspheres. A double-antibody sandwich immunochromatographic kit was prepared using conventional methods. The concentration of antibody 3E67 was 1.2 μg / mL, and the concentration of antibody 5T68 was 1.5 μg / mL. This kit was used to detect fecal samples and digestive tract tissue samples, and the results are shown in Table 1.

[0130] Table 2 shows the test results of fecal samples.

[0131]

[0132] Note: "++" indicates a strong positive result, "++" indicates a moderate positive result, "+" indicates a weak positive result, and "-" indicates a negative result.

[0133] The present invention has been disclosed above with reference to preferred embodiments, but it is not intended to limit the invention. Any equivalent substitutions are permitted.

[0134] All technical solutions obtained by means of equivalent transformation fall within the protection scope of this invention.

Claims

1. A kit for detecting Helicobacter pylori CagA subtype, characterized in that, The kit includes: An antibody or antigen-binding fragment that binds to the antigenic properties of the CagA subtype; the antibody or antigen-binding fragment includes a heavy chain variable region and a light chain variable region: the heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3, and the light chain variable region VL includes LCDR1, LCDR2, and LCDR3; the antibody or antigen-binding fragment is: Antibody 3E67: The amino acid sequences of HCDR1 are shown in SEQ ID NO:9, HCDR2 in SEQ ID NO:10, and HCDR3 in SEQ ID NO:11; the amino acid sequences of LCDR1 are shown in SEQ ID NO:12, LCDR2 in SEQ ID NO:13, and LCDR3 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 amino acid sequence of LCDR3 is 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 variable region of the heavy chain of the antibody 5T68 is shown in SEQ ID NO: 7, and the nucleotide sequence of the variable region of the light chain 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 reagent kits, enzyme-linked immunosorbent assay (ELISA) kits, and immunochromatographic reagent kits.

5. The kit for detecting Helicobacter pylori CagA subtype according to claim 4, characterized in that: The chemiluminescence kit is a chemiluminescence kit based on the NMHL reaction system, including enzyme markers, biotin markers, adjuvants, acridinium ester derivative markers, and calibrators.

6. The kit for detecting Helicobacter pylori CagA subtype according to claim 5, characterized in that: The calibrator contains CagA and 0.1M phosphate buffer; The enzyme label is streptavidin labeled with peroxidase and 0.05M phosphate buffer; The biotin label is a biotin-labeled anti-CagA monoclonal antibody 3E67 and 0.05M phosphate buffer. The acridinium ester derivative label is an acridinium ester derivative-labeled anti-CagA monoclonal antibody 5T68 and 0.05M Tris buffer. The auxiliary agents include a luminescent auxiliary agent and a citrate buffer solution; 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 (ELISA) kit includes an ELISA plate, antibody 3E67, mouse anti-human-HRP, chromogenic solution, stop solution, diluent, washing solution, and standards; the ELISA plate is coated with antibody 5T68.

8. The kit for detecting Helicobacter pylori CagA subtype according to claim 7, characterized in that: The concentrations of antibody 3E67 and antibody 5T68 were 1 μg / ml to 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 comprising: 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 coated 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. The use of the kit for detecting Helicobacter pylori CagA subtype according to any one of claims 1-2 in the preparation of products for detecting Helicobacter pylori CagA subtype.

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