Anti-Helicobacter pylori CagA subtype antibody detection kit and its application

Abstract

The present invention provides an anti-Helicobacter pylori CagA subtype antibody detection kit and its application. CagA subtype proteins are optimized and screened to obtain truncated fragments containing two dominant epitope targets. These fragments are expressed in different systems to obtain CagA subtype recombinant antigen fragments. The antigen fragments are used to screen antibodies as quality control standards, and a chemiluminescence detection kit is developed to quickly detect whether an organism is infected with Helicobacter pylori.

Description

Technical Field

[0001] This invention belongs to the field of Helicobacter pylori detection technology, specifically relating to an anti-Helicobacter pylori CagA subtype antibody detection kit and its application. Background Technology

[0002] Helicobacter pylori is a spiral-shaped, flagellated, microaerophilic Gram-negative bacillus, first discovered and documented by Marshall and Warren in 1982. It colonizes the gastric mucosa of over 50% of the global population. The acidic environment of the stomach does not allow viruses, bacteria, and other microorganisms to survive, but Helicobacter pylori has evolved unique abilities to overcome this harsh environment. It secretes urease, an enzyme that converts urea into ammonia and bicarbonate to neutralize stomach acid, making the stomach a more suitable environment for its survival. Having acquired this survival ability, the stomach provides a special environment for Helicobacter pylori. Host immune cells, and antibodies that normally recognize and attack invading bacteria, cannot reach the freely moving Helicobacter pylori in the gastric mucosa. Instead, an ineffective host immune response continues to react to the site of infection, with immune cells and epithelial cells dying and releasing nutrients to feed the gastric pathogen. Helicobacter pylori infection is primarily acquired in childhood and is mainly transmitted within families via oral-oral or fecal-oral routes.

[0003] However, most people infected with Helicobacter pylori are asymptomatic, but it can lead to chronic gastritis; only 10% of infected individuals experience symptomatic disease. Furthermore, experimental and epidemiological studies have shown that Helicobacter pylori infection does indeed increase the risk of stomach cancer. Therefore, the International Agency for Research on Cancer (IARC) of the World Health Organization classified Helicobacter pylori as a Group 1 carcinogen in 1994.

[0004] Individual differences in the risk of Helicobacter pylori-induced gastric diseases involve significant heterogeneity in host genetics and virulence factors of Helicobacter pylori strains. Several strain-specific virulence factors have been reported in the pathogenic mechanisms of Helicobacter pylori-related diseases, such as CagA (cytotoxin-associated gene A), VacA (vacuole cytotoxin A), HpaA (Helicobacter pylori adhesin A), BabA (blood group antigen-binding adhesin), DupA (duodenal ulcer-promoting gene A), and IceA (epithelial contact-induced gene). Among these, CagA is one of the major virulence factors, which is associated with a high risk of gastric cancer and peptic ulcers. CagA protein can interact with intercellular proteins, activating signaling pathways through tyrosine phosphorylation-dependent or independent mechanisms.

[0005] Based on the presence or absence of CagA (the gene encoding the CagA protein) in the bacterial genome, *H. pylori* can be divided into CagA-positive and CagA-negative strains. Due to structural polymorphism in the C-terminal region, the size of the CagA protein varies from 130 to 145 kDa. The CagA gene is located at one end of the Cag pathogenic island (cagPAI). Approximately 30-40% of strains isolated from Western countries do not carry CagPAI and are CagA-negative, while almost all *H. pylori* isolates from East Asian countries contain the structure and cancer-promoting function of CagA.

[0006] Therefore, early screening for Helicobacter pylori can effectively prevent the incidence of gastric cancer. 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 typical limitation is that although the test result is positive, it cannot determine whether an infection with the specific strain is present, causing some confusion in clinical medication. Summary of the Invention

[0007] In order to overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides an anti-Helicobacter pylori CagA subtype antibody detection kit and its application, which can quickly determine whether the body is infected with Helicobacter pylori strains.

[0008] To address the aforementioned technical problems, this invention provides a detection kit for anti-Helicobacter pylori CagA subtype antibodies. Specifically, it is a detection kit for anti-CagA-IgM and anti-CagA-IgG antibodies, comprising a CagA subtype recombinant antigen fragment. The amino acid sequence of the CagA subtype recombinant antigen fragment is shown in SEQ ID NO:1.

[0009] SAKNGVNGTLIGNGLSKAEATTLSKNFSDIQKELNAELGNFNNNNNGLKNEPIYAKVNKKKAGQAASPEEPIYAQVAKKVNAKIDRLNQIASGLGGVGQAAGFPLKRHDKVDDLSKVGRSVSPE PIYATIDDLGGPFPLKRHDKVDDLSKVGRSVSPEPIYATIDDLGGPFPLKRHDKVDDLSKVWLSRNQELAQKIDNLNQAVSEAKAGFFGNLEQTIDKLKDSAKNNPVNLWAEGAKKCLLVCQRN.

[0010] The recombinant antigen fragment for the CagA subtype includes two target sites, with amino acid sequences shown in SEQ ID NO:2: SVSPEPIYA and SEQ ID NO:3: SVSPEPIYA, respectively. This recombinant antigen fragment for the CagA subtype is obtained by recombinantly expressing a truncated fragment of the Helicobacter pylori CagA subtype protein containing the dominant antigenic epitopes (i.e., two target sites) in different systems, followed by immunoselection to obtain high-titer antigens for use in Helicobacter pylori antibody detection.

[0011] Furthermore, the above-mentioned CagA subtype recombinant antigen fragment was converted into a nucleotide sequence according to the translation principles of E. coli codon, Homo sapiens codon, and Spodoptera frugiperda codon, respectively, to obtain the edited nucleotide sequence of the CagA subtype recombinant antigen fragment as shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0012] The sequence of SEQ ID NO:4 is as follows:

[0013] Agcgcgaaaaacggcgtgaacggcaccctgattggcaacggcctgagcaaagcggaagcgaccaccctgagcaaaaactttagcgatattcagaaagaactgaacgcggaactgggcaactttaacaacaacaacaacaacggcctgaaaaacgaaccgatttatgcgaaagtgaacaaaaaaaaagcgggccaggcggcgagcccggaagaaccgatttatgcgcaggtggcgaaaaaagtgaacgcgaaaattgatcgcctgaaccagattgcgagcggcctgggcggcgtgggccaggcggcgggctttccgctgaaacgccatgataaagtggatgatctgagcaaagtgggccgcagcgtgagcccggaaccgatttatgcgaccattgatgatctgggcggcccgtttccgctgaaacgccatgataaagtggatgatctgagcaaagtgggccgcagcgtgagcccggaaccgatttatgcgaccattgatgatctgggcggcccgtttccgctgaaacgccatgataaagtggatgatctgagcaaagtgtggctgagccgcaaccaggaactggcgcagaaaattgataacctgaaccaggcggtgagcgaagcgaaagcgggcttttttggcaacctggaacagaccattgataaactgaaagatagcgcgaaaaacaacccggtgaacctgtgggcggaaggcgcgaaaaaatgcctgctggtgtgccagcgcaac。

[0014] The sequence of SEQ ID NO:5 is as follows:

[0015] Agcgccaagaacggcgtgaacggcaccctgatcggcaacggcctgagcaaggccgaggccaccaccctgagcaagaacttcagcgacatccagaaggagctgaacgccgagctgggcaacttcaacaacaacaacaacaacggcctgaagaacgagcccatctacgccaaggtgaacaagaagaaggccggccaggccgccagccccgaggagcccatctacgcccaggtggccaagaaggtgaacgccaagatcgacagactgaaccagatcgccagcggcctgggcggcgtgggccaggccgccggcttccccctgaagagacacgacaaggtggacgacctgagcaaggtgggcagaagcgtgagccccgagcccatctacgccaccatcgacgacctgggcggccccttccccctgaagagacacgacaaggtggacgacctgagcaaggtgggcagaagcgtgagccccgagcccatctacgccaccatcgacgacctgggcggccccttccccctgaagagacacgacaaggtggacgacctgagcaaggtgtggctgagcagaaaccaggagctggcccagaagatcgacaacctgaaccaggccgtgagcgaggccaaggccggcttcttcggcaacctggagcagaccatcgacaagctgaaggacagcgccaagaacaaccccgtgaacctgtgggccgagggcgccaagaagtgcctgctggtgtgccagagaaac。

[0016] The sequence of SEQ ID NO:6 is as follows:

[0017] .

[0018] Different expression systems for preparing recombinant antigen fragments of the CagA subtype were Escherichia coli, baculovirus, or 293F cells.

[0019] Furthermore, the kit is a chemiluminescent immunoassay kit, comprising positive and negative controls for anti-CagA-IgG antibody, positive and negative controls for anti-IgM antibody, horseradish peroxidase (HRP)-labeled streptavidin, biotin-coated magnetic beads labeled with recombinant CagA subtype antigen fragments, acrid ester derivatives labeled with anti-human IgG or IgM antibodies, adjuvants, and substrate solution.

[0020] Biotin-labeled CagA subtype recombinant antigen fragments are coated onto magnetic beads. When added serum samples containing anti-CagA-IgM or anti-CagA-IgG antibodies, these antibodies bind to the recombinant antigen fragments, forming a biotin-antigen-antibody conjugate. After washing away non-specific antibodies from the serum, monoclonal antibodies labeled with acridil ester derivatives against human IgG or IgM and streptavidin labeled with horseradish peroxidase (HRP) are added sequentially, forming an HRP-streptavidin-biotin-antigen-antibody-anti-human monoclonal antibody-acrididil ester derivative complex. Spatially, horseradish peroxidase (HRP) and acridil ester derivatives are close to each other. Triggering the reaction with the addition of an auxiliary agent and substrate solution produces a flash-type chemiluminescence. The intensity of the luminescence is directly proportional to the antibody content in the serum sample being tested.

[0021] Furthermore, the concentration of the CagA subtype recombinant antigen fragment is 0.1 μg / mL to 5 μg / mL.

[0022] Furthermore, the present invention also provides the application of the above-described detection kit in the non-disease diagnosis of Helicobacter pylori detection.

[0023] The beneficial technical effects achieved by this invention are as follows: This invention provides an anti-Helicobacter pylori CagA subtype antibody detection kit and its application. The CagA subtype protein is optimized and screened to obtain a truncated fragment containing two dominant epitope targets. The CagA subtype recombinant antigen fragment is obtained by expression in different systems. Antibodies are screened using this antigen fragment as quality control standards, and a chemiluminescent detection kit is developed, which can quickly detect whether the body is infected with Helicobacter pylori. Attached Figure Description

[0024] Figure 1 This is a specific embodiment of the purification results of the CagA subtype recombinant antigen according to the present invention;

[0025] Figure 2 This is the result of purification of anti-CagA-IgM antibody and anti-CagA-IgG antibody in a specific embodiment of the present invention. Detailed Implementation

[0026] 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.

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0028] Example 1: Preparation and optimization of CagA subtype recombinant antigen fragments

[0029] I. Constructing plasmids

[0030] First, the dominant CagA antigenic epitope region 34-282AA (GenBank: BBJ21835.1) was screened and extracted. Its amino acid sequence is shown in SEQ ID NO:1.

[0031] SAKNGVNGTLIGNGLSKAEATTLSKNFSDIQKELNAELGNFNNNNNNGLKNEPIYAKVNKKKAGQAASPEEPIYAQVAKKVNAKIDRLNQIASGLGGVGQAAGFPLKRHDKVDDLSKVGRSVSPEPIYATI DDLGGPFPLKRHDKVDDLSKVGRSVSPEPIYATIDDLGGPFPLKRHDKVDDLSKVWLSRNQELAQKIDNLNQAVSEAKAGFFGNLEQTIDKLKDSAKNNPVNLWAEGAKKCLLVCQRN, including 2 target SEQ ID NO:2: SSVPEPIYA and SEQ ID NO:3: SSVPEPIYA.

[0032] The above amino acid sequences were converted into nucleotide sequences according to the translation principles of the E. coli codon, Homo sapiens codon, and Spodopterafrugiperda codon, respectively, and then sent to a gene synthesis company (GenScript Biotechnology Co., Ltd.) for synthesis. The optimized nucleotide sequences are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively:

[0033] SEQ ID NO:4

[0034] Agcgcgaaaaacggcgtgaacggcaccctgattggcaacggcctgagcaaagcggaagcgaccaccctgagcaaaaactttagcgatattcagaaagaactgaacgcggaactgggcaactttaacaacaacaacaacaacggcctgaaaaacgaaccgatttatgcgaaagtgaacaaaaaaaaagcgggccaggcggcgagcccggaagaaccgatttatgcgcaggtggcgaaaaaagtgaacgcgaaaattgatcgcctgaaccagattgcgagcggcctgggcggcgtgggccaggcggcgggctttccgctgaaacgccatgataaagtggatgatctgagcaaagtgggccgcagcgtgagcccggaaccgatttatgcgaccattgatgatctgggcggcccgtttccgctgaaacgccatgataaagtggatgatctgagcaaagtgggccgcagcgtgagcccggaaccgatttatgcgaccattgatgatctgggcggcccgtttccgctgaaacgccatgataaagtggatgatctgagcaaagtgtggctgagccgcaaccaggaactggcgcagaaaattgataacctgaaccaggcggtgagcgaagcgaaagcgggcttttttggcaacctggaacagaccattgataaactgaaagatagcgcgaaaaacaacccggtgaacctgtgggcggaaggcgcgaaaaaatgcctgctggtgtgccagcgcaac。

[0035] SEQ ID NO:5

[0036] Agcgccaagaacggcgtgaacggcaccctgatcggcaacggcctgagcaaggccgaggccaccaccctgagcaagaacttcagcgacatccagaaggagctgaacgccgagctgggcaacttcaacaacaacaacaacaacggcctgaagaacgagcccatctacgccaaggtgaacaagaagaaggccggccaggccgccagccccgaggagcccatctacgcccaggtggccaagaaggtgaacgccaagatcgacagactgaaccagatcgccagcggcctgggcggcgtgggccaggccgccggcttccccctgaagagacacgacaaggtggacgacctgagcaaggtgggcagaagcgtgagccccgagcccatctacgccaccatcgacgacctgggcggccccttccccctgaagagacacgacaaggtggacgacctgagcaaggtgggcagaagcgtgagccccgagcccatctacgccaccatcgacgacctgggcggccccttccccctgaagagacacgacaaggtggacgacctgagcaaggtgtggctgagcagaaaccaggagctggcccagaagatcgacaacctgaaccaggccgtgagcgaggccaaggccggcttcttcggcaacctggagcagaccatcgacaagctgaaggacagcgccaagaacaaccccgtgaacctgtgggccgagggcgccaagaagtgcctgctggtgtgccagagaaac。

[0037] SEQ ID NO:6

[0038] Tccgctaagaacggtgtgaacggtaccctgatcggtaacggtctgtccaaggctgaggctaccaccctgtccaagaacttctccgacatccagaaggagctgaacgctgagctgggtaacttcaacaacaacaacaacaacggtctgaagaacgagcctatctacgctaaggtgaacaagaagaaggctggtcaggctgcttcccctgaggagcctatctacgctcaggtggctaagaaggtgaacgctaagatcgaccgcctgaaccagatcgcttccggtctgggtggtgtgggtcaggctgctggtttccctctgaagcgccacgacaaggtggacgacctgtccaaggtgggtcgctccgtgtcccctgagcctatctacgctaccatcgacgacctgggtggtcctttccctctgaagcgccacgacaaggtggacgacctgtccaaggtgggtcgctccgtgtcccctgagcctatctacgctaccatcgacgacctgggtggtcctttccctctgaagcgccacgacaaggtggacgacctgtccaaggtgtggctgtcccgcaaccaggagctggctcagaagatcgacaacctgaaccaggctgtgtccgaggctaaggctggtttcttcggtaacctggagcagaccatcgacaagctgaaggactccgctaagaacaaccctgtgaacctgtgggctgagggtgctaagaagtgcctgctggtgtgccagcgcaac。

[0039] The above gene sequence was ligated to an expression vector by restriction enzyme digestion to construct expression plasmids: pCold-CagA (34-282), pcDNA3.1-CagA (34-282), pFastBac-CagA (34-282). All three expression plasmids carry an N-terminal GST tag.

[0040] II. Plasmid Expression

[0041] The pCold-CagA(34-282) and pcDNA3.1-CagA(34-282) plasmids were transformed into E. coli DH5α plates and incubated overnight at 37°C. Colonies on the plates were picked to scale up the culture system stepwise. Finally, the bacterial culture was collected, centrifuged, and the supernatant was removed. The expression plasmid was extracted using the TaKaRa MiniBEST Plasmid Purification Kit.

[0042] The pFastBac-CagA(34-282) plasmid was transformed into DH10Bac baculovirus and cultured at 37°C for 48 hours. Recombinant strains were screened using blue-white screening on LB agar plates (containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline, 40 μg / ml X-gal, and 40 μg / ml IPTG). White clones were picked and re-stripened to confirm that the obtained white clones were genuine white clones. Recombinant bacmid DNA was extracted using the Beyotime baculovirus shuttle vector bacmid mini-extraction kit.

[0043] III. Expression and purification in different systems

[0044] 1. *E. coli* Expression System: The validated pCold-CagA(34-282) plasmid was transformed into *E. coli* BL21 strain plates. Single colonies were picked and cultured in liquid medium for expansion. The culture was incubated at 37°C with shaking until the OD600 reached 0.6-0.8. IPTG was added to a final concentration of 1 mM to induce expression. After 16-18 h of induction, the bacterial culture was collected and centrifuged at 5000 rpm for 20 min. The bacterial pellet was resuspended in lysis buffer (50 mM Tris, pH 8.0, 0.15 M NaCl, 10 mM imidazole, 1 mM PMSF) and sonicated in ice water for 3 s intervals followed by a 6 s pause, for a total sonication time of 40 min. The mixture was then centrifuged at 13000 rpm for 40 min, and the supernatant was purified.

[0045] 2. 293F cell expression system: The validated pcDNA3.1-CagA(34-282) plasmid was transfected into the 293F cell line using PEIMax transfection reagent at a rate of 1×10⁻⁶. 6Transfection was performed at a cell concentration of [cells / ml]. 300 μg of pcDNA3.1-CagA(34-282) plasmid (filtered and sterilized) was added to 30 mL of PBS, followed by 600 μg of filtered and sterilized PEI solution. After standing at room temperature for 20 min, the mixture was added to 293F cell culture medium. The cells were incubated at 37°C, 120 rpm, and 5% CO2 for 24 h. The mixture was then centrifuged at 4000 rpm for 5 min, and the pellet was collected. The pellet was then sonicated in ice water with a 2-second sonication pause followed by a 6-second pause, for a total sonication time of 30 min. Finally, the cells were centrifuged at 12000 rpm for 40 min, and the supernatant was collected for purification.

[0046] 3. Insect Baculovirus Expression System: Extracted pFastBac-CagA bacmid DNA was transfected into Sf-9 insect cells using LipoInsect transfection reagent (Beyotime). The cells were cultured at 28℃ for 72-96 hours, and the culture supernatant was collected. The cells were centrifuged at 500g for 5 minutes to precipitate cells and cell debris; the supernatant was the P1 generation insect baculovirus. The P1 generation insect baculovirus was then used to infect Sf-9 insect cells again to obtain the P2 generation virus. Sf-9 insect cells were then infected with the P2 generation virus during the logarithmic growth phase, maintaining a cell density of 1×10⁻⁶ cells / year. 6 / ml -2×10 6 Infect cells at / ml, and collect cells 72 hours after infection. Centrifuge at 4000rpm for 20min at 4℃, collect the pellet for purification. Sonicate in ice water for 30min, sonicating for 2s followed by a 6s pause. Centrifuge at 12000rpm for 40min, and collect the supernatant for purification.

[0047] Wash the glutathione-agarose resin and place it in a reagent bottle. Mix the purified material collected from the three expression systems with 50% glutathione-agarose resin and incubate with shaking at room temperature for 30 min. Centrifuge at 3000 rpm for 5 min at 4°C, discard the supernatant, add 10 volumes of 1×PBS to the precipitate, vortex and incubate for 5 min, wash away impurities, repeat washing 3 times, then add an appropriate amount of glutathione elution buffer to the precipitate, incubate for 10 min, centrifuge at 3000 rpm for 5 min at 4°C, collect the target protein, concentrate it to approximately 7 mg / ml using a 10 kD ultrafiltration tube, and perform electrophoresis. The bands are all around 53 kDa, as shown in the attached image. Figure 1 As shown. From Figure 1 As shown, CagA antigen protein was obtained in all three expression systems.

[0048] Example 2 Preparation of calibrator antibodies

[0049] (a) Animal Immunization

[0050] 50 μg of CagA antigen protein obtained from the 293F cell expression system was emulsified with an equal mass of adjuvant and diluted with 1×PBS buffer. For the first immunization, Freund's complete adjuvant was used for emulsification. Before use, the orange-yellow precipitate at the bottom was stirred, mixed with the CagA antigen protein, and then emulsified using an ultrasonic homogenizer. 6–8 week old BalB / C mice were selected, and 400 μL was injected subcutaneously and intraperitoneally at multiple sites. Two weeks later, a second immunization was performed, followed by immunizations every two weeks until the fifth immunization. Three days before cell fusion, a booster immunization of 200 μL of CagA antigen protein was administered via tail vein injection.

[0051] (ii) Cell fusion

[0052] Resuscitate myeloma cells SP2 / 0 and culture them to the logarithmic growth phase. Blood was collected from immunized mice by enucleation, and serum was separated as a positive control. After euthanizing the mice, the cells were washed with 75% ethanol. Under aseptic conditions, the spleen was removed, washed once with incomplete culture medium, and the spleen capsule was removed with forceps. The cells were repeatedly ground into a suspension, counted, and prepared into a cell suspension. 100 mL of logarithmic growth phase myeloma cells SP2 / 0 were taken and washed twice with 1640 culture medium by centrifugation. The spleen cells and myeloma cells SP2 / 0 were mixed, with a ratio of spleen cells to myeloma cells SP2 / 0 at 2:1. The mixture was then centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and the cell clumps were gently broken up at the bottom of the tube. Preheated 50% PEG was added dropwise over 1 min, and the reaction was carried out at 37°C for 1 min. After the reaction was complete, 10 mL of 1640 culture medium was added to terminate the reaction.

[0053] After fusion, centrifuge at low speed and discard the supernatant. Resuspend the cells in 1640 medium containing 10% fetal bovine serum to prepare a cell suspension. Take out a 96-well culture plate and add 200 μL of the cell suspension to each well. Incubate at 37°C in a 5% CO2 incubator.

[0054] (III) Screening for positive hybridoma cells

[0055] A 96-well ELISA plate was coated with recombinant CagA antigen protein. The plate was incubated with fusion cell supernatant, 1:1000 diluted positive serum from immunized mice, and 1:1000 diluted negative serum from mice. Then, it was incubated with horseradish peroxide (HRP)-labeled goat anti-mouse IgG and goat anti-mouse IgM. Finally, TMB substrate was added, and the OD450nm value was measured using an ELISA reader. Wells with an OD450nm value ≤0.1 and an OD value more than twice the OD value of the negative wells were considered positive wells.

[0056] Positive wells were sampled and counted, diluted to 100 cells / 10 mL of culture medium, and the diluted cell suspension was added to a 96-well cell culture plate and incubated at 37°C in a 5% CO2 cell culture incubator. After 6-7 days, the wells where clonal cells formed were observed under a microscope, and the cell supernatant was collected for ELISA detection to select positive monoclonal cells. After three limiting dilutions, a hybridoma cell line that stably secreted anti-CagA antibodies was finally obtained.

[0057] (iv) Purification of different subtype antibodies

[0058] Hybridoma-positive cell lines stably expressing anti-CagA antibodies were screened and cultured extensively. After 7-9 days, the cells were centrifuged at 12000 rpm for 20 min, and the supernatant was collected. The supernatant was dialyzed into deionized water at 4℃ for 24 h, and then centrifuged at 12000 rpm for 50 min to collect the precipitate. The precipitate was dissolved in 5 mL of borate buffer and purified by SE gel chromatography. The precipitate was eluted with borate buffer and collected, and the IgM protein peak was collected according to molecular weight. The IgM protein concentration was determined to be 4.2 mg / mL by UV spectrophotometer at OD 280 nm. The purity of IgM was identified by SDS-PAGE. The results are attached. Figure 2 As shown.

[0059] Hybridoma-positive cell lines stably expressing anti-CagA antibodies were screened and cultured extensively. After 7-9 days, the cells were centrifuged at 12000 rpm for 20 min, and the supernatant was collected. The supernatant was dialyzed against 20 mM PB, 150 mM NaCl, pH 7.4 at 4°C for 24 h. The supernatant was then adsorbed onto a protein A affinity chromatography column and eluted with 0.1 M glycine solution to obtain the target antibody, IgG antibody. The IgM protein concentration was determined to be 5.1 mg / mL using a UV spectrophotometer at OD 280 nm. The purity of IgG was identified by SDS-PAGE. The results are shown in the attached figure. Figure 2 As shown.

[0060] (v) Antibody titer testing

[0061] CagA recombinant antigen was diluted with 0.05 mol / L pH 9.6 carbonate buffer and coated onto 96-well plates. The plates were blocked with BSA, washed with 1×PBST, and the diluted monoclonal antibody samples were added to the plates for incubation. Then, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG or goat anti-mouse IgM diluted 1:4000 was added for incubation, followed by TMB substrate for color development. The OD value at 450 nm was measured using a microplate reader. Results showed that the titers of both monoclonal IgG and IgM antibodies were greater than 1:640,000.

[0062] Example 3: Preparation of Anti-Helicobacter pylori CagA Subtype Antibody Detection Kit

[0063] (a) The reagent contains the following components:

[0064] Table 1: Kit Contents

[0065]

[0066] (II) Preparation process

[0067] 1. Preparation of Quality Control 1 and Quality Control 2

[0068] The anti-CagA-IgG and CagA-IgM antibodies obtained in Example 2 were diluted to concentrations of 0.05 ng / mL and 5 ng / mL, respectively, using 0.1 M phosphate buffer as positive controls and stored at 2-8°C for later use.

[0069] 2. Biotin-labeled CagA recombinant antigen-coated magnetic beads: Add 0.5 ml of commercially available active magnetic beads (JSR LifeSciences Magnosphere 2023MC 03) to a 5 mL reaction vessel, place it in a dedicated test tube rack, and wash thoroughly with 0.1 mol / L PB buffer at pH 9.5. Repeat this process three times. Add the biotin-labeled CagA subtype recombinant antigen solution to the magnetic beads, mix well, and react at room temperature for 4 hours. Wash the coated magnetic beads three times with 0.1 mol / L phosphate buffer at pH 7.2. Then, prepare a 0.05% biotin-labeled CagA recombinant antigen-coated magnetic bead solution using magnetic bead preservation solution for later use.

[0070] 3. The preparation process of reagents 1, 3, 4 and 5 is not limited, and the preparation process in the existing technology can be realized, so it will not be described in detail here.

[0071] (III) Detection Methods

[0072] The steps for detecting CagA-IgG and CagA-IgM antibodies are as follows:

[0073] 1. Sample addition: Add 25 μL of positive control 1 or positive control 2 and 225 μL of the test sample and reagent to the reaction tube respectively, shake to mix, and incubate at 37°C for 10 min;

[0074] 2. Washing: Wash three times with washing solution (0.05M Tris buffer).

[0075] 3. Add reagent 1 and reagent 3, shake to mix, and incubate at 37°C for 5 minutes;

[0076] 3. Detection: Add 4-5 μL of reagent, vortex to mix, and let stand for 1-2 minutes; add 5-75 μL of reagent, detect immediately, and read the signal value. Read the luminescence values ​​of the positive control and the sample respectively.

[0077] 4. Calculation: Calculate the sample concentration using the sample RLU. Result Interpretation:

[0078] Positive: The ratio of the sample OD value to the cut-off OD value > 1;

[0079] Negative: The ratio of the sample OD value to the cut-off OD value is < 0.9;

[0080] Gray area: The ratio of the sample OD value to the cut-off OD value is between 0.9 and 1.0.

[0081] 5. Clinical interpretation of results:

[0082] 5.1 Anti-CagA-IgM

[0083] Positive for anti-CagA-IgM: indicates recent infection with the CagA strain.

[0084] CagA-IgM negative: indicates that there has been no recent infection with the CagA strain.

[0085] Anti-CagA-IgM gray zone: Combine with clinical diagnosis, or regular follow-up examination.

[0086] 5.2 Anti-CagA-IgG

[0087] Positive anti-CagA-IgG indicates previous infection with a CagA subtype strain.

[0088] Anti-CagA-IgG negative: CagA subtype strains have never been infected.

[0089] Anti-CagA-IgG gray zone: Combine with clinical diagnosis, or regular follow-up examination.

[0090] (iv) Reagent kit performance evaluation

[0091] 1. Experiments on interference from endogenous substances

[0092] Clinically abnormal samples mainly included hyperlipidemia, jaundice, and hemolysis; among these, triglycerides, bilirubin, and hemoglobin were also the main endogenous substances interfering with experimental results. We used standardized interfering substances to conduct interference experiments. The specific procedure is as follows:

[0093] 1) Preparation of basic samples: Clinical negative samples, weak positive samples, and positive samples were used as basic samples, labeled as 1, 2, and 3 respectively.

[0094] 2) Preparation of interfering solutions: Prepare stock solutions of interfering substances using pure solvents at a concentration at least 20 times that of the experimental concentration to reduce dilution of the base sample matrix. The preparation of each stock solution of interfering substance is shown in Table 1.

[0095] Table 1

[0096]

[0097] 3) Sample preparation: Prepare experimental samples (T) and control samples (C) for each interfering substance according to Table 2, and repeat each sample 3 times.

[0098] Table 2

[0099]

[0100] The above samples were tested using the kit prepared in Example 3, and the results are shown in Tables 3-5.

[0101] Table 3

[0102]

[0103] Table 4

[0104]

[0105] Table 5

[0106]

[0107] In the table above, "-" indicates negative; "+" indicates positive.

[0108] As shown in Table 3-5, the detection results are unaffected when the sample contains certain concentrations of bilirubin (<20 mg / dL), hemoglobin (<500 mg / dL), and triglycerides (<1000 mg / dL). This indicates that the kit in this application has excellent anti-interference properties.

[0109] 2. Stability test

[0110] Three different batches of reagent kits were taken and stored at 2–8°C after opening. Positive and negative controls were tested at 0, 4, 8, 12, and 13 months from the date of storage, with each test repeated three times. After each test, the bottle caps were tightened and the kits were returned to their original locations for future use. This was to assess the stability of the reagent kits. The results are shown in Table 6 below; the test results were stable and met the requirements.

[0111] Table 6

[0112]

[0113] In the table above, "-" indicates negative; "+" indicates positive.

[0114] 3. Specificity test

[0115] Antibodies against pathogenic microorganisms such as bacteria, mycoplasma, viruses, and Toxoplasma gondii were selected for specific analysis. The antibodies are shown in Table 7.

[0116] Table 7

[0117]

[0118] The antibody concentration was adjusted to 1000 ng / mL as the application solution. This application solution was used as the test sample and detected using the kit from Example 3. Positive and negative controls were also tested simultaneously. No false positive results were observed. The results are shown in Table 8.

[0119] Table 8

[0120]

[0121] The present invention has been disclosed above with reference to preferred embodiments, but it is not intended to limit the present invention. All technical solutions obtained by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the present invention.

Claims

1. Anti-Helicobacter pylori CagA subtype antibody detection kit, characterized by: It includes a CagA subtype recombinant antigen fragment, the amino acid sequence of which is shown in SEQ ID NO: 1; the CagA subtype recombinant antigen fragment includes two target sites, the amino acid sequences of which are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

2. The anti-Helicobacter pylori CagA subtype antibody detection kit according to claim 1, characterized in that: The edited nucleotide sequence of the CagA subtype recombinant antigen fragment is shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:

6.

3. The anti-Helicobacter pylori CagA subtype antibody detection kit according to claim 1, characterized in that: The kits detect anti-CagA-IgG antibodies and anti-CagA-IgM antibodies, respectively.

4. The anti-Helicobacter pylori CagA subtype antibody detection kit according to claim 1, characterized in that: The expression system for preparing the CagA subtype recombinant antigen fragment is Escherichia coli, insect baculovirus or 293F cells.

5. The anti-Helicobacter pylori CagA subtype antibody detection kit according to claim 1, characterized in that: The kit is a chemiluminescence immunoassay kit.

6. The anti-Helicobacter pylori CagA subtype antibody detection kit according to claim 5, characterized in that: It includes anti-CagA-IgG antibody positive quality control and negative quality control, anti-CagA-IgM antibody positive quality control and negative quality control, horseradish peroxidase HRP-labeled streptavidin, biotin-coated magnetic beads labeled with CagA subtype recombinant antigen fragments, acridinium ester derivatives labeled with anti-human IgG or IgM antibodies, auxiliary agents and substrate solution.

7. The anti-Helicobacter pylori CagA subtype antibody detection kit according to claim 6, characterized in that: The concentration of the CagA subtype recombinant antigen fragment is 0.1 μg / mL-5 μg / mL.

8. Use of the detection kit according to any one of claims 1 to 7 in non-disease diagnosis for Helicobacter pylori detection.

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