Anti-helicobacter pylori CagA subtype antibody detection kit and application thereof

Abstract

According to the anti-helicobacter pylori CagA subtype antibody detection kit and the application thereof provided by the invention, CagA subtype protein is optimized and screened to obtain a truncated fragment containing two dominant epitope targets, expression of different systems is performed to obtain a CagA subtype recombinant antigen fragment, the antigen fragment is utilized to screen an antibody as a quality control standard substance, and the detection kit is used for detecting the helicobacter pylori CagA subtype antibody. A chemiluminescence detection kit is developed, and whether a body is infected with helicobacter pylori or not can be rapidly detected.

Description

Technical Field

[0001] The invention belongs to the technical field of Helicobacter pylori detection, and particularly relates to an anti-Helicobacter pylori CagA subtype antibody detection kit and application thereof. Background Art

[0002] Helicobacter pylori is a spiral-shaped, flagellated, microaerophilic Gram-negative bacillus first discovered and documented by Marshall and Warren in 1982. H. pylori colonizes the gastric mucosa of over 50% of the world's population, with higher prevalence in developing countries worldwide. The acidic environment of the stomach is inhospitable to viruses, bacteria, and other microorganisms, but H. pylori has evolved unique abilities to overcome this harsh environment. H. pylori secretes urease, an enzyme that converts urea into ammonia and bicarbonate, neutralizing gastric acid and making the stomach a more hospitable environment for H. pylori. Having acquired this ability to survive, the stomach provides a unique habitat for H. pylori. Host immune cells and antibodies, which normally recognize and attack invading bacteria, are unable to reach H. pylori, which freely roam in the gastric mucosa. Instead, the ineffective host immune response continues to respond to the site of infection, with immune cells and epithelial cells dying and releasing nutrients that feed the gastric pathogen. H. pylori infection is primarily acquired during childhood and is primarily transmitted within families via the oral-oral or fecal-oral route.

[0003] However, most H. pylori infections are asymptomatic, but they can lead to chronic gastritis, with only 10% of infected individuals experiencing symptomatic disease. Furthermore, experimental and epidemiological studies have shown that H. pylori infection does increase the risk of gastric cancer. Consequently, the World Health Organization's International Agency for Research on Cancer classified H. pylori as a Class I carcinogen in 1994.

[0004] Individual differences in the risk of Helicobacter pylori-induced gastric disease are related to significant heterogeneity in host genetics and virulence factors among H. pylori strains. Several strain-specific virulence factors have been reported in the pathogenesis of H. pylori-related diseases, including CagA (cytotoxin-associated gene A), CagA (vacuolating cytotoxin A), HpaA (Helicobacter pylori adhesin A), BabA (blood group antigen-binding adhesin), DupA (duodenal ulcer-promoting gene A), and IceA (epithelial contact-induced) genes. Among these, CagA is a major virulence factor, which is associated with a higher risk of gastric cancer and peptic ulcers. The CagA protein can interact with intercellular proteins and activate signaling pathways through tyrosine phosphorylation-dependent or -independent mechanisms.

[0005] H. pylori can be divided into CagA-positive and CagA-negative strains based on the presence or absence of CagA (the gene encoding the CagA protein) in the bacterial genome. Due to structural polymorphism in the carboxyl-terminal (C-terminal) region, the size of the CagA protein encoded by CagA varies from 130 to 145 kDa. The CagA gene is located at one end of the Cag pathogenicity island (cag PAI). Approximately 30-40% of strains isolated in Western countries do not carry the Cag PAI and are CagA-negative, whereas nearly all H. pylori isolates from East Asian countries contain the structural and cancer-promoting functions of CagA.

[0006] Therefore, early screening for Helicobacter pylori can effectively prevent the incidence of gastric cancer. Numerous methods exist for detecting Helicobacter pylori infection, including direct bacterial examination, urease activity assays, immunological testing, and polymerase chain reaction. However, these methods all have their limitations. The most typical one is that even if the test result is positive, it cannot be confirmed whether the infection is caused by a virulent strain, which can lead to confusion in clinical medication. Summary of the Invention

[0007] In order to solve 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 a body is infected with Helicobacter pylori strains.

[0008] To solve the above technical problems, the present invention provides an anti-Helicobacter pylori CagA subtype antibody detection kit, specifically, an anti-CagA-IgM and anti-CagA-IgG antibody detection kit, comprising a CagA subtype recombinant antigen fragment, the amino acid sequence of which is shown in SEQ ID NO: 1:

[0009] SAKNGVNGTLIGNGLSKAEATTLSKNFSDIQKELNAELGNFNNNNNGLKNEPIYAKVNKKKAGQAASPEEPIYAQVAKKVNAKIDRLNQIASGLGGVGQAAGFPLKRHDKVDDLSKVGRSVSPE PIYATIDDLGGPFPLKRHDKVDDLSKVGRSVSPEPIYATIDDLGGPFPLKRHDKVDDLSKVWLSRNQELAQKIDNLNQAVSEAKAGFFGNLEQTIDKLKDSAKNNPVNLWAEGAKKCLLVCQRN.

[0010] The CagA subtype recombinant antigen fragment includes two targets, with amino acid sequences shown as SEQ ID NO: 2: SVSPEPIYA and SEQ ID NO: 3: SVSPEPIYA, respectively. The CagA subtype recombinant antigen fragment utilizes a truncated fragment of the H. pylori CagA subtype protein containing the dominant antigenic epitopes, namely the two targets, which are recombinantly expressed in different systems and then immunologically screened to obtain high-titer antigens for use in H. pylori antibody detection.

[0011] Further, the above-mentioned CagA subtype recombinant antigen fragment was converted into a nucleotide sequence according to the translation principles of E. coli codons, Homo sapiens codons, and Spodoptera frugiperda codons, 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:

[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 CagA subtypes include Escherichia coli, insect baculovirus or 293F cells.

[0019] Furthermore, the kit is a chemiluminescent immunoassay kit, comprising anti-CagA-IgG antibody positive quality control and negative quality control, anti-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.

[0020] Recombinant CagA subtype antigen fragments are labeled with biotin and coated with magnetic beads. When a serum sample containing anti-CagA-IgM or anti-CagA-IgG antibodies is added, they bind to the recombinant antigen fragments, forming a biotin-antigen-antibody conjugate. After washing to remove nonspecific antibodies from the serum, an anti-human IgG or IgM monoclonal antibody labeled with an acridinium ester derivative and streptavidin labeled with horseradish peroxidase (HRP) are added sequentially to form an HRP-streptavidin-biotin-antigen-antibody-antihuman monoclonal antibody-acridinium ester derivative complex. The HRP-streptavidin and acridinium ester derivative are spatially close to each other. Addition of an adjuvant and substrate solution triggers the reaction, producing flash chemiluminescence. The intensity of the luminescence is proportional to the antibody content in the serum sample.

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

[0022] In addition, the present invention also provides the use of the above detection kit in non-disease diagnosis for Helicobacter pylori detection.

[0023] The beneficial technical effects achieved by the present invention are as follows: the present invention provides an anti-Helicobacter pylori CagA subtype antibody detection kit and its application, optimizes and screens CagA subtype proteins to obtain truncated fragments containing two dominant epitope targets, expresses them in different systems to obtain CagA subtype recombinant antigen fragments, uses the antigen fragments to screen antibodies as quality control standards, and develops a chemiluminescence detection kit that can quickly detect whether the body is infected with Helicobacter pylori. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the purification result of CagA subtype recombinant antigen in a specific embodiment of 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 DESCRIPTION

[0026] 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.

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

[0028] Example 1 Preparation and Optimization of CagA Subtype Recombinant Antigen Fragments

[0029] 1. Plasmid Construction

[0030] First, the dominant antigen epitope segment 34-282AA of CagA (GenBank: BBJ21835.1) was screened and intercepted, and its amino acid sequence is shown in SEQ ID NO: 1:

[0031] SAKNGVNGTLIGNGLSKAEATTLSKNFSDIQKELNAELGNFNNNNNNGLKNEPIYAKVNKKKAGQAASPEEPIYAQVAKKVNAKIDRLNQIASGLGGVGQAAGFPLKRHDKVDDLSKVGRSVSPEPIYATI DDLGGPFPLKRHDKVDDLSKVGRSVSPEPIYATIDDLGGPFPLKRHDKVDDLSKVWLSRNQELAQKIDNLNQAVSEAKAGFFGNLEQTIDKLKDSAKNNPVNLWAEGAKKCLLVCQRN, including 2 target SEQ ID NO: 1: SSVPEPIYA and SEQ ID NO: 2: SSVPEPIYA.

[0032] The amino acid sequences were converted into nucleotide sequences according to the translation rules for E. coli, Homo sapiens, and Spodoptera frugiperda, respectively, and sent to a gene synthesis company (performed by GenScript Biotech). 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] [[ID=?]]II. Plasmid expression

[0041] It should be noted that there seems to be an error in the original text where the tag [[ID=?]] is used instead of in the translation of the relevant text. It has been corrected in the translation.The pCold-CagA(34-282) and pcDNA3.1-CagA(34-282) plasmids were transformed into E. coli DH5α plates and cultured overnight at 37°C. Colonies were picked from the plates and the culture system was gradually expanded. Finally, the bacterial suspension 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 insect baculovirus and cultured at 37°C for 48 hours. Recombinant strains were screened by blue-white spots growing on LB agarose 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 spots were picked and restreaked to confirm that the obtained white colonies were authentic. Recombinant bacmid DNA was extracted using the Biyuntian Baculovirus Shuttle Vector Bacmid Miniprep Kit.

[0043] 3. Expression and purification in different systems

[0044] 1. E. coli expression system: Transform the verified pCold-CagA(34-282) plasmid into E. coli BL21 plates and select a single colony for expansion in liquid culture. Cultivate at 37°C with shaking until the OD600 reaches 0.6-0.8. Add IPTG to a final concentration of 1 mM to induce expression. After 16-18 hours of induction, collect the bacterial suspension and centrifuge at 5000 rpm for 20 minutes. Resuspend the bacterial pellet in lysis buffer (50 mM Tris, pH 8.0, 0.15 M NaCl, 10 mM imidazole, 1 mM PMSF) and sonicate in ice water for 3 seconds, 6 seconds, for a total of 40 minutes. Subsequently, centrifuge at 13000 rpm for 40 minutes, and collect the supernatant for purification.

[0045] 2.293F cell expression system: The verified correct pcDNA3.1-CagA (34-282) plasmid was transfected into 293F cell line using PEI Max transfection reagent at a rate of 1×10 6Cells were transfected at a concentration of 10 cells / ml. 300 μg of pcDNA3.1-CagA (34-282) plasmid (filter-sterilized) was added to 30 mL of PBS, and 600 μg of filter-sterilized PEI solution was added. After standing at room temperature for 20 minutes, the mixture was added to the 293F cell suspension. The mixture was incubated in a shaking incubator at 37°C, 120 rpm, and 5% carbon dioxide for 24 hours, and then centrifuged at 4000 rpm for 5 minutes. The precipitate was collected and ultrasonically disrupted in ice water, with a 6-second pause for every 2 seconds of ultrasonication, for a total of 30 minutes of ultrasonication. The mixture was centrifuged at 12000 rpm for 40 minutes, and the supernatant was collected for purification.

[0046] 3. Insect baculovirus expression system: The extracted pFastBac-CagAbacmid DNA was transfected into insect cells Sf-9 using the insect transfection reagent LipoInsect produced by Biyuntian and cultured at 28°C. The culture supernatant was collected after 72-96 hours. Centrifuge 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 re-infected with insect cells Sf-9 to obtain the P2 generation virus. Insect cells Sf-9 were infected with the P2 generation virus during the logarithmic growth phase, and the cell density was maintained at 1×10 6 / ml-2×10 6 Infect with 500 μl of PBS and harvest cells 72 hours after infection. Centrifuge at 4000 rpm for 20 minutes at 4°C, and collect the precipitate for purification. Ultrasonicate in ice water for 2 seconds, 6 seconds pause, for a total of 30 minutes. Centrifuge at 12000 rpm for 40 minutes, and collect the supernatant for purification.

[0047] The glutathione-agarose resin was cleaned and placed in a reagent bottle. The substances to be purified collected from the three expression systems were mixed with 50% glutathione-agarose resin and incubated at room temperature for 30 minutes; centrifuged at 4°C and 3000 rpm for 5 minutes, the supernatant was discarded, 10 times the volume of 1×PBS was added to the precipitate and mixed by shaking, incubated for 5 minutes, and the impurities were washed away. The washing was repeated 3 times, and then an appropriate amount of glutathione elution buffer was added to the precipitate, incubated for 10 minutes, centrifuged at 4°C and 3000 rpm for 5 minutes, and the target protein was collected. The solution was exchanged with a 10kD ultrafiltration tube and concentrated to about 7 mg / ml. The bands were all around 53 kDa, as shown in the attached figure. Figure 1 As shown. Figure 1 As shown, CagA antigen protein was obtained in all three expression systems.

[0048] Example 2 Preparation of Calibrator Antibodies

[0049] (1) Animal immunization

[0050] Take 50 μg of CagA antigen protein obtained by the 293F cell expression system, emulsify it with an equal mass of adjuvant, and dilute it with a 1× PBS buffer. For the first immunization, use Freund's complete adjuvant for emulsification. When used, stir up the orange-yellow precipitate at the bottom, mix it with the CagA antigen protein, and emulsify it using the ultrasonic core of an ultrasonic disruptor. Select 6-8 week old BalB / C mice and inject 400 μL subcutaneously and intraperitoneally at multiple points. Two weeks later, perform the second immunization, and immunize once every two weeks thereafter. After the fifth immunization, perform a tail vein injection of 200 μL of CagA antigen protein three days before cell fusion for booster immunization.

[0051] (2) Cell fusion

[0052] Myeloma SP2 / 0 cells were revived and cultured to the logarithmic growth phase. Immunized mice were eyeballed and blood was collected, and serum was isolated as a positive control. After killing the mice, the spleens were washed with 75% ethanol. The spleens were removed under sterile conditions and washed once with incomplete culture medium. The splenic capsule was removed with forceps and repeatedly ground into a cell suspension, which was then counted to prepare a cell suspension. 100 mL of logarithmically growing myeloma SP2 / 0 cells was collected and washed twice by centrifugation in 1640 culture medium. Splenocytes and myeloma SP2 / 0 cells were mixed at a ratio of 2:1. The mixture was then centrifuged at 1000 rpm for 10 minutes, the supernatant discarded, and cell clumps were evenly broken up at the bottom of the tube. Preheated 50% PEG was added dropwise over 1 minute. The reaction was allowed to react at 37°C for 1 minute. After completion, 10 mL of 1640 culture medium was added to terminate the reaction.

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

[0054] (3) Screening of positive hybridoma cells

[0055] A 96-well ELISA plate was coated with recombinant CagA antigen protein and incubated with fusion cell supernatant, 1:1000 diluted immune mouse positive serum and 1:1000 diluted mouse negative serum, respectively. The plates were then incubated with horseradish peroxide (HRP)-labeled goat anti-mouse IgG and goat anti-mouse IgM. TMB substrate was added and the OD450nm value was measured using a microplate reader. When the OD450nm value of the negative serum was ≤0.1 and the detected OD value was more than twice that of the negative well, the well was considered a positive well.

[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 in a 37°C, 5% CO2 incubator. After 6-7 days, the wells were observed under a microscope, and the wells where clones had formed were marked. The cell supernatant was removed and analyzed by ELISA to select positive monoclonal cells. After three limiting dilutions, a hybridoma cell line that stably secreted anti-CagA antibodies was obtained.

[0057] (IV) Purification of antibodies of different subtypes

[0058] The hybridoma positive cell lines that stably express anti-CagA antibodies were screened out and cultured. After 7-9 days, the cells were centrifuged at 12,000 rpm for 20 minutes and the supernatant was collected. The supernatant was dialyzed into deionized water at 4°C. After 24 hours of dialysis, the cells were centrifuged at 12,000 rpm for 50 minutes and the precipitate was collected. The precipitate was dissolved with 5 ml of boric acid buffer and purified by chromatography using an SE gel column. The precipitate was eluted and collected with boric acid buffer, and the IgM protein peak was collected according to the 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 shown in the attached figure. Figure 2 shown.

[0059] Hybridoma-positive cell lines stably expressing anti-CagA antibodies were screened and cultured. After 7-9 days, the cells were centrifuged at 12,000 rpm for 20 minutes and the supernatant was collected. The supernatant was dialyzed into 20 mM PB, 150 mM NaCl, pH 7.4, at 4°C for 24 hours. The cells were then adsorbed onto an equilibrated protein A affinity column and the target antibody was eluted with 0.1 M glycine solution to obtain IgG antibodies. The IgM protein concentration was determined to be 5.1 mg / mL by UV spectrophotometry at OD 280 nm. The IgG purity was determined by SDS-PAGE. The results are shown in the attached figure. Figure 2 shown.

[0060] (V) Antibody titer detection

[0061] CagA recombinant antigen was diluted in 0.05 mol / L pH 9.6 carbonate buffer and coated onto a 96-well plate. The plate was blocked with BSA and washed with 1× PBST. The diluted monoclonal antibody sample was then added to the plate and incubated. A 1:4000 dilution of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG or goat anti-mouse IgM was then added, followed by color development with TMB substrate. OD 450 nm was measured using a microplate reader. The titers of both the 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] (1) The reagent contains the following components:

[0064] Table 1: Kit Contents

[0065]

[0066]

[0067] (2) Preparation process

[0068] 1. Preparation of Quality Control 1 and Quality Control 2

[0069] The anti-CagA-IgG and CagA-IgM antibodies obtained in Example 2 were diluted with 0.1 M phosphate buffer to concentrations of 0.05 ng / mL and 5 ng / mL, respectively, as positive quality controls and stored at 2-8°C for later use.

[0070] 2. Biotin-labeled CagA recombinant antigen-coated magnetic beads: Add 0.5 ml of commercially available activated magnetic beads (JSR LifeSciences Magnosphere 2023MC 03) to a 5 mL reaction cup, place in a dedicated test tube rack, mix and wash with 0.1 mol / L PB buffer, pH 9.5, and repeat this operation three times. Add the biotin-labeled CagA subtype recombinant antigen solution to the above magnetic beads, mix and react at room temperature for 4 hours, wash the coated magnetic beads three times with 0.1 mol / L phosphate buffer, pH 7.2, and then prepare a 0.05% biotin-labeled CagA recombinant antigen-coated magnetic bead solution with magnetic bead storage buffer and set aside.

[0071] 3. The preparation process of reagent 1, reagent 3, reagent 4, and reagent 5 is not limited and can be realized by any preparation process in the prior art, which will not be described in detail here.

[0072] (3) Detection method

[0073] The steps for detecting CagA-IgG and CagA-IgM antibodies are as follows:

[0074] 1. Sample addition: Add 25uL of positive control 1 or positive control 2 and the sample to be tested, and 225uL of reagent into the reaction tube, shake and mix, and incubate at 37°C for 10 minutes;

[0075] 2. Washing: Wash three times with washing solution (0.05 M tris buffer).

[0076] 3. Add reagent 1 and reagent 3, shake and mix, and incubate at 37°C for 5 minutes;

[0077] 3. Detection: Add 45 μL of reagent, shake and mix thoroughly, let stand for 1-2 minutes; then add 575 μL of reagent, detect immediately, and read the signal value. Read the luminescence value of the positive quality control and the sample separately.

[0078] 4. Calculation: Calculate the sample concentration by using the sample RLU. Result judgment:

[0079] Positive: the ratio of sample OD value to Cut-off OD value is greater than 1;

[0080] Negative: the ratio of sample OD value to Cut-off OD value is less than 0.9;

[0081] Gray area: the ratio of sample OD value to Cut-off OD value is between 0.9 and 1.0.

[0082] 5. Clinical interpretation of the results:

[0083] 5.1 Anti-CagA-IgM

[0084] Anti-CagA-IgM positive: indicates recent infection with CagA strain.

[0085] Anti-CagA-IgM negative: indicates no recent infection with CagA strain.

[0086] Anti-CagA-IgM gray zone: Combined with clinical diagnosis, or regular review.

[0087] 5.2 Anti-CagA-IgG

[0088] Anti-CagA-IgG positive: indicates previous infection with CagA subtype strain.

[0089] Anti-CagA-IgG negative: No infection with CagA subtype strain.

[0090] Anti-CagA-IgG gray zone: Combined with clinical diagnosis, or regular review.

[0091] (IV) Kit performance evaluation

[0092] 1. Endogenous substance interference experiment

[0093] Clinical abnormalities include lipemia, icterus, and hemolysis. Triglycerides, bilirubin, and hemoglobin are also major endogenous substances that interfere with experimental results. We use standardized interfering substances for interference experiments. The specific process is as follows:

[0094] 1) Preparation of basic samples: Clinical negative samples, weakly positive samples, and positive samples are used as basic samples, numbered 1, 2, and 3 respectively.

[0095] 2) Preparation of Interferor Solutions: Prepare interferor stock solutions using pure solvents at a concentration at least 20 times the experimental concentration to minimize dilution of the underlying sample matrix. The preparation of each interferor stock solution is shown in Table 1.

[0096] Table 1

[0097]

[0098] 3) Sample preparation: Prepare the experimental sample (T) and control sample (C) of each interfering substance according to Table 2, and repeat each sample 3 times.

[0099] Table 2

[0100]

[0101] The above samples were tested using the kit prepared in Example 3, and the results are shown in Table 3-5.

[0102] Table 3

[0103]

[0104] Table 4

[0105]

[0106] Table 5

[0107]

[0108] In the above table: “-” indicates negative; “+” indicates positive.

[0109] As can be seen from Tables 3-5, when the sample contains certain concentrations of bilirubin (<20 mg / dL), hemoglobin (<500 mg / dL), and triglyceride (<1000 mg / dL), the test results are not affected, indicating that the kit of the present application has good anti-interference performance.

[0110] 2. Stability test

[0111] Three different batches of test kits were opened and stored at 2-8°C. Positive and negative controls were tested three times at 0, 4, 8, 12, and 13 months from the date of storage. After each test, the bottles were capped and replaced for the next test. This was done to assess the stability of the test kits. The results, shown in Table 6 below, were stable and met the requirements.

[0112] Table 6

[0113]

[0114] In the above table: “-” indicates negative; “+” indicates positive.

[0115] 3. Specificity test

[0116] Antibodies to pathogenic microorganisms such as bacteria, mycoplasma, virus and Toxoplasma were selected for specificity analysis. The antibodies are shown in Table 7.

[0117] Table 7

[0118]

[0119] The above antibody concentration was adjusted to 1000 ng / mL as a working solution, and the working solution was used as the test sample for detection using the kit of Example 3. At the same time, positive and negative controls were tested, and no false positive results were found. The results are shown in Table 8.

[0120] Table 8

[0121]

[0122]

[0123] The present invention has been disclosed above with preferred embodiments, which are not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation solutions fall within the protection scope of the present invention.

Claims

1. Anti-Helicobacter pylori CagA subtype antibody detection kit, characterized in that: 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 chemiluminescent 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-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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