A reagent combination for typing helicobacter pylori and its preparation method and application

Abstract

The application relates to the field of biotechnology, in particular to a Helicobacter pylori antibody typing reagent combination and a preparation method and application thereof, a magnetic particle chemiluminescence reagent combination for Helicobacter pylori antibody typing, which comprises an acridinium ester mouse anti-human IgG complex solution, a biotin-Ure recombinant protein complex solution, a biotin-CVfp fusion protein complex solution and a streptavidin magnetic bead mother liquor; the biotin-Ure recombinant protein complex solution is obtained by marking Ure recombinant protein with biotin, the amino acid sequence of the Ure recombinant protein is shown as SEQ ID No. 1; the biotin-CVfp fusion protein complex solution is obtained by marking CVfp fusion protein with biotin, the CVfp fusion protein is fused and expressed to antigen epitopes of CagA and VacA, the amino acid sequence of the CVfp fusion protein is shown as SEQ ID No. 2, and the problems of complex detection indexes, high reagent cost and low detection flux for current Hp virulence factor typing are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a reagent combination for Helicobacter pylori antibody typing and a preparation method and application thereof. BACKGROUND

[0002] Helicobacter pylori (Hp) is a pathogenic gram-negative bacterium that colonizes the human stomach, can be transmitted between populations, and can cause chronic gastritis, peptic ulcer and even gastric cancer.

[0003] After Helicobacter pylori enters the stomach, it will be subjected to an extremely acidic environment, and will secrete various virulence factor proteins, including urease (Ure), cytotoxin-associated protein A (CagA) and vacuole toxin A (VacA), etc. The pathogenicity of Hp is closely related to the virulence factors it secretes, and the presence of Hp can be determined by detecting Ure, and Hp can be typed by detecting CagA and VacA, so as to distinguish the virulence level of the strain that infects the human body. Among them, the type I strain is a strong virulent strain of Hp with strong pathogenicity, which expresses Ure and one or two of CagA or VacA; the type II strain is a weak virulent strain of Hp with weak pathogenicity, which only expresses Ure and does not express CagA and VacA. The Expert Consensus on Serological Detection of Helicobacter pylori in Physical Examination Population published in China proposes that Hp typing detection can provide important clinical basis for precise eradication of Hp.

[0004] The existing Hp typing reagent products on the market mainly include immunoblotting and immunochromatography, among which immunoblotting is recommended for serological Hp antibody typing detection in the Expert Consensus on Serological Detection of Helicobacter pylori in Physical Examination Population. However, for the diagnosis and virulence typing of Hp, immunoblotting needs to use two indicators of UreA and UreB to determine the presence of Hp infection, and then use two indicators of CagA and VacA to determine the virulence factor typing result, which increases the complexity and misjudgment probability of result determination, reduces the detection efficiency and increases the reagent cost. In addition, the existing Hp virulence factor typing detection reagent products on the market mostly need manual loading, and have low automation degree, which cannot meet the demand of high-throughput screening. SUMMARY

[0005] The purpose of the present application is to provide a reagent combination for Helicobacter pylori antibody typing and a preparation method and application thereof, to solve the problems of complex detection indicators, high reagent cost and low detection throughput of the current Hp virulence factor typing.

[0006] To achieve this purpose, the following technical solutions are adopted in the present application:

[0007] A Helicobacter pylori antibody typing magnetic particle chemiluminescence reagent combination, comprising acridine ester mouse anti-human IgG complex solution, biotin-Ure recombinant protein complex solution, biotin-CVfp fusion protein complex solution, and streptavidin magnetic bead mother liquor;

[0008] The biotin-Ure recombinant protein complex solution is obtained by biotin labeling Ure recombinant protein, and the amino acid sequence of the Ure recombinant protein is shown as SEQ ID No. 1;

[0009] The biotin-CVfp fusion protein complex solution is obtained by biotin labeling CVfp fusion protein, and the CVfp fusion protein is fused and expressed to antigen epitopes of CagA and VacA, and the amino acid sequence of the CVfp fusion protein is shown as SEQ ID No. 2.

[0010] According to the preparation method of the Helicobacter pylori antibody typing magnetic particle chemiluminescence reagent combination, the following steps are included:

[0011] S1, Ure protein and CVfp fusion protein expression: the amino acid sequences of Ure recombinant protein and CVfp fusion protein are respectively translated into DNA sequences, the corresponding gene fragments are artificially synthesized, and then the gene fragments are introduced into a vector to construct a plasmid, the plasmid is transformed into competent cells of Escherichia coli BL21 (DE3) for culture, and then expression is induced to obtain bacterial liquid of each induction completion;

[0012] S2, purification of Ure protein and CVfp fusion protein: the bacterial liquid of each induction completion is centrifuged for the first time to collect bacterial precipitate and perform ultrasonic crushing to collect supernatant, saturated ammonium sulfate is added to the supernatant to make the final concentration reach 50%, and then the second centrifugation is performed to collect the precipitate, the precipitate is dissolved by using a phosphate buffer solution to obtain a corresponding protein redissolution solution, the protein redissolution solution is centrifuged for the third time, the supernatant is filtered to obtain a crude protein of each, and then the crude protein is purified by Ni 2+ NTA affinity chromatography, and the protein molecular weight is detected by SDS-PAGE, and finally the required Ure protein and CVfp fusion protein are obtained respectively;

[0013] S3, biotin labeling Ure recombinant protein and CVfp fusion protein: the Ure protein and the CVfp fusion protein obtained in step S2 are respectively treated by dialysis with a carbonate buffer solution with a pH of 9.6 overnight, mixed uniformly with a Biotin-PEG4-NHS solution, placed at room temperature for 1 h, then transferred into a phosphate buffer solution with a pH of 7.4 for dialysis overnight at 4°C, and then mixed with an equal volume of glycerol to obtain biotin-Ure recombinant protein complex solution and biotin-CVfp fusion protein complex solution respectively, and stored at -20°C;

[0014] S4, the acridinium ester mouse anti-human IgG complex solution is obtained by using acridinium ester to label the mouse anti-human IgG antibody, and is stored at -20°C.

[0015] Preferably, in step S1, the used carrier is one of pET-28a, pET-21(+), pET-24(+) or pET-23(+).

[0016] Isopropyl-β-d-thiogalactopyranoside is added for inducing expression.

[0017] Preferably, in step S2, the speed of the second centrifugation is 12000 rpm, the speed of the third centrifugation is 13000 rpm, the temperature is 4°C, and the centrifugation time is 30 min.

[0018] Preferably, in step S3, the concentration of the Biotin-PEG4-NHS solution is 10 mg / mL, and the solvent is anhydrous dimethyl sulfoxide.

[0019] 0.5 mg of the dialyzed protein is mixed with 5 μL of the Biotin-PEG4-NHS solution.

[0020] Preferably, in step S2, the crude protein is obtained by filtering through a 0.45 μm filter membrane after the third centrifugation.

[0021] A non-diagnostic detection method for H. pylori antibody typing, using the H. pylori antibody typing magnetic particle chemiluminescence reagent combination, comprises the following steps:

[0022] (1) The acridinium ester mouse anti-human IgG complex solution is diluted to 0.05-0.2 μg / mL to obtain R1 reagent working solution;

[0023] The biotin-Ure recombinant protein complex solution is diluted to 0.5-2 μg / mL to obtain Ure-R2 reagent working solution, and the biotin-CVfp fusion protein complex solution is diluted to 0.5-2 μg / mL to obtain CVfp-R2 reagent working solution;

[0024] The streptavidin magnetic bead mother liquor is diluted to 0.3-0.5 μg / mL to obtain R3 reagent working solution;

[0025] (2) The reagent working solution obtained in step (1) is divided into reagent bottles, the R1 reagent working solution, the Ure-R2 reagent working solution and the R3 reagent working solution are installed on the full-automatic chemiluminescence analyzer, the sample is added, and Ure-CLIA test is performed; the R1 reagent working solution, the CVfp-R2 reagent working solution and the R3 reagent working solution are installed on the full-automatic chemiluminescence analyzer, the sample is added, and CVfp-CLIA test is performed;

[0026] (3) judging according to the chemiluminescence signal obtained by testing.

[0027] Preferably, in step (2), the sample volume is 10-30 μL, and the addition amount of R1 reagent working solution, Ure-R2 reagent working solution, CVfp-R2 reagent working solution and R3 reagent working solution is 30-70 μL.

[0028] Further, in the testing process of step (2), incubation and washing are required twice, the incubation time is 5-10 min, and the washing times are 3-6 times.

[0029] Then 100 μL of pre-priming solution is added for short-term incubation for 1 min, followed by addition of 100 μL of priming solution, and the chemiluminescence signal is immediately collected.

[0030] The technical solution provided by the present application can include the following beneficial effects:

[0031] 1. The technical solution is based on Ure recombinant protein with an amino acid sequence as shown in SEQ ID No. 1 and CVfp fusion protein with an amino acid sequence as shown in SEQ ID No. 2, a magnetic micro-particle chemiluminescence reagent combination is constructed, the detection index of Helicobacter pylori virulence factor typing is effectively reduced, the reagent cost is reduced, and the degree of automation is improved by using a full-automatic luminescence analyzer to meet the demand of high-throughput detection.

[0032] 2. The CVfp fusion protein with an amino acid sequence as shown in SEQ ID No. 2 can be better applied to a full-automatic luminescence analyzer, the signal-to-noise ratio is high, and the detection accuracy is improved. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 is a detection principle schematic diagram of the present application.

[0034] Figure 2 is a detection result diagram of example group 1 of the present application.

[0035] Figure 3 is a detection result diagram of comparative example group 1-1 of the present application.

[0036] Figure 4 is a detection result diagram of comparative example group 1-2 of the present application.

[0037] Figure 5 is a detection result diagram of comparative example group 1-3 of the present application.

[0038] Figure 6 is an ELISA detection result diagram of example group 2-1 of the present application.

[0039] Figure 7This is a graph showing the ELISA detection results of Embodiment 2-2 of the present invention.

[0040] Figure 8 These are ELISA detection results for Examples 2-3 of the present invention.

[0041] Figure 9 These are ELISA detection results for Examples 2-4 of the present invention.

[0042] Figure 10 This is a chemiluminescence signal result diagram of Example 4-1 of the present invention.

[0043] Figure 11 This is the ROC curve of embodiment group 4-1 of the present invention.

[0044] Figure 12 This is a chemiluminescence signal result diagram of embodiment group 4-2 of the present invention.

[0045] Figure 13 This is the ROC curve of embodiment group 4-2 of the present invention. Detailed Implementation

[0046] The technical solution of the present invention will be further illustrated below through specific embodiments.

[0047] To facilitate understanding of the present invention, a more complete description is provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0048] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0049] A magnetic microparticle chemiluminescent reagent combination for Helicobacter pylori antibody typing includes acridinium ester mouse anti-human IgG complex solution, biotin-Ure recombinant protein complex solution, biotin-CVfp fusion protein complex solution, and streptavidin magnetic bead mother solution;

[0050] The biotin-Ure recombinant protein complex solution was obtained by biotin-labeling Ure recombinant protein, and the amino acid sequence of the Ure recombinant protein is shown in SEQ ID No. 1;

[0051] The biotin-CVfp fusion protein complex solution is obtained by biotin labeling CVfp fusion protein, which expresses the antigenic epitopes of CagA and VacA through fusion. The amino acid sequence of the CVfp fusion protein is shown in SEQ ID No. 2.

[0052] To address the problems existing in the prior art, this invention proposes a reagent combination for Helicobacter pylori antibody typing. Compared to the immunoblotting method, which uses UreA and UreB indicators to determine the presence of Helicobacter pylori infection and then uses CagA and VacA indicators to determine the virulence factor typing results, this invention only requires the determination of two indicators. The corresponding indicator combinations are shown in Table 1 below:

[0053] Table 1

[0054]

[0055] This invention utilizes recombinant Ure protein to simultaneously fuse and express the antigenic epitopes of CagA and VacA, constructing a fusion protein CVfp (CagA+VacA fusion protein). Biotinylation is then applied to both the Ure recombinant protein and the CVfp fusion protein using bioconjugation technology to obtain biotin-Ure recombinant protein complex and biotin-CVfp fusion protein complex, respectively. These are then combined with acrid ester-labeled mouse anti-human IgG and streptavidin magnetic beads for magnetic microparticle chemiluminescent immunoassay. As long as either CagA or VacA antibody target is present in the sample, it can be captured by the CVfp fusion protein, allowing for the simultaneous detection of two indicators in a single immunoassay. Furthermore, by using the Ure recombinant protein to capture urease antibody targets, it enables the typing of Helicobacter pylori virulence factors, greatly simplifying the criteria for H. pylori typing. This improves detection efficiency, reduces testing costs, minimizes misinterpretations, and addresses the current problems of complex detection indicators and high reagent costs in H. pylori virulence factor typing.

[0056] Furthermore, this reagent combination is a magnetic microparticle chemiluminescence reagent, used in a fully automated chemiluminescence analyzer, and the corresponding detection principle is as follows: Figure 1As shown, during use, streptavidin magnetic beads bind to biotin-labeled Ure recombinant protein or biotin-labeled CVfp fusion protein. The corresponding protein then binds to the corresponding antibody present in the sample, which in turn binds to acridinium ester mouse anti-human IgG. Acridinium ester is a chemiluminescent substrate that releases a high-intensity signal under specific conditions, thereby detecting the corresponding antibody concentration in the sample. To ensure detection accuracy, the amino acid sequence of the CVfp fusion protein is shown in SEQ ID No. 2. This CVfp fusion protein exhibits a high signal-to-noise ratio, reducing the likelihood of false negatives in positive samples. Therefore, this CVfp fusion protein is used in the preparation of magnetic microparticle chemiluminescent reagents, meeting the requirements of chemiluminescent immunoassay and achieving higher detection rates and accuracy in subsequent tests. Using a fully automated chemiluminescence analyzer increases automation and throughput, addressing the current problem of low throughput in Hp virulence factor typing.

[0057] The CagA+VacA fusion protein (CVfp) sequence was constructed by using flexible links to intercalate the DNA sequences of CagA and VacA proteins. The specific amino acid sequence of the flexible linker is as follows: ggsggggsggrs.

[0058] The preparation method of the magnetic microparticle chemiluminescent reagent combination for Helicobacter pylori antibody typing, as described above, includes the following steps:

[0059] S1, Ure protein and CVfp fusion protein expression: The amino acid sequences of Ure recombinant protein and CVfp fusion protein were translated into DNA sequences, respectively. The corresponding gene fragments were artificially synthesized from the DNA sequences and then transferred into vectors to construct plasmids. The plasmids were transformed into Escherichia coli BL21(DE3) competent cells and cultured. After induction, the expression was obtained and the bacterial culture of each was obtained.

[0060] Purification of S2, Ure, and CVfp fusion proteins: The induced bacterial cultures were centrifuged once to collect the cell pellet, followed by sonication and supernatant collection. Saturated ammonium sulfate was added to the supernatant to achieve a final concentration of 50%, followed by a second centrifugation and pellet collection. The pellet was dissolved in phosphate buffer to obtain the corresponding protein reconstitution solutions. These solutions were then centrifuged a third time, and the supernatant was filtered to obtain the crude proteins. Finally, the proteins were purified using Ni... 2+ Purified by NTA affinity chromatography and the molecular weight of the protein was detected by SDS-PAGE, the desired Ure protein and CVfp fusion protein were finally obtained respectively.

[0061] S3, Biotin-labeled Ure recombinant protein and CVfp fusion protein: The Ure protein and CVfp fusion protein obtained in step S2 were dialyzed overnight in carbonate buffer at pH 9.6, then mixed with Biotin-PEG4-NHS solution, reacted at room temperature for 1 h, and then transferred to phosphate buffer at pH 7.4 and dialyzed overnight at 4°C. After that, an equal volume of glycerol was added and mixed well to obtain biotin-Ure recombinant protein complex solution and biotin-CVfp fusion protein complex solution, respectively, which were stored at -20°C.

[0062] S4. Acridinium ester-labeled mouse anti-human IgG antibody was used to obtain acridinium ester mouse anti-human IgG complex solution, which was stored at -20℃.

[0063] Specifically, the DNA sequence of the Ure recombinant protein is shown in SEQ ID No. 3, and the DNA sequence of the CVfp fusion protein is shown in SEQ ID No. 4.

[0064] Ure recombinant protein and CVfp fusion protein with the desired amino acid sequences were obtained through protein expression and purification. Biotin was then used to label the Ure recombinant protein and CVfp fusion protein. Currently, the commonly used Biotin-LC-NHS has a strong hydrophobic structure, leading to protein structural instability. However, the biotin used in step S3 is Biotin-PEG4-NHS, which is selective. The PEG chain is hydrophilic and biocompatible, reducing the changes in hydrophilicity and hydrophobicity after labeling the Ure recombinant protein and CVfp fusion protein. Furthermore, the PEG4 chain length is moderate, not significantly altering the protein structure, and can extend the biotin, reducing steric hindrance. This results in the prepared biotin-Ure recombinant protein complex and biotin-CVfp fusion protein complex having better stability, meeting the requirements of fully automated chemiluminescence analyzers, thus improving detection throughput and automation.

[0065] In step S1, the induced bacterial culture contains Ure recombinant protein and CVfp fusion protein within the bacterial cells. In step S2, a first centrifugation separates the bacterial cells from other substances such as the culture medium. The collected bacterial precipitate is then sonicated to release intracellular substances, including the desired Ure recombinant protein or CVfp fusion protein. Centrifugation separates soluble and insoluble components, and the collected supernatant is a crude extract containing Ure recombinant protein or CVfp fusion protein. Saturated ammonium sulfate is added to this supernatant to achieve a final concentration of 50%, ensuring specific precipitation of the Ure recombinant protein and CVfp fusion protein. A second centrifugation achieves preliminary separation from some still-dissolved impurities. The protein is then dissolved in phosphate buffer to obtain a protein reconstitution solution. A subsequent third centrifugation removes undissolved impurities from the previous step, and filtration further purifies the protein to obtain a crude product. This crude protein is then processed using Ni... 2+ -NTA affinity chromatography was used to purify the protein to obtain the desired Ure recombinant protein or CVfp fusion protein. SDS-PAGE was used to determine the protein molecular weight, ensuring that the obtained protein was the desired one.

[0066] Specifically, in step S4, the operation of labeling mouse anti-human IgG antibody with acridine ester involves dialyzing the mouse anti-human IgG antibody in phosphate buffer at pH 8.0 at 4°C overnight to replace the buffer. The antibody concentration is then measured using nanodrop. The acridine ester derivative NSP-SA-NHS is dissolved in anhydrous DMSO (dimethyl sulfoxide) and the concentration is adjusted to 5 mg / mL. 10 μL of the dissolved acridine ester derivative solution is mixed with 0.5 mg of mouse anti-human IgG antibody and reacted at room temperature in the dark for 1 h. 10% glycine solution is added to the reaction solution and reacted at room temperature in the dark for 30 min. The reacted solution is transferred to a centrifugal desalting column, centrifuged at 1500g for 1 min, and the filtrate is collected as the labeled product. An equal volume of glycerol is added and mixed to obtain the acridine ester mouse anti-human IgG complex solution.

[0067] In step S3, the buffer is replaced by dialyzing with carbonate buffer at pH 9.6, and unbound biotin molecules are removed by dialyzing with phosphate buffer at pH 7.4.

[0068] Specifically, the streptavidin magnetic bead mother liquor can be commercially available.

[0069] Preferably, in step S1, the carrier used is one of pET-28a, pET-21(+), pET-24(+), or pET-23(+);

[0070] Isopropyl-β-d-thiogalactoside was added to induce expression.

[0071] Specifically, pET-28a, pET-21(+), pET-24(+), or pET-23(+) are all pET series prokaryotic expression vectors. These vectors are all designed based on the same core pET system, have a strong T7 promoter system, use the replication origin derived from pBR322, carry the ampicillin resistance gene, and are widely used for the efficient expression of recombinant proteins in Escherichia coli.

[0072] Furthermore, expression was induced using isopropyl-β-d-thiogalactoside (IPTG), which, in conjunction with the aforementioned vector, is a lactose analogue that can bind to the lac repressor protein of E. coli, thereby relieving its inhibition of the lac repressor protein on the T7 promoter, thus initiating transcription and translation, and inducing the synthesis of Ure recombinant protein and CVfp fusion protein.

[0073] The specific procedure for inducing expression is to add 0.5 mM isopropyl-β-d-thiogalactoside to induce expression for 6 h, which can ensure the full expression of the required Ure recombinant protein and CVfp fusion protein, while reducing protein degradation caused by excessive induction.

[0074] Specifically, in step S1, the plasmid is transformed into Escherichia coli BL21(DE3) competent cells for initial culture, and the transformed bacterial culture is inoculated into LB liquid medium containing kanamycin and cultured at 37°C until OD600 is 1.0 before induction culture.

[0075] Preferably, in step S2, the second centrifugation speed is 12000 rpm, the third centrifugation speed is 13000 rpm, the temperature is 4℃, and the centrifugation time is 30 min.

[0076] Specifically, protein denaturation is reduced by maintaining a low temperature of 4°C. This involves limiting the rotation speeds of the second and third centrifugations to ensure complete sedimentation of the protein precipitate generated by ammonium sulfate precipitation during the second centrifugation, separating it from other proteins in the supernatant; and ensuring the removal of undissolved impurities during reconstitution to prevent interference with subsequent purification.

[0077] Preferably, in step S3, the concentration of the Biotin-PEG4-NHS solution is 10 mg / mL, and the solvent is anhydrous dimethyl sulfoxide;

[0078] Each 0.5 mg of dialyzed protein was mixed with 5 μL of Biotin-PEG4-NHS solution.

[0079] Specifically, Biotin-PEG4-NHS was dissolved in anhydrous DMSO and the concentration was adjusted to 10 mg / mL to ensure biotin activity. Each 0.5 mg of dialyzed protein was mixed with 5 μL of Biotin-PEG4-NHS solution to ensure labeling efficiency and meet the requirements for subsequent magnetic bead binding, while avoiding over-labeling that would affect protein activity.

[0080] Preferably, in step S2, after the third centrifugation, the crude protein is obtained by filtration through a 0.45 μm filter membrane.

[0081] Specifically, the 0.45μm filter membrane can trap tiny particles remaining in the supernatant from the third centrifugation, such as submicron-sized cell debris and insoluble protein aggregates, for further purification, ensuring that the packing material of the subsequent affinity chromatography column is not contaminated and improving purification efficiency.

[0082] A non-diagnostic detection method for Helicobacter pylori antibody typing, using the above-mentioned combination of magnetic microparticle chemiluminescent reagents for Helicobacter pylori antibody typing, includes the following steps:

[0083] (1) Dilute the acridinium ester mouse anti-human IgG complex solution to 0.05-0.2 μg / mL to obtain the working solution of reagent R1;

[0084] Dilute the biotin-Ure recombinant protein complex solution to 0.5-2 μg / mL to obtain the Ure-R2 reagent working solution, and dilute the biotin-CVfp fusion protein complex solution to 0.5-2 μg / mL to obtain the CVfp-R2 reagent working solution;

[0085] Dilute the streptavidin magnetic bead stock solution to 0.3-0.5 μg / mL to obtain the working solution of reagent R3;

[0086] (2) Dispense the reagent working solution obtained in step (1) into reagent bottles, install the R1 reagent working solution, Ure-R2 reagent working solution and R3 reagent working solution on the fully automated chemiluminescence analyzer, add samples, and perform Ure-CLIA test; install the R1 reagent working solution, CVfp-R2 reagent working solution and R3 reagent working solution on the fully automated chemiluminescence analyzer, add samples, and perform CVfp-CLIA test;

[0087] (3) Make a judgment based on the chemiluminescence signal obtained from the test.

[0088] Specifically, the reagent working solution is diluted to obtain the required concentration to ensure that the chemiluminescence signal value can be collected and the detection rate is guaranteed.

[0089] In step (2), for the same sample, Ure-CLIA test and CVfp-CLIA test need to be performed. The specific difference is that the working solution of R2 reagent is different, that is, different proteins are used to bind the corresponding antibodies in the sample.

[0090] Specifically, the sample to be tested is serum.

[0091] Preferably, in step (2), the sample aspiration volume is 10-30 μL, and the addition amounts of R1 reagent working solution, Ure-R2 reagent working solution, CVfp-R2 reagent working solution and R3 reagent working solution are all 30-70 μL.

[0092] Specifically, under these limits on sample volume and reagent addition, it is ensured that the final chemiluminescence signal value can be accurately collected and detected.

[0093] Preferably, during the test in step (2), two incubation and cleaning processes are required, with an incubation time of 5-10 minutes and 3-6 cleaning cycles.

[0094] Then, after adding 100 μL of pre-excitation solution and incubating briefly for 1 min, 100 μL of excitation solution was added, and the chemiluminescence signal was immediately collected.

[0095] Ensure that the final chemiluminescence signal value can be accurately collected and detected.

[0096] The pre-activation solution is composed of hydrogen peroxide, and the activation solution is composed of sodium hydroxide.

[0097] The technical solution of the present invention will be further illustrated below through specific embodiments.

[0098] Preparation of CVfp fusion protein according to this technical solution:

[0099] S1. Expression of CVfp fusion protein: The amino acid sequence of CVfp fusion protein is shown in SEQ ID No. 2. The amino acid sequence of CVfp fusion protein is translated into DNA sequence. The corresponding gene fragment is artificially synthesized from the DNA sequence and then transformed into pET-28a vector to construct plasmid. The plasmid is transformed into Escherichia coli BL21(DE3) competent cells for initial culture. The transformed bacterial culture is inoculated into LB liquid medium containing kanamycin and cultured at 37℃ until OD600 is 1.0. Then, 0.5 mM isopropyl-β-d-thiogalactoside is added to induce expression for 6 h to obtain the induced bacterial culture.

[0100] S2. Purification of CVfp fusion protein: After the induction of the bacterial culture, the bacterial cell pellet was collected by centrifugation and ultrasonically disrupted. The supernatant was collected, and saturated ammonium sulfate was added to the supernatant to achieve a final concentration of 50%. The mixture was then centrifuged at 12,000 rpm for 30 min at 4°C to collect the pellet. The pellet was dissolved in phosphate buffer to obtain a CVfp fusion protein reconstituted solution. Subsequently, the solution was centrifuged at 13,000 rpm for 30 min, and the supernatant was filtered through a 0.45 μm filter membrane to obtain the crude protein. The crude protein was then purified using Ni... 2+ The crude protein was purified by NTA affinity chromatography, and the molecular weight of the purified protein was determined by SDS-PAGE to obtain the desired CVfp fusion protein.

[0101] Preparation of Ure recombinant protein according to this technical solution:

[0102] S1. Expression of Ure recombinant protein: The amino acid sequence of Ure recombinant protein is shown in SEQ ID No. 1. The amino acid sequence of Ure recombinant protein was translated into DNA sequence. The corresponding gene fragment was artificially synthesized from the DNA sequence and then transformed into pET-28a vector to construct plasmid. The plasmid was transformed into Escherichia coli BL21(DE3) competent cells for initial culture. The transformed bacterial culture was inoculated into LB liquid medium containing kanamycin and cultured at 37°C until OD600 reached 1.0. Then, 0.5 mM isopropyl-β-d-thiogalactoside was added to induce expression for 6 h to obtain the induced bacterial culture.

[0103] S2. Purification of Ure recombinant protein: After the induction of the bacterial culture, the bacterial cell pellet was collected by centrifugation and ultrasonically disrupted. The supernatant was then collected, and saturated ammonium sulfate was added to the supernatant to achieve a final concentration of 50%. The mixture was then centrifuged at 12,000 rpm for 30 min at 4°C to collect the pellet. The pellet was dissolved in phosphate buffer to obtain a reconstituted Ure recombinant protein solution, which was then centrifuged at 13,000 rpm for 30 min. The supernatant was filtered through a 0.45 μm filter membrane to obtain the crude protein. The crude protein was then purified using Ni... 2+ The crude protein was purified by NTA affinity chromatography, and the molecular weight of the purified protein was determined by SDS-PAGE to obtain the desired Ure recombinant protein.

[0104] Example Group 1

[0105] S1-S2: CVfp fusion protein was prepared according to this technical solution;

[0106] S3. Biotin-labeled CVfp fusion protein: The CVfp fusion protein was dialyzed overnight at 4°C in carbonate buffer (pH 9.6) to displace the buffer. The protein concentration was then measured using nanodrop. Biotin-PEG4-NHS was dissolved in anhydrous DMSO and the concentration was adjusted to 10 mg / mL. 5 μL of the dissolved Biotin-PEG4-NHS solution was mixed with 0.5 mg of dialyzed CVfp fusion protein and reacted at room temperature for 1 hour. The mixture was then transferred to phosphate buffer (pH 7.4) and dialyzed overnight at 4°C. Finally, an equal volume of glycerol was added and mixed to obtain the biotin-CVfp fusion protein complex solution, which was stored at -20°C.

[0107] S4. The mouse anti-human IgG antibody was dialyzed overnight at 4°C in phosphate buffer at pH 8.0 to replace the buffer. The antibody concentration was then measured using nanodrop. The acrid ester derivative NSP-SA-NHS was dissolved in anhydrous DMSO (dimethyl sulfoxide) and the concentration was adjusted to 5 mg / mL. 10 μL of the dissolved acrid ester derivative solution was mixed with 0.5 mg of mouse anti-human IgG antibody and reacted at room temperature in the dark for 1 h. 10% glycine solution was added to the reaction solution and reacted at room temperature in the dark for 30 min. The reacted solution was transferred to a centrifugal desalting column, centrifuged at 1500 g for 1 min, and the filtrate was collected as the labeled product. An equal volume of glycerol was added and mixed to obtain the acrid ester mouse anti-human IgG complex solution.

[0108] CVfp-CLIA Test:

[0109] (1) Dilute the acridinium ester mouse anti-human IgG complex solution to 0.1 μg / mL to obtain the working solution of reagent R1;

[0110] The biotin-CVfp fusion protein complex was diluted to 0.5 μg / mL to obtain the CVfp-R2 reagent working solution;

[0111] The streptavidin magnetic bead stock solution was diluted to 0.45 μg / mL to obtain the working solution of reagent R3;

[0112] (2) Dispense the prepared reagent working solution into reagent bottles, install them on the fully automated chemiluminescence analyzer, add serum samples, set the reaction sequence, aspirate 20 μL, add 50 μL of reagent working solution for R1, R2, and R3, the first incubation time is 5 min, the number of washes is 4, the second incubation time is 10 min, after washing 4 more times, add 100 μL of pre-excitation solution, incubate briefly for 1 min, add 100 μL of excitation solution and immediately collect chemiluminescence signals; multiple positive and negative serum samples are taken for detection.

[0113] (3) Based on the corresponding chemiluminescence signals, obtain the P / N value (mean signal of the positive sample group / mean signal of the negative sample group), such as Figure 2 As shown.

[0114] Comparative Example 1

[0115] Comparative Example 1-1

[0116] The difference between Comparative Example Group 1-1 and Example Group 1 lies in the amino acid sequence of the CVfp fusion protein used. Specifically, the amino acid sequence is consistent with the chimeric antigen amino acid sequence in patent CN110846335B. The results are as follows... Figure 3 As shown.

[0117] Comparative example group 1-2

[0118] The difference between Comparative Examples 1-2 and Example 1 is that the CVfp fusion protein used is the commercially available CagA+VacA fusion protein from Company A. The results are as follows: Figure 4 As shown.

[0119] Comparative examples 1-3

[0120] The difference between comparative examples 1-3 and example group 1 lies in the inconsistent amino acid sequence of the CVfp fusion protein used. The results are as follows: Figure 5 As shown.

[0121] Example Group 2

[0122] Validation of immune activity:

[0123] Example Group 2-1

[0124] S1-S2: Ure recombinant protein was prepared according to this technical solution:

[0125] A. Dilute the Ure recombinant protein to a concentration of 5 μg / mL using carbonate buffer, add 100 μL / well to each well of the microplate, and incubate overnight at 4 ℃.

[0126] B. After patting dry and washing 3 times with PBST (phosphate buffer containing Tween 20), add blocking buffer at a rate of 250 μL / well and block at 37 °C for 2 h.

[0127] C. After patting dry, wash three times with PBST, add 100 μL of commercial antibody, and react at 37 °C for 30 min; wherein, the commercial antibody is commercial UreA antibody of different concentrations;

[0128] D. After patting dry again, wash three times with PBST, add 100 μL of HRP (horseradish peroxidase) labeled mouse anti-human secondary antibody (diluted 1:8000 with phosphate buffer), and react at 37 ℃ for 30 min.

[0129] E. After patting dry, wash three times with PBST, add 100 μL of chromogenic substrate to each well, react at room temperature for 10 min, then add 50 μL of stop solution and measure the absorbance at 450 nm using a microplate reader. The results are as follows: Figure 6 As shown.

[0130] Example Group 2-2

[0131] The difference from Example Group 2-1 is that the commercially available antibodies used were commercially available UreB antibodies of different concentrations, and the results are as follows: Figure 7 As shown.

[0132] Example Group 2-3

[0133] The difference from Example Group 2-1 is that the CVfp fusion protein prepared according to the method of the present invention was used, and the commercially available antibodies used were commercially available CagA antibodies of different concentrations, as shown in the following figures. Figure 8 As shown.

[0134] Example Groups 2-4

[0135] The difference from Examples 2-3 is that the commercial antibodies used are commercial VacA antibodies of different concentrations, and the absorbance results are as follows. Figure 9 As shown.

[0136] Example Group 3

[0137] (1) Preparation of biotin-CVfp fusion protein complex:

[0138] S1-S2: CVfp fusion protein was prepared according to this technical solution;

[0139] S3. Biotin-labeled CVfp fusion protein: The CVfp fusion protein was dialyzed overnight at 4°C in carbonate buffer (pH 9.6) to displace the buffer. The protein concentration was then measured using nanodrop. Biotin-PEG4-NHS was dissolved in anhydrous DMSO and the concentration was adjusted to 10 mg / mL. 5 μL of the dissolved Biotin-PEG4-NHS solution was mixed with 0.5 mg of dialyzed CVfp fusion protein and reacted at room temperature for 1 hour. The mixture was then transferred to phosphate buffer (pH 7.4) and dialyzed overnight at 4°C. Finally, an equal volume of glycerol was added and mixed to obtain the biotin-CVfp fusion protein complex solution, which was stored at -20°C.

[0140] (2) Preparation of biotin-Ure recombinant protein complex solution:

[0141] S1-S2: Ure recombinant protein was prepared according to this technical solution;

[0142] S3. Biotin-labeled Ure recombinant protein: The Ure recombinant protein was dialyzed overnight at 4°C in carbonate buffer at pH 9.6 to replace the buffer. The protein concentration was then determined using nanodrop. Biotin-PEG4-NHS was dissolved in anhydrous DMSO and the concentration was adjusted to 10 mg / mL. 5 μL of the dissolved Biotin-PEG4-NHS solution was mixed with 0.5 mg of dialyzed Ure recombinant protein and reacted at room temperature for 1 hour. The mixture was then transferred to phosphate buffer at pH 7.4 and dialyzed overnight at 4°C. Finally, an equal volume of glycerol was added and mixed to obtain the biotin-Ure recombinant protein complex solution, which was stored at -20°C.

[0143] (3) Preparation of acridinium ester mouse anti-human IgG complex solution:

[0144] Mouse anti-human IgG antibody was dialyzed overnight at 4°C in phosphate buffer (pH 8.0) to replace the buffer. The antibody concentration was then measured using nanodrop. Acridinium ester derivative NSP-SA-NHS was dissolved in anhydrous DMSO (dimethyl sulfoxide) and the concentration was adjusted to 5 mg / mL. 10 μL of the dissolved acridinium ester derivative solution was mixed with 0.5 mg of mouse anti-human IgG antibody and reacted at room temperature in the dark for 1 h. 10% glycine solution was added to the reaction solution and reacted at room temperature in the dark for 30 min. The reacted solution was transferred to a centrifugal desalting column, centrifuged at 1500 g for 1 min, and the filtrate was collected as the labeled product. An equal volume of glycerol was added and mixed to obtain the acridinium ester mouse anti-human IgG complex solution.

[0145] The biotin-CVfp fusion protein complex, biotin-Ure recombinant protein complex, and acridinium ester mouse anti-human IgG complex prepared above were sealed and subjected to accelerated aging treatment. CVfp-CLIA and Ure-CLIA tests were then performed using chemiluminescent reagent combinations that were not accelerated aging, accelerated aging at 37°C for 3 days, accelerated aging at 37°C for 7 days, and accelerated aging at 37°C for 11 days, respectively.

[0146] CVfp-CLIA Test:

[0147] (1) Dilute the acridinium ester mouse anti-human IgG complex solution to 0.1 μg / mL to obtain the working solution of reagent R1;

[0148] The biotin-CVfp fusion protein complex was diluted to 0.5 μg / mL to obtain the CVfp-R2 reagent working solution;

[0149] The streptavidin magnetic bead stock solution was diluted to 0.45 μg / mL to obtain the working solution of reagent R3;

[0150] (2) Dispense the prepared reagent working solution into reagent bottles, install them on the fully automated chemiluminescence analyzer, add serum samples, set the reaction sequence, aspirate 20 μL, add 50 μL of reagent working solution for R1, R2 and R3, the first incubation time is 5 min, the number of washes is 4, the second incubation time is 10 min, after washing 4 more times, add 100 μL of pre-excitation solution, incubate briefly for 1 min, add 100 μL of excitation solution and immediately collect chemiluminescence signal; the serum sample includes one negative serum sample and one positive serum sample;

[0151] (3) Make a judgment based on the chemiluminescence signal obtained from the test.

[0152] Ure-CLIA Test:

[0153] (1) Dilute the acridinium ester mouse anti-human IgG complex solution to 0.1 μg / mL to obtain the working solution of reagent R1;

[0154] The biotin-Ure recombinant protein complex solution was diluted to 0.5 μg / mL to obtain the Ure-R2 reagent working solution;

[0155] The streptavidin magnetic bead stock solution was diluted to 0.45 μg / mL to obtain the working solution of reagent R3;

[0156] (2) Dispense the prepared reagent working solution into reagent bottles, install them on the fully automated chemiluminescence analyzer, add serum samples, set the reaction sequence, aspirate 20 μL, add 50 μL of reagent working solution for R1, R2 and R3, the first incubation time is 5 min, the number of washes is 4, the second incubation time is 10 min, after washing 4 more times, add 100 μL of pre-excitation solution, incubate briefly for 1 min, add 100 μL of excitation solution and immediately collect chemiluminescence signal; the serum sample includes one negative serum sample and one positive serum sample;

[0157] (3) Make a judgment based on the chemiluminescence signal obtained from the test.

[0158] The test results for this embodiment group 3 are shown in Table 2:

[0159] Table 2

[0160]

[0161] COI is the ratio between the chemiluminescence signal value RLU measured for each serum sample and the cut-off value at which the Youden index is at its maximum.

[0162] Example Group 4

[0163] Example Group 4-1

[0164] S1-S2: Ure recombinant protein was prepared according to this technical solution;

[0165] S3. Biotin-labeled Ure recombinant protein: The Ure recombinant protein was dialyzed overnight at 4°C in carbonate buffer at pH 9.6 to replace the buffer. The protein concentration was then determined using nanodrop. Biotin-PEG4-NHS was dissolved in anhydrous DMSO and the concentration was adjusted to 10 mg / mL. 5 μL of the dissolved Biotin-PEG4-NHS solution was mixed with 0.5 mg of dialyzed Ure recombinant protein and reacted at room temperature for 1 hour. The mixture was then transferred to phosphate buffer at pH 7.4 and dialyzed overnight at 4°C. Finally, an equal volume of glycerol was added and mixed to obtain the biotin-Ure recombinant protein complex solution, which was stored at -20°C.

[0166] S4. The mouse anti-human IgG antibody was dialyzed overnight at 4°C in phosphate buffer at pH 8.0 to replace the buffer. The antibody concentration was then measured using nanodrop. The acrid ester derivative NSP-SA-NHS was dissolved in anhydrous DMSO (dimethyl sulfoxide) and the concentration was adjusted to 5 mg / mL. 10 μL of the dissolved acrid ester derivative solution was mixed with 0.5 mg of mouse anti-human IgG antibody and reacted at room temperature in the dark for 1 h. 10% glycine solution was added to the reaction solution and reacted at room temperature in the dark for 30 min. The reacted solution was transferred to a centrifugal desalting column, centrifuged at 1500 g for 1 min, and the filtrate was collected as the labeled product. An equal volume of glycerol was added and mixed to obtain the acrid ester mouse anti-human IgG complex solution.

[0167] Ure-CLIA Test:

[0168] (1) Dilute the acridinium ester mouse anti-human IgG complex solution to 0.1 μg / mL to obtain the working solution of reagent R1;

[0169] The biotin-Ure recombinant protein complex solution was diluted to 0.5 μg / mL to obtain the Ure-R2 reagent working solution;

[0170] The streptavidin magnetic bead stock solution was diluted to 0.45 μg / mL to obtain the working solution of reagent R3;

[0171] (2) Dispense the prepared reagent working solution into reagent bottles, install them on the fully automated chemiluminescence analyzer, add serum samples, set the reaction sequence, aspirate 20 μL, add 50 μL of reagent working solution for R1, R2 and R3, the first incubation time is 5 min, the number of washes is 4, the second incubation time is 10 min, after washing 4 more times, add 100 μL of pre-activation solution, incubate briefly for 1 min, add 100 μL of activation solution and immediately collect chemiluminescence signal; the number of serum samples is 143, and the detection results of each serum sample are known according to the immunoblotting method (using the Helicobacter pylori antibody typing kit of Shenzhen Bolaote Biotechnology);

[0172] (3) Obtain the corresponding chemiluminescence signal value, such as Figure 10 As shown, and ROC curves are plotted based on the detection results, as follows. Figure 11 As shown.

[0173] Example 4-2

[0174] S1-S2: CVfp fusion protein was prepared according to this technical solution;

[0175] S3. Biotin-labeled CVfp fusion protein: The CVfp fusion protein was dialyzed overnight at 4°C in carbonate buffer (pH 9.6) to displace the buffer. The protein concentration was then measured using nanodrop. Biotin-PEG4-NHS was dissolved in anhydrous DMSO and the concentration was adjusted to 10 mg / mL. 5 μL of the dissolved Biotin-PEG4-NHS solution was mixed with 0.5 mg of dialyzed CVfp fusion protein and reacted at room temperature for 1 hour. The mixture was then transferred to phosphate buffer (pH 7.4) and dialyzed overnight at 4°C. Finally, an equal volume of glycerol was added and mixed to obtain the biotin-CVfp fusion protein complex solution, which was stored at -20°C.

[0176] S4. The mouse anti-human IgG antibody was dialyzed overnight at 4°C in phosphate buffer at pH 8.0 to replace the buffer. The antibody concentration was then measured using nanodrop. The acrid ester derivative NSP-SA-NHS was dissolved in anhydrous DMSO (dimethyl sulfoxide) and the concentration was adjusted to 5 mg / mL. 10 μL of the dissolved acrid ester derivative solution was mixed with 0.5 mg of mouse anti-human IgG antibody and reacted at room temperature in the dark for 1 h. 10% glycine solution was added to the reaction solution and reacted at room temperature in the dark for 30 min. The reacted solution was transferred to a centrifugal desalting column, centrifuged at 1500 g for 1 min, and the filtrate was collected as the labeled product. An equal volume of glycerol was added and mixed to obtain the acrid ester mouse anti-human IgG complex solution.

[0177] CVfp-CLIA Test:

[0178] (1) Dilute the acridinium ester mouse anti-human IgG complex solution to 0.1 μg / mL to obtain the working solution of reagent R1;

[0179] The biotin-CVfp fusion protein complex was diluted to 0.5 μg / mL to obtain the CVfp-R2 reagent working solution;

[0180] The streptavidin magnetic bead stock solution was diluted to 0.45 μg / mL to obtain the working solution of reagent R3;

[0181] (2) Dispense the prepared reagent working solution into reagent bottles, install them on the fully automated chemiluminescence analyzer, add serum samples, set the reaction sequence, aspirate 20 μL, add 50 μL of reagent working solution for R1, R2 and R3, the first incubation time is 5 min, the number of washes is 4, the second incubation time is 10 min, after washing 4 more times, add 100 μL of pre-activation solution, incubate briefly for 1 min, add 100 μL of activation solution and immediately collect chemiluminescence signal; the number of serum samples is 143, and the detection results of each serum sample are known according to the immunoblotting method (using the Helicobacter pylori antibody typing kit of Shenzhen Bolaote Biotechnology);

[0182] (3) Obtain the corresponding chemiluminescence signal value, such as Figure 12 As shown, and ROC curves are plotted based on the detection results, as follows. Figure 13 As shown.

[0183] Example Group 5

[0184] Example Group 5-1

[0185] S1-S2: Ure recombinant protein was prepared according to this technical solution;

[0186] S3. Biotin-labeled Ure recombinant protein: The Ure recombinant protein was dialyzed overnight at 4°C in carbonate buffer at pH 9.6 to replace the buffer. The protein concentration was then determined using nanodrop. Biotin-PEG4-NHS was dissolved in anhydrous DMSO and the concentration was adjusted to 10 mg / mL. 5 μL of the dissolved Biotin-PEG4-NHS solution was mixed with 0.5 mg of dialyzed Ure recombinant protein and reacted at room temperature for 1 hour. The mixture was then transferred to phosphate buffer at pH 7.4 and dialyzed overnight at 4°C. Finally, an equal volume of glycerol was added and mixed to obtain the biotin-Ure recombinant protein complex solution, which was stored at -20°C.

[0187] S4. The mouse anti-human IgG antibody was dialyzed overnight at 4°C in phosphate buffer at pH 8.0 to replace the buffer. The antibody concentration was then measured using nanodrop. The acrid ester derivative NSP-SA-NHS was dissolved in anhydrous DMSO (dimethyl sulfoxide) and the concentration was adjusted to 5 mg / mL. 10 μL of the dissolved acrid ester derivative solution was mixed with 0.5 mg of mouse anti-human IgG antibody and reacted at room temperature in the dark for 1 h. 10% glycine solution was added to the reaction solution and reacted at room temperature in the dark for 30 min. The reacted solution was transferred to a centrifugal desalting column, centrifuged at 1500 g for 1 min, and the filtrate was collected as the labeled product. An equal volume of glycerol was added and mixed to obtain the acrid ester mouse anti-human IgG complex solution.

[0188] Ure-CLIA Test:

[0189] (1) Dilute the acridinium ester mouse anti-human IgG complex solution to 0.1 μg / mL to obtain the working solution of reagent R1;

[0190] The biotin-Ure recombinant protein complex solution was diluted to 0.5 μg / mL to obtain the Ure-R2 reagent working solution;

[0191] The streptavidin magnetic bead stock solution was diluted to 0.45 μg / mL to obtain the working solution of reagent R3;

[0192] (2) Dispense the prepared reagent working solution into reagent bottles, install them on the fully automated chemiluminescence analyzer, add serum samples, set the reaction sequence, aspirate 20 μL, add 50 μL of reagent working solution for R1, R2 and R3, the first incubation time is 5 min, the number of washes is 4, the second incubation time is 10 min, after washing 4 more times, add 100 μL of pre-excitation solution, incubate briefly for 1 min, add 100 μL of excitation solution and immediately collect chemiluminescence signal;

[0193] The serum samples consisted of one negative serum sample and one positive serum sample, and each serum sample was tested 10 times.

[0194] (3) Calculate the ratio of the coefficient of variation of the chemiluminescence signal to the mean of the signal based on the corresponding chemiluminescence signal.

[0195] Example Group 5-2

[0196] S1-S2: CVfp fusion protein was prepared according to this technical solution;

[0197] S3. Biotin-labeled CVfp fusion protein: The CVfp fusion protein was dialyzed overnight at 4°C in carbonate buffer (pH 9.6) to displace the buffer. The protein concentration was then measured using nanodrop. Biotin-PEG4-NHS was dissolved in anhydrous DMSO and the concentration was adjusted to 10 mg / mL. 5 μL of the dissolved Biotin-PEG4-NHS solution was mixed with 0.5 mg of dialyzed CVfp fusion protein and reacted at room temperature for 1 hour. The mixture was then transferred to phosphate buffer (pH 7.4) and dialyzed overnight at 4°C. Finally, an equal volume of glycerol was added and mixed to obtain the biotin-CVfp fusion protein complex solution, which was stored at -20°C.

[0198] S4. The mouse anti-human IgG antibody was dialyzed overnight at 4°C in phosphate buffer at pH 8.0 to replace the buffer. The antibody concentration was then measured using nanodrop. The acrid ester derivative NSP-SA-NHS was dissolved in anhydrous DMSO (dimethyl sulfoxide) and the concentration was adjusted to 5 mg / mL. 10 μL of the dissolved acrid ester derivative solution was mixed with 0.5 mg of mouse anti-human IgG antibody and reacted at room temperature in the dark for 1 h. 10% glycine solution was added to the reaction solution and reacted at room temperature in the dark for 30 min. The reacted solution was transferred to a centrifugal desalting column, centrifuged at 1500 g for 1 min, and the filtrate was collected as the labeled product. An equal volume of glycerol was added and mixed to obtain the acrid ester mouse anti-human IgG complex solution.

[0199] CVfp-CLIA Test:

[0200] (1) Dilute the acridinium ester mouse anti-human IgG complex solution to 0.1 μg / mL to obtain the working solution of reagent R1;

[0201] The biotin-CVfp fusion protein complex was diluted to 0.5 μg / mL to obtain the CVfp-R2 reagent working solution;

[0202] The streptavidin magnetic bead stock solution was diluted to 0.45 μg / mL to obtain the working solution of reagent R3;

[0203] (2) Dispense the prepared reagent working solution into reagent bottles, install them on the fully automated chemiluminescence analyzer, add serum samples, set the reaction sequence, aspirate 20 μL, add 50 μL of reagent working solution for R1, R2 and R3, the first incubation time is 5 min, the number of washes is 4, the second incubation time is 10 min, after washing 4 more times, add 100 μL of pre-excitation solution, incubate briefly for 1 min, add 100 μL of excitation solution and immediately collect chemiluminescence signal;

[0204] The serum samples consisted of one negative serum sample and one positive serum sample, and each serum sample was tested 10 times.

[0205] (3) Replace the corresponding chemiluminescence signal with the COI value and calculate the ratio of the coefficient of variation of COI to the mean of COI.

[0206] The data results obtained from Example Group 5 are shown in Table 3 below:

[0207] Table 3

[0208]

[0209] Where CV is the coefficient of variation, SD is the standard deviation, and COI is the ratio between the chemiluminescence signal value RLU measured for each serum sample and the cut-off value at which the Youden index is at its maximum.

[0210] Example Group 6

[0211] Example Group 6-1

[0212] S1-S2: Ure recombinant protein was prepared according to this technical solution;

[0213] S3. Biotin-labeled Ure recombinant protein: The Ure recombinant protein was dialyzed overnight at 4°C in carbonate buffer at pH 9.6 to replace the buffer. The protein concentration was then determined using nanodrop. Biotin-PEG4-NHS was dissolved in anhydrous DMSO and the concentration was adjusted to 10 mg / mL. 5 μL of the dissolved Biotin-PEG4-NHS solution was mixed with 0.5 mg of dialyzed Ure recombinant protein and reacted at room temperature for 1 hour. The mixture was then transferred to phosphate buffer at pH 7.4 and dialyzed overnight at 4°C. Finally, an equal volume of glycerol was added and mixed to obtain the biotin-Ure recombinant protein complex solution, which was stored at -20°C.

[0214] S4. The mouse anti-human IgG antibody was dialyzed overnight at 4°C in phosphate buffer at pH 8.0 to replace the buffer. The antibody concentration was then measured using nanodrop. The acrid ester derivative NSP-SA-NHS was dissolved in anhydrous DMSO (dimethyl sulfoxide) and the concentration was adjusted to 5 mg / mL. 10 μL of the dissolved acrid ester derivative solution was mixed with 0.5 mg of mouse anti-human IgG antibody and reacted at room temperature in the dark for 1 h. 10% glycine solution was added to the reaction solution and reacted at room temperature in the dark for 30 min. The reacted solution was transferred to a centrifugal desalting column, centrifuged at 1500 g for 1 min, and the filtrate was collected as the labeled product. An equal volume of glycerol was added and mixed to obtain the acrid ester mouse anti-human IgG complex solution.

[0215] Ure-CLIA Test:

[0216] (1) Dilute the acridinium ester mouse anti-human IgG complex solution to 0.1 μg / mL to obtain the working solution of reagent R1;

[0217] The biotin-Ure recombinant protein complex solution was diluted to 0.5 μg / mL to obtain the Ure-R2 reagent working solution;

[0218] The streptavidin magnetic bead stock solution was diluted to 0.45 μg / mL to obtain the working solution of reagent R3;

[0219] (2) Dispense the prepared reagent working solution into reagent bottles, install them on the fully automated chemiluminescence analyzer, add serum samples, set the reaction sequence, aspirate 20 μL, add 50 μL of reagent working solution for R1, R2 and R3, the first incubation time is 5 min, the number of washes is 4, the second incubation time is 10 min, after washing 4 more times, add 100 μL of pre-activation solution, incubate briefly for 1 min, add 100 μL of activation solution and immediately collect chemiluminescence signal; the number of serum samples is 150, and each serum sample is a serum sample of the subject who has been tested for 13C urea breath test;

[0220] (3) Based on the corresponding chemiluminescence signal, calculate the ratio (COI) between the RLU value of the chemiluminescence signal measured for each serum sample and the cut-off value at the maximum Youden index. Determine the positive or negative result based on the COI value. A COI ≥ 1.0 indicates a positive result, and a COI < 1.0 indicates a negative result. Calculate the concordance rate with the 13C urea breath test results, as shown in Table 4 below:

[0221] Table 4

[0222]

[0223] UBT refers to the 13C urea breath test.

[0224] Example Group 6-2

[0225] S1-S2: CVfp fusion protein was prepared according to this technical solution;

[0226] S3. Biotin-labeled CVfp fusion protein: The CVfp fusion protein was dialyzed overnight at 4°C in carbonate buffer (pH 9.6) to displace the buffer. The protein concentration was then measured using nanodrop. Biotin-PEG4-NHS was dissolved in anhydrous DMSO and the concentration was adjusted to 10 mg / mL. 5 μL of the dissolved Biotin-PEG4-NHS solution was mixed with 0.5 mg of dialyzed CVfp fusion protein and reacted at room temperature for 1 hour. The mixture was then transferred to phosphate buffer (pH 7.4) and dialyzed overnight at 4°C. Finally, an equal volume of glycerol was added and mixed to obtain the biotin-CVfp fusion protein complex solution, which was stored at -20°C.

[0227] S4. The mouse anti-human IgG antibody was dialyzed overnight at 4°C in phosphate buffer at pH 8.0 to replace the buffer. The antibody concentration was then measured using nanodrop. The acrid ester derivative NSP-SA-NHS was dissolved in anhydrous DMSO (dimethyl sulfoxide) and the concentration was adjusted to 5 mg / mL. 10 μL of the dissolved acrid ester derivative solution was mixed with 0.5 mg of mouse anti-human IgG antibody and reacted at room temperature in the dark for 1 h. 10% glycine solution was added to the reaction solution and reacted at room temperature in the dark for 30 min. The reacted solution was transferred to a centrifugal desalting column, centrifuged at 1500 g for 1 min, and the filtrate was collected as the labeled product. An equal volume of glycerol was added and mixed to obtain the acrid ester mouse anti-human IgG complex solution.

[0228] CVfp-CLIA Test:

[0229] (1) Dilute the acridinium ester mouse anti-human IgG complex solution to 0.1 μg / mL to obtain the working solution of reagent R1;

[0230] The biotin-CVfp fusion protein complex was diluted to 0.5 μg / mL to obtain the CVfp-R2 reagent working solution;

[0231] The streptavidin magnetic bead stock solution was diluted to 0.45 μg / mL to obtain the working solution of reagent R3;

[0232] (2) Dispense the prepared reagent working solution into reagent bottles, install them on the fully automated chemiluminescence analyzer, add serum samples, set the reaction sequence, aspirate 20 μL, add 50 μL of reagent working solution for R1, R2 and R3, the first incubation time is 5 min, the number of washes is 4, the second incubation time is 10 min, after washing 4 more times, add 100 μL of pre-activation solution, incubate briefly for 1 min, add 100 μL of activation solution and immediately collect chemiluminescence signal; the number of serum samples is 248, and each serum sample is a serum sample of a subject who has been tested with the immunoblotting Helicobacter pylori antibody typing kit (Shenzhen Bolaite Biotechnology);

[0233] (3) Based on the corresponding chemiluminescence signal, calculate the ratio (COI) between the RLU value of the chemiluminescence signal measured for each serum sample and the cut-off value at the maximum Youden index. Determine the positive or negative result based on the COI value. A COI ≥ 1.0 indicates a positive result, and a COI < 1.0 indicates a negative result. Calculate the concordance rate with the 13C urea breath test results, as shown in Table 5 below:

[0234] Table 5

[0235]

[0236] Table 5 shows the detection compliance rate of the CVfp-CLIA detection method.

[0237] like Figure 1 As shown, multiple serum samples were tested according to the method in Example Group 1. The P / N value was 14.35, indicating a large difference between the average signal value of the positive sample group and the average signal value of the negative sample group. This demonstrates that the reagent prepared using the CVfp fusion protein proposed in this invention exhibits excellent discrimination ability when used in a fully automated chemiluminescence analyzer, achieving high-throughput detection while maintaining high detection efficiency. In contrast, although the fusion proteins used in Comparative Example Group 1 were constructed by chimerizing the DNA sequences of CagA and VacA proteins, the fusion proteins constructed based on these sequences, when applied to a fully automated chemiluminescence analyzer for magnetic particle chemiluminescence immunoassay, showed... Figures 2 to 5 As shown, its P / N value is 1.44-3.06, which is much smaller than the P / N value of Example Group 1. That is, the distinguishing ability of the fusion protein in Example Group 1 is far less than that of the CVfp fusion protein proposed in this invention. In other words, the CVfp fusion protein used in this invention reduces the detection indicators while being compatible with magnetic microparticle chemiluminescence immunoassay, meeting the requirements of high-throughput detection while having high detection accuracy and detection rate.

[0238] Regarding Example Group 2, the ELISA (enzyme-linked immunosorbent assay) results of Example Group 2-1, which involved incubating microplates coated with Ure recombinant protein with different concentrations of UreA antibody, are as follows: Figure 6 As shown, the absorbance increases with increasing UreA antibody concentration, demonstrating the immunoreactivity between the recombinant Ure protein and the UreA antibody. Similarly, as... Figure 7 As shown, the measured absorbance increases with increasing UreB antibody concentration, demonstrating that the Ure recombinant protein of this invention exhibits good immunoreactivity with UreB antibody, indicating that the Ure recombinant protein proposed in this invention can effectively detect UreA and UreB antibodies. Similarly, according to... Figure 8 and Figure 9 This demonstrates the immunoreactivity of the CVfp fusion protein of this technical solution with CagA and VacA antibodies, proving that the Ure recombinant protein and CVfp fusion protein proposed in this invention can specifically bind to the corresponding antibodies, meeting the requirements for detecting the corresponding antibodies in serum samples.

[0239] Examples 1 and 2 illustrate that the Ure recombinant protein and CVfp fusion protein proposed in this invention are suitable for magnetic microparticle chemiluminescent immunoassay.

[0240] Example 3 involved accelerated aging of the prepared magnetic microparticle chemiluminescence reagent combination for Helicobacter pylori antibody typing. Reagent combinations with different aging periods were used for magnetic microparticle chemiluminescence detection, specifically CVfp-CLIA and Ure-CLIA tests. Table 2 shows that after accelerated aging, the COI value and positive / negative results did not significantly change. According to the Arrhenius equation, the magnetic microparticle chemiluminescence reagent combination equivalent to this Helicobacter pylori antibody typing can be stored at 4°C for 18 months, demonstrating good stability and meeting application requirements.

[0241] Regarding Example Group 4, as Figure 10 As shown, the Youden index of the Ure-CLIA test method in Example Group 4-1 reaches a maximum cut-off value (critical value) of 480954. Figure 11 As shown, the area under the operating characteristic curve (AUC) of the Ure-CLIA test method compared with that of the immunoblotting method was 0.953, the sensitivity was 85.84%, and the specificity was 93.83%. The Youden index of the CVfp-CLIA test method in Example Group 4-2 was 382283 at its maximum cut-off value. Figure 13As shown, the area under the operating characteristic curve (ROC curve) of the CVfp-CLIA test method compared with that of the immunoblotting method is 0.995, the sensitivity is 96.08%, and the specificity is 100%. This indicates that the Ure-CLIA test method and the CVfp-CLIA test method proposed in this invention have excellent specificity and sensitivity and can be applied to the virulence factor typing of Helicobacter pylori.

[0242] Example 5 uses the Ure-CLIA and CVfp-CLIA testing methods to perform multiple tests on the samples. As shown in Table 3, after testing the same sample ten times using these two methods and judging by the COI value, the coefficient of variation of the COI value is 1.22-6.01%, all less than 10%. This shows that the Ure-CLIA and CVfp-CLIA testing methods proposed in this invention have good repeatability.

[0243] Example 6-1 used the Ure-CLIA test method to detect serum samples from 150 subjects who had undergone the 13C urea breath test. The ratio (COI) between the chemiluminescence signal value RLU measured for each serum sample and the cut-off value at which the Youden index was at its maximum was calculated. The positive or negative result was determined by the COI value; a COI ≥ 1.0 was considered positive, and a COI < 1.0 was considered negative. The concordance rate between the Ure-CLIA test method and the 13C urea breath test results was calculated, as shown in Table 4. The positive concordance rate between the Ure-CLIA test method and the 13C urea breath test was 90.59%, the negative concordance rate was 90.77%, and the overall concordance rate was 90.67%. Example 6-2 used the CVfp-CLIA test method to detect serum samples from 248 subjects who had previously been tested with the Western blot Helicobacter pylori antibody typing kit (Shenzhen Bolout Biotechnology). The ratio (COI) between the chemiluminescence signal value (RLU) and the cut-off value at which the Youden index was at its maximum was calculated for each serum sample. The COI value was used to determine whether the result was positive or negative; a COI ≥ 1.0 was considered positive, and a COI < 1.0 was considered negative. The concordance rate between the CVfp-CLIA test method and the Western blot Helicobacter pylori antibody typing kit results was calculated, as shown in Table 5. The positive concordance rate was 91.58%, the negative concordance rate was 95.21%, and the overall concordance rate was 93.55%. Therefore, as shown in Example 6, the Ure-CLIA test method and the CVfp-CLIA test method proposed in this invention have good concordance rates, i.e., high accuracy.

[0244] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0245] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A magnetic microparticle chemiluminescent reagent combination for Helicobacter pylori antibody typing, characterized in that: Including acridinium ester mouse anti-human IgG complex solution, biotin-Ure recombinant protein complex solution, biotin-CVfp fusion protein complex solution, and streptavidin magnetic bead mother solution; The biotin-Ure recombinant protein complex solution was obtained by biotin-labeling Ure recombinant protein, and the amino acid sequence of the Ure recombinant protein is shown in SEQ ID No. 1; The biotin-CVfp fusion protein complex solution is obtained by biotin labeling CVfp fusion protein, which expresses the antigenic epitopes of CagA and VacA through fusion. The amino acid sequence of the CVfp fusion protein is shown in SEQ ID No.

2.

2. The preparation method of the magnetic microparticle chemiluminescent reagent combination for Helicobacter pylori antibody typing according to claim 1, characterized in that, Includes the following steps: S1, Ure protein and CVfp fusion protein expression: The amino acid sequences of Ure recombinant protein and CVfp fusion protein were translated into DNA sequences, respectively. The corresponding gene fragments were artificially synthesized from the DNA sequences and then transferred into vectors to construct plasmids. The plasmids were transformed into Escherichia coli BL21(DE3) competent cells and cultured. After induction, the expression was obtained and the bacterial culture of each was obtained. Purification of S2, Ure, and CVfp fusion proteins: The induced bacterial cultures were centrifuged once to collect the cell pellet, followed by sonication and supernatant collection. Saturated ammonium sulfate was added to the supernatant to achieve a final concentration of 50%, followed by a second centrifugation and pellet collection. The pellet was dissolved in phosphate buffer to obtain the corresponding protein reconstitution solutions. These solutions were then centrifuged a third time, and the supernatant was filtered to obtain the crude proteins. Finally, the proteins were purified using Ni... 2+ Purified by NTA affinity chromatography and the molecular weight of the protein was detected by SDS-PAGE, the desired Ure protein and CVfp fusion protein were finally obtained respectively. S3, Biotin-labeled Ure recombinant protein and CVfp fusion protein: The Ure protein and CVfp fusion protein obtained in step S2 were dialyzed overnight in carbonate buffer at pH 9.6, then mixed with Biotin-PEG4-NHS solution, reacted at room temperature for 1 h, and then transferred to phosphate buffer at pH 7.4 and dialyzed overnight at 4°C. After that, an equal volume of glycerol was added and mixed well to obtain biotin-Ure recombinant protein complex solution and biotin-CVfp fusion protein complex solution, respectively, which were stored at -20°C. S4. Acridinium ester-labeled mouse anti-human IgG antibody was used to obtain acridinium ester mouse anti-human IgG complex solution, which was stored at -20℃.

3. The method for preparing a magnetic microparticle chemiluminescent reagent combination for Helicobacter pylori antibody typing according to claim 2, characterized in that: In step S1, the carrier used is one of pET-28a, pET-21(+), pET-24(+) or pET-23(+); Isopropyl-β-d-thiogalactoside was added to induce expression.

4. The preparation method of the magnetic microparticle chemiluminescent reagent combination for Helicobacter pylori antibody typing according to claim 2, characterized in that: In step S2, the second centrifugation speed was 12000 rpm, the third centrifugation speed was 13000 rpm, the temperature was 4℃, and the centrifugation time was 30 min.

5. The method for preparing a magnetic microparticle chemiluminescent reagent combination for Helicobacter pylori antibody typing according to claim 2, characterized in that: In step S3, the concentration of the Biotin-PEG4-NHS solution is 10 mg / mL, and the solvent is anhydrous dimethyl sulfoxide. Each 0.5 mg of dialyzed protein was mixed with 5 μL of Biotin-PEG4-NHS solution.

6. The method for preparing a magnetic microparticle chemiluminescent reagent combination for Helicobacter pylori antibody typing according to claim 2, characterized in that: In step S2, after the third centrifugation, the crude protein is obtained by filtration through a 0.45 μm filter membrane.

7. A non-diagnostic detection method for Helicobacter pylori antibody typing, characterized in that, The magnetic microparticle chemiluminescent reagent combination for Helicobacter pylori antibody typing as described in claim 1 includes the following steps: (1) Dilute the acridinium ester mouse anti-human IgG complex solution to 0.05-0.2 μg / mL to obtain the working solution of reagent R1; Dilute the biotin-Ure recombinant protein complex solution to 0.5-2 μg / mL to obtain the Ure-R2 reagent working solution, and dilute the biotin-CVfp fusion protein complex solution to 0.5-2 μg / mL to obtain the CVfp-R2 reagent working solution; Dilute the streptavidin magnetic bead stock solution to 0.3-0.5 μg / mL to obtain the working solution of reagent R3; (2) Dispense the reagent working solution obtained in step (1) into reagent bottles, install the R1 reagent working solution, Ure-R2 reagent working solution and R3 reagent working solution on the fully automated chemiluminescence analyzer, add samples, and perform Ure-CLIA test; install the R1 reagent working solution, CVfp-R2 reagent working solution and R3 reagent working solution on the fully automated chemiluminescence analyzer, add samples, and perform CVfp-CLIA test; (3) Make a judgment based on the chemiluminescence signal obtained from the test.

8. The non-diagnostic detection method for Helicobacter pylori antibody typing according to claim 7, characterized in that: In step (2), the sample volume is 10-30 μL, and the amount of R1 reagent working solution, Ure-R2 reagent working solution, CVfp-R2 reagent working solution and R3 reagent working solution added is 30-70 μL.

9. The non-diagnostic detection method for Helicobacter pylori antibody typing according to claim 8, characterized in that: During the test in step (2), two incubation and cleaning processes are required, with an incubation time of 5-10 minutes and 3-6 cleaning cycles. Then, after adding 100 μL of pre-excitation solution and incubating briefly for 1 min, 100 μL of excitation solution was added, and the chemiluminescence signal was immediately collected.

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