Antibodies targeting Helicobacter pylori type B urease and their applications

Abstract

The present invention relates to the field of immunoassay technology, and in particular to antibodies targeting Helicobacter pylori type B urease and their applications. The present invention provides an antibody that targets and recognizes Helicobacter pylori type B urease (ureB), specifically targeting an antigen epitope with a highly conserved amino acid sequence, and having high specificity and high affinity characteristics. The present invention further uses the antibody to prepare a Helicobacter pylori colloidal gold immunochromatographic test strip, which has a low detection limit and high sensitivity, reduces detection costs, shortens detection time, and improves detection efficiency, and is of great significance to units or regions that lack professional equipment and well-trained technicians.

Description

Technical Field

[0001] The present invention relates to the technical field of immune detection, and in particular to an antibody targeting Helicobacter pylori type B urease and an application thereof. Background Art

[0002] Helicobacter pylori is a common bacterium that lives in the human stomach. It is considered one of the main causes of diseases such as gastritis, gastric ulcers and gastric cancer. In H. pylori, urease is considered to be a key enzyme, and its structure plays a vital role in the survival and reproduction of this bacterium in the stomach.

[0003] Helicobacter pylori urease is an enzyme that can decompose urea into carbon dioxide and ammonia. The ammonia produced in this process can neutralize gastric acid, help bacteria survive in the gastric acid environment, and provide a large amount of energy source for Helicobacter pylori. The importance of urease in this process is self-evident, so understanding its structure is of great significance for studying the treatment and prevention of Helicobacter pylori-related diseases. Helicobacter pylori urease is a trimeric enzyme composed of mostly six subunits. Each subunit contains a catalytic active site, which contains two important nickel ions. These two nickel ions form coordination bonds with two glutamic acid residues in the subunit, respectively, so that urease obtains its catalytic activity.

[0004] There are some other important residues in the structure of urease. Among them, glutamic acid residue is considered to be the key residue in the catalytic center of urease. It can bind to urea and hydrolyze urea through a series of covalent and non-covalent interactions. In addition, histidine residue is also an important component in the structure of urease. It can bind to ammonia to form ammonia fragments, which are then excreted from the body. The activity of Helicobacter pylori urease can be regulated by many factors. Among them, pH value is an important factor. Urease shows high activity under neutral or alkaline conditions, while the activity is significantly reduced under acidic conditions. This regulatory mechanism enables Helicobacter pylori to survive in the acid environment of the stomach and use urea as an energy source. In addition, the activity of urease is also affected by other molecules. For example, some metal ions and inhibitors can affect the catalytic activity of urease. By regulating these factors, Helicobacter pylori can adapt and survive in different environments.

[0005] Due to the important role of urease in Helicobacter pylori, studying and understanding its structure is of great significance for the treatment and prevention of diseases caused by Helicobacter pylori. The structural information of urease can provide a basis for the design and development of specific inhibitors, thereby inhibiting the activity of urease and alleviating the diseases caused by Helicobacter pylori infection. In addition, urease can also be used as the basis of biosensors for the detection and monitoring of Helicobacter pylori infection. By detecting the specific activity or structural changes of urease, the goal of portable rapid detection of Helicobacter pylori can be achieved.

[0006] The structure of Helicobacter pylori urease is the key to the survival and reproduction of the bacteria in the stomach. By understanding the structural characteristics of urease and its regulatory mechanism, we can better study the treatment and prevention of Helicobacter pylori-related diseases. Urease also has a wide range of applications, including the development of inhibitors and biosensors. The comprehensive application of various means will help to better understand and deal with Helicobacter pylori-related issues.

[0007] In summary, the development of monoclonal antibodies targeting Helicobacter pylori urease is of great significance for the diagnosis of Helicobacter pylori and the discovery of antibody drugs with strong efficacy against Helicobacter pylori. Summary of the invention

[0008] The present invention provides an antibody or a functional fragment thereof targeting Helicobacter pylori type B urease (ureB), wherein the antibody or the functional fragment thereof comprises the following complementary determining regions:

[0009] CDR-VH1: amino acid sequence is DYPVH;

[0010] CDR-VH2: amino acid sequence IIWAAGSSNYNSWLMS;

[0011] CDR-VH3: amino acid sequence is AYYVSSDWFAY;

[0012] CDR-VL1: amino acid sequence is KSSRSLLTSSNQKNHLA;

[0013] CDR-VL2: amino acid sequence is WTSTRDS;

[0014] CDR-VL3: The amino acid sequence is QQYHNYSWT.

[0015] Preferably, the amino acid sequences of the light chain variable region and the heavy chain variable region of the antibody are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0016] In some embodiments, the antibody further comprises a constant region;

[0017] Preferably, the constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD;

[0018] Preferably, the species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human.

[0019] Preferably, the light chain constant region sequence of the constant region is as shown in SEQ ID NO.3, and the heavy chain constant region sequence of the constant region is as shown in SEQ ID NO.4.

[0020] Preferably, the amino acid sequence of the light chain of the antibody is shown as SEQ ID NO.5, and the amino acid sequence of the heavy chain of the antibody is shown as SEQ ID NO.6.

[0021] In some embodiments, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv, Fd, dAb, and scFv of the antibody.

[0022] The functional fragments of the above antibodies usually have the same binding specificity as the antibodies from which they are derived. It is easy for a person skilled in the art to understand based on the contents described in the present invention that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, a person skilled in the art can easily obtain the above functional fragments.

[0023] The functional fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems.

[0024] In some embodiments of the present invention, a nucleic acid molecule encoding the antibody or a functional fragment thereof is introduced into a host cell to obtain a recombinant cell, and the recombinant cell is cultured to obtain the antibody or a functional fragment thereof through separation and purification.

[0025] Furthermore, the present invention provides a nucleic acid encoding the antibody or a functional fragment thereof.

[0026] According to the amino acid sequence and codon rules of the above-mentioned antibody or its functional fragment, a person skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the above-mentioned antibody or its functional fragment. Due to the degeneracy of codons, the nucleotide sequence of the nucleic acid molecule encoding the above-mentioned antibody or its functional fragment is not unique, and all nucleic acid molecules capable of encoding the above-mentioned antibody or its functional fragment are within the protection scope of the present invention.

[0027] Preferably, the nucleic acid sequence is shown as SEQ ID NO.7 and SEQ ID NO.8.

[0028] The sequence shown in SEQ ID NO.7 encodes the light chain of the antibody, and the sequence shown in SEQ ID NO.8 encodes the heavy chain of the antibody.

[0029] Furthermore, the present invention provides a product, which contains the antibody or its functional fragment, or the nucleic acid;

[0030] Preferably, the product is any one of an antibody conjugate, a biological material, a drug, a detection reagent or a kit, and a test strip;

[0031] Preferably, in the product, the antibody or its functional fragment is labeled with a detectable marker;

[0032] Preferably, the product is used for detecting Helicobacter pylori (type B urease).

[0033] Preferably, the antibody conjugate is obtained by conjugating the antibody or a functional fragment thereof with a detectable marker.

[0034] Preferably, the medicine is used for treating diseases caused by Helicobacter pylori.

[0035] When the antibody of the present invention is applied to the design of therapeutic targets for Helicobacter pylori, therapeutic drugs for Helicobacter pylori can be designed. The therapeutic target design includes but is not limited to the target design of therapeutic antibodies.

[0036] In some embodiments, the detection reagent or kit is a quantitative or qualitative detection reagent or kit for Helicobacter pylori.

[0037] In some embodiments, the detection reagent or kit is used for ELISA detection, immunochemiluminescence detection or immunofluorescence detection.

[0038] In some embodiments, the test strip is a colloidal gold immunochromatographic test strip.

[0039] In some embodiments, the test strip is a lateral flow immunochromatographic test strip.

[0040] In some embodiments, the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or non-renewable cells or tissues.

[0041] In some embodiments, the detectable marker refers to a class of substances that have properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0042] In an optional embodiment, the detectable markers include, but are not limited to, fluorescent dyes, enzymes that catalyze the color development of substrates, radioactive isotopes, chemiluminescent agents, and nanoparticle markers.

[0043] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0044] In an optional embodiment, the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0045] In an optional embodiment, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxidase.

[0046] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0047] In an optional embodiment, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0048] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0049] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0050] Furthermore, the present invention provides the use of the antibody or its functional fragment, or the nucleic acid, or the product, or the Helicobacter pylori colloidal gold immunochromatographic test strip in at least one of the following aspects:

[0051] (1) Application in the preparation of products for detecting Helicobacter pylori;

[0052] (2) Application in the preparation of products for diagnosing diseases caused by Helicobacter pylori infection;

[0053] (3) Application in the detection of Helicobacter pylori for non-diagnostic and non-therapeutic purposes;

[0054] (4) Application in detecting the antigen content of Helicobacter pylori vaccine;

[0055] (5) Application in quality control of Helicobacter pylori vaccines.

[0056] Preferably, the product is any one of an antibody conjugate, a biological material, a medicine, a detection reagent or a kit, and a test strip.

[0057] In the above applications, the sample may be a sample from a living human or animal (including blood, excrement, oral and nasal secretions, etc.), or may be a sample not from a living human or animal, such as in vitro cultured cells or cell culture fluid.

[0058] In the above applications, vaccine quality control includes testing whether the quality, content, stability, etc. of the antigens in the vaccine are qualified.

[0059] In the above applications, enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, etc. can be used.

[0060] Furthermore, the present invention provides a Helicobacter pylori (type B urease) colloidal gold immunochromatographic test strip, comprising a sample pad, a colloidal gold binding pad, a reaction membrane, a water absorbent pad and a base plate; the reaction membrane is provided with a detection line and a quality control line, and the sample pad, colloidal gold binding pad, reaction membrane and water absorbent pad are sequentially pasted on the base plate; the detection line is coated with the antibody.

[0061] Preferably, the colloidal gold binding pad is coated with a colloidal gold-labeled antibody targeting Helicobacter pylori type B urease; more preferably, the amino acid sequences of the light chain and heavy chain of the colloidal gold-labeled antibody targeting Helicobacter pylori type B urease are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0062] Preferably, the quality control line is coated with goat anti-mouse polyclonal antibody.

[0063] Preferably, the concentration of the antibody coated on the colloidal gold binding pad is 10-15 μg / mL.

[0064] Preferably, the concentration of the antibody coated on the detection line is 1-2 mg / mL.

[0065] The Helicobacter pylori colloidal gold immunochromatographic test strip of the present invention belongs to a lateral flow immunochromatographic test strip, which is generally in the shape of a narrow strip, usually with a width of 4-6 mm and a length of 6-7 cm.

[0066] Preferably, the material of the sample pad is cellulose; the material of the colloidal gold binding pad is glass fiber; the material of the reaction membrane is nitrocellulose membrane (NC membrane); and the material of the water absorbent pad is cellulose.

[0067] When the lateral flow immunochromatography test strip is inserted into the solution to be tested, the solution will flow from the sample pad area to the binding pad area. When the solution contains the substance to be tested, the substance to be tested will bind to the colloidal gold (coupled) antigen or colloidal gold (coupled) antibody in the binding pad area. The colloidal gold (coupled) antibody or colloidal gold (coupled) antigen rehydrated by the solution or the complex formed with the sample to be tested will pass through the detection area (reaction membrane) and the absorbent pad area in turn under the action of capillary force. At this time, the lateral flow immunochromatography test strip will have two result display models, namely the standard model ( Figure 1 a) and the competition model ( Figure 1 b).

[0068] Figure 1aWhen the lateral flow immunochromatographic test paper uses the standard model (detection of type B urease antigen of Helicobacter pylori), the test line (containing specific antibodies targeting type B urease antigen of Helicobacter pylori) can only capture the complex of the antigen to be tested and the colloidal gold (coupled) primary antibody: If the antigen to be tested exists in the solution, the test line can capture the complex formed by the antigen to be tested and the colloidal gold (coupled) primary antibody. Since the colloidal gold (coupled) primary antibody itself will always be captured by the quality control line (containing the corresponding secondary antibody), the color of both the test line and the quality control line will change at this time, and the result is judged as a positive reaction. If the antigen to be tested does not exist in the solution, only the quality control line (containing the corresponding secondary antibody) can capture the colloidal gold (coupled) primary antibody and change color. At this time, the quality control line will be colored while the test line will not be colored, and the result is judged as a negative reaction.

[0069] Figure 1 bWhen the lateral flow test paper adopts a competitive model (detecting IgM / IgG antibodies targeting the B-type urease antigen of Helicobacter pylori), the antibody to be tested binds to the colloidal gold (coupled) B-type urease antigen coated on the binding pad area, and the antibody to be tested and the colloidal gold (coupled) B-type urease antigen can each be captured by the test line (containing anti-IgG / IgM antibodies). When the antibody to be tested is present, it can be captured by the test line while the colloidal gold (coupled) antigen cannot be captured by the test line (due to the competitive blocking of the antibody to be tested), and thus no color change can occur, but the quality control line (containing the B-type urease antigen paired antibody) can still capture the colloidal gold (coupled) antigen, at which time the test line does not develop color while the quality control line develops color, and the result is determined as a positive reaction; if the antibody to be tested does not exist in the solution, both the test line and the quality control line can capture the colloidal gold (coupled) antigen, at which time both the test line and the quality control line will change color, and the result is determined as a negative reaction.

[0070] The Helicobacter pylori colloidal gold immunochromatographic test strip provided by the present invention can achieve the general standards required for rapid diagnosis, such as sensitivity, stability, economy, user-friendliness and no need for equipment, and is of great significance to units or regions lacking professional equipment and untrained technicians.

[0071] Compared with the prior art, the present invention has the following beneficial effects:

[0072] The present invention provides an antibody for targeting and recognizing Helicobacter pylori type B urease (ureB), specifically targeting an antigen epitope with a highly conserved amino acid sequence, and having high specificity and high affinity characteristics. The present invention further uses the antibody to prepare a Helicobacter pylori colloidal gold immunochromatographic test strip, which has a low detection limit and high sensitivity, reduces detection costs, shortens detection time, and improves detection efficiency, and is of great significance to units or regions that lack professional equipment and well-trained technicians. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 This is a schematic diagram of the detection principle of the lateral flow immunochromatographic test strip; wherein, a: standard model; b: competitive model.

[0074] Figure 2 It is the relevant information about the antigenic epitopes and functional localization of Helicobacter pylori type B urease.

[0075] Figure 3 This is the SDS-PAGE identification result of recombinant Helicobacter pylori type B urease.

[0076] Figure 4 The graphs are the binding curves of the antibody of the present invention with the recombinant Helicobacter pylori type B urease antigen at gradient concentrations; wherein 0.5-g is the binding curve when each well is coated with 50 ng of antigen (100-L per well), and 1-g is the binding curve when each well is coated with 100 ng of antigen (100-L per well).

[0077] Figure 5 This is a graph showing the test results of Helicobacter pylori colloidal gold immunochromatographic test strips detecting different concentrations of recombinant Helicobacter pylori type B urease.

[0078] Figure 6 This is a graph showing the test results of the sensitivity of Helicobacter pylori colloidal gold immunochromatographic test strip in detecting recombinant Helicobacter pylori type B urease.

[0079] Figure 7 This is a graph showing the test results of Helicobacter pylori colloidal gold immunochromatographic test strips in detecting normal human feces samples. DETAILED DESCRIPTION

[0080] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0081] If no specific techniques or conditions are specified in the examples, all are carried out according to conventional methods or the techniques or conditions described in the literature in the field, or according to the product instructions. If no manufacturer is specified for the reagents and instruments used, all are conventional products that can be purchased through regular channels.

[0082] Example 1 Preparation of hybridomas producing monoclonal antibodies

[0083] 1. Design of Helicobacter pylori type B urease antigen

[0084] According to the amino acid sequence obtained by translating the Helicobacter pylori type B urease gene (GenBank: M60398.1) on GenBank (SEQ ID NO.11; the optimized gene sequence is shown in SEQ ID NO.12), the amino acid sequence of type B urease of Helicobacter pylori (ATCC 700392 / 26695) was selected for sequence comparison, and it was found that the sequence had a high homology with other Helicobacter pylori type B ureases. After immunogenicity, hydrophilicity and surface accessibility analysis, the full-length 569 amino acids of the CDS region of type B urease of Helicobacter pylori (ATCC 700392 / 26695) were finally selected as the subsequent recombinant protein, i.e., the sequence of the antigen (SEQ ID NO.13).

[0085] Related information on the antigenic epitopes and functional localization of Helicobacter pylori type B urease Figure 2 As shown (IEDB analysis software).

[0086] 2. Preparation of recombinant Helicobacter pylori type B urease

[0087] After gene synthesis and molecular cloning design, the recombinant Helicobacter pylori type B urease was finally expressed in the E. coli system. The protein is the antigen. The SDS-PAGE identification results are as follows Figure 3 shown.

[0088] 3. Immunization of mice

[0089] 1 mg / mL recombinant Helicobacter pylori type B urease was mixed with Freund's complete adjuvant at a ratio of 1:1 to 500 μL for emulsification, and then subcutaneously injected into 6-8 week old female Balb / c mice at multiple points, with each mouse inoculated with 100 μg of recombinant Helicobacter pylori type B urease (antigen). Three weeks later, 1 mg / mL antigen was mixed with Freund's incomplete adjuvant at a ratio of 1:1 to 500 μL for emulsification, and then subcutaneously injected at multiple points, with each mouse receiving 50 μg of antigen as a booster immunization. After three, six, and nine weeks, the mice were immunized again according to the aforementioned booster immunization process, for a total of 4 booster immunizations.

[0090] 4. Immune serum titer determination

[0091] 10 days after the fourth booster immunization, blood was collected from the tail vein of mice, and the titer of immune serum was determined by indirect ELISA. 50 μg of synthetic antigen recombinant Helicobacter pylori type B urease was dissolved in 10 mL 0.05 M pH 9.6 phosphate buffer, coated on a polystyrene 96-well plate, 100 μL / well, overnight at 4°C. The plate was washed three times with PBST (0.02 M PBS containing 0.05% v / v Tween-20), blocked with 100 μl / well of 10 mM PBS containing 1% BSA blocking solution at 37°C for 2 hours, washed three times with PBST (0.02 M PBS containing 0.05% v / v Tween-20), and prepared for use. Mouse immune serum was incubated with 10 mM PBS containing 1% BSA for 10 2 ~10 6 The plate was diluted 1:10000 and added to a 96-well plate, 100 μL / well, and then incubated at 37°C for 1 hour. After that, the plate was washed three times with PBST (0.02MPBS containing 0.05% v / vTween-20), and then 100 μL / well of horseradish peroxidase-labeled goat anti-mouse IgG (Sigma, INC.) was added, and then incubated at 37°C for 30 minutes, and the plate was washed as above. Then, 100 μL / well of TMB was added to each well for color development, and the reaction was stopped at 37°C at room temperature and away from light for 10 minutes, and then 2M H2SO4 was added at 50 μL / well to terminate the reaction. The absorbance at 450 nm was measured, and the pre-immune mouse serum was used as the negative control. The ratio of the measured value to the control value ≥ 2:1 was used as the positive judgment value to determine the immune serum titer. The results are shown in Table 1.

[0092] Table 1

[0093] Dilution multiple Immunization of mice 1 Immunization of mice 2 Immunization of mice 3 Immunization of mice 4 10000 3.22 3.12 3.16 3.3 50000 2.31 2.25 2.3 2.35 100000 1.40 1.34 1.44 1.43 500000 0.76 0.72 0.79 0.78 Pre-immune mouse serum 0.11 0.08 0.12 0.15

[0094] 5. Preparation of hybridomas

[0095] Take serum with a titer greater than 1:10 5For mice, 3 days before fusion, take the synthetic antigen Helicobacter pylori type B urease and mix it with an equal volume of PBS, and then inject it intraperitoneally into BALB / c mice to be fused at a volume of 50μg / 500μL per mouse for booster immunization. Feeder layer cell preparation process: Balb / c mice (about 4 weeks old) are killed by orbital vein bleeding, soaked in 75% ethanol for 3 minutes, with the ventral side facing up; open the chest cavity to separate the thymus, grind it with a cell sieve, and use preheated basal medium to suspend the cells. Preparation of peritoneal macrophages: On the day of fusion, select one healthy mouse (one spleen), remove its eyeball and bleed until the blood stops dripping. Kill by cervical dislocation, soak and disinfect in 75% alcohol for 5 minutes, move to the clean bench, and fix it on the dissection board with the abdomen facing up. Lift the mouse abdominal skin with tweezers, cut a small incision with scissors (be careful not to damage the peritoneum to prevent the outflow of peritoneal fluid), fully expose the peritoneum by blunt dissection, and wipe with alcohol for disinfection. Use a disposable sterile syringe to draw 5-10mL of basal culture medium and inject it into the mouse peritoneal cavity. Hold the syringe in the right hand and keep it still. Use tweezers in the left hand to pick up an alcohol cotton ball and gently rub the mouse abdomen for 1-2 minutes to encourage macrophages to migrate out. Use a syringe to draw out the culture medium in the peritoneal cavity and transfer it to a 15mL centrifuge tube. After mixing the peritoneal macrophages and thymocytes, centrifuge at 1200r / min for 10 minutes and discard the supernatant. Use preheated 20% FBS1×HAT medium (hypoxanthine (H), aminopterin (A) and thymidine (T) (HAT, Sigma)) to resuspend and store at 37°C for use.

[0096] On the morning of the day of cell fusion, the preparation process of spleen cells is as follows: take mice that have been boosted for 3 to 4 days, remove the eyeballs to collect blood, and collect the separated serum as the positive control serum for anti-detection. At the same time, kill the mice by cervical dislocation, soak them in 75% alcohol for 5 minutes for disinfection, and immediately put them in the clean bench. Fix the mice on the dissection table, open the abdomen aseptically, and then lift the skin on the right side of the abdomen to see the spleen. Change the ophthalmic scissors, cut the peritoneum with sterile surgical scissors, remove the spleen with forceps, rinse the spleen with saline, use scissors to cut the spleen, place it in a disposable cell sieve, gently squeeze the spleen with the inner core of the syringe, and repeatedly rinse the cell sieve with saline until only connective tissue is left in the cell sieve, and then use the disposable cell sieve to filter the cell suspension again. Harvest the spleen cell suspension, centrifuge at 1200r / min for 10 minutes, and wash once with 30-40mL of resuspension centrifugation (try to remove red blood cell clusters). The spleen cells were resuspended and added to the basic culture medium containing 10% FBS, and then placed in a T75 cell flask and cultured in a 37° C., 5% CO 2 incubator for 2-3 hours to allow the macrophages in the cell suspension to adhere to the wall.

[0097] In the afternoon of the day of cell fusion, the preparation process of myeloma cells was as follows: 3 bottles of T75 myeloma cells, discard the supernatant, blow down with 50mL preheated saline, centrifuge together with spleen cells, and centrifuge at 1200r / min for 10 minutes. The spleen cells and 3 bottles of T75 myeloma cells were fully mixed, the supernatant was discarded, and resuspended with 40mL preheated saline, and centrifuged at 1200r / min for 10 minutes.

[0098] Cell fusion process: discard the supernatant, discard the supernatant as much as possible, use a dropper to suck out the remaining liquid to avoid affecting the concentration of the cell fusion agent, and try to remove red blood cells. Use your fingers to gently tap the bottom of the centrifuge tube to mix and make the precipitated cells loose and even into a paste. Fusion at room temperature: The cell fusion agent and the culture medium containing the feeding layer need to be placed in an incubator for warmth when preparing spleen cells. Use a Pasteur pipette to absorb the 1mL cell fusion agent solution (try to add it to the centrifuge tube wall as close to the cells as possible). Gently mix within 60s-90s. After the timing is completed, add 30mL of preheated 37℃ basal culture medium at one time to dilute the cell fusion agent and lose its fusion-promoting effect, and let it stand at 37℃ for 5min. Centrifuge at 800r / min for 6 minutes and discard the supernatant. Add preheated 20% FBS1×HAT culture medium and gently blow and suck the precipitated cells to suspend and mix them (mix thoroughly to reduce cell clusters), and move gently. In this experiment, 10 96-well cell culture plates were prepared, with 200 μL per well, requiring 200 mL of 20% FBS 1× HAT medium.

[0099] The cells were cultured in an incubator at 37°C and 5% CO2. The cells were observed under a microscope 7 days after cell fusion. The culture wells in the 96-well cell culture plate with obvious cell clones were counted and the fusion rate was calculated. Fusion rate = (number of fused cells / total number of cells) × 100%.

[0100] 6. Screening of hybridoma cells secreting monoclonal antibodies against Helicobacter pylori type B urease

[0101] The cell culture supernatant was screened by indirect ELISA, and the positive cloned hybridoma cells with higher titers were selected for subcloning, and the limiting dilution method was used for continuous cloning 2-3 times until the cell positive rate reached 100%. The culture supernatants of the 20 hybridoma cell lines with higher titers were tested by indirect ELISA, and 0.02M PBS was used for dilution testing. The results are shown in Table 2.

[0102] Table 2

[0103]

[0104] As can be seen from Table 2, by comparing the detection data of the ELISA method of the hybridoma cell line culture supernatant, a cell line that stably secretes anti-Helicobacter pylori type B urease monoclonal antibodies with a high antibody titer can be further selected, labeled as 2D7. The cells with a positive rate of 100% after cloning were expanded and cultured and then frozen in liquid nitrogen.

[0105] 7. Preparation and purification of ascites

[0106] Hybridoma cell line 2D7 was cultured at 1×10 6 / mouse was injected into the peritoneal cavity of 8-10 week old BALB / c female mice pretreated with liquid paraffin, and ascites was extracted after 10-14 days of feeding and observation when the abdomen of the mice was swollen. Monoclonal antibodies were purified by affinity chromatography Protein G Sepharose Fast Flow, and the purity of monoclonal antibodies was determined by SDS-PAGE, and the purity reached more than 90%.

[0107] Example 2 Characterization of Monoclonal Antibodies

[0108] 1. Determination of antibody concentration: The ascites prepared by hybridoma cell 2D7 was purified to obtain monoclonal antibodies against Helicobacter pylori type B urease, and the concentration was determined by Nanodrop nucleic acid protein analyzer produced by Thermofisher, and was >1 mg / mL.

[0109] 2. Antibody subtype identification: The Thermofisher mouse monoclonal antibody subtype identification kit was used to identify the subtype of the hybridoma cell line. The subtype of the antibody secreted by 2D7 was IgG1, and the light chain was κ chain.

[0110] 3. Identification of the potency of purified antibodies: 50 μg of synthetic Helicobacter pylori type B urease was dissolved in 10 mL of 0.05 M carbonate coating buffer at pH 9.6, added to a 96-well plate, 100 μL per well, and incubated at 4°C overnight. The plate was washed three times with PBS (containing 0.05% v / v Tween-20), blocked with 10 mM PBS containing 1% BSA blocking solution 150 μL / well at 37°C for 2 h, washed three times with PBS (containing 0.05% v / v Tween-20), 100 μL of purified antibody was added to each well, incubated at 37°C for 1 h, washed three times with PBS (containing 0.05% v / v Tween-20), horseradish peroxidase-labeled goat anti-mouse IgG polyclonal antibody was added as secondary antibody, incubated at 37°C for 30 min, washed three times with PBS (containing 0.05% v / v Tween-20), 100 μL was added to each well, TMB was used for color development, incubated at 37°C for 15 min, 2M H2SO4 solution was added to terminate the reaction, and the absorbance was detected at 450 nm with an enzyme reader.

[0111] 4. Antibody binding test:

[0112] Helicobacter pylori type B urease was diluted to 0.5μg / mL and 1μg / mL with 1×CB, and added to the wells of the ELISA plate at a volume of 100μL / well, and duplicate wells were made, and placed at 4°C overnight or 37°C for adsorption for 2 hours. The coated microplate was dried, washed once according to the operating program set by the plate washer (AFP program), and the blocking solution was added at a volume of 200μL / well, placed in a 37°C incubator for 2h, and then placed at 4°C overnight. Before use, the blocked microplate was taken out from 4°C, dried, and the cleaning solution (1×PBS-T) was added to wet the ELISA plate; the monoclonal antibody of the present invention was pre-diluted to 30μg / mL with 1×PBS, and the pre-dilution multiple m was recorded, and the highest concentration (S1) was 3μg / mL after dilution 10 times, and then diluted in a 1:3 gradient (diluted in a 96-deep well plate), with a total of 8 dilution gradients (S1-S8).

[0113] Add 100 μL of diluted antibody to a 96-well microplate that has been tapped clean on absorbent paper and incubate at 37°C for 30 min. After incubation, spin dry the plate and tap dry on absorbent paper. Wash the plate three times with a plate washer. Add 100 μL of 1×PBS to each well in columns 1-4. Add 200 μL of urea treatment solution to each well in columns 5 and 6 and incubate at 37°C for 30 min. After incubation, spin dry the plate and tap dry on absorbent paper. , wash the ELISA plate 3 times with a plate washer, add 100μL of GAM-HRP enzyme-labeled secondary antibody diluted 10,000 times with secondary antibody diluent to each well, and incubate at 37°C for 30 minutes; after incubation, spin dry the ELISA plate, pat dry on absorbent paper, wash the ELISA plate 3 times with a plate washer, take TMB color development solution, add 100μL color development solution to each well, and incubate at 37°C for 5-10 minutes; after color development, add 50μL stop solution to each well. Set the ELISA reader to read at 450nm / 630nm.

[0114] The ELISA antigen-antibody binding experiment was tested under the conditions of 0.5μg / mL and 1μg / mL antigen coating, and the data were obtained by testing the absorbance value OD ( Figure 4 ), and the corresponding polynomial curve was fitted. The corresponding binding showed an obvious gradient with the change of antibody dilution ratio. At the same time, the affinity binding reflected by the two curves under the conditions of 0.5μg / mL and 1μg / mL antigen coating reflected the specificity of binding and reflected that the concentration corresponding to the antibody binding force reached 6.60E-10mol / L (under the condition of 0.5μg / mL antigen coating, the highest OD reading was 50%, that is, the concentration of antigen-antibody complex accounted for half of the total concentration, and the antibody concentration value K 0.5) and 5.97E-10 mol / L (the concentration value K1 corresponding to 50% of the highest OD reading under 1 μg / mL antigen coating conditions, that is, when the concentration of the antigen-antibody complex accounts for half of the total concentration).

[0115] Binding strength calculation: Calculate the average OD readings of the duplicate wells of S1-S8 antibody concentrations before and after urea treatment under 0.5μg / mL and 1μg / mL antigen coating conditions. Substitute the readings into the binding strength calculation formula:

[0116]

[0117] 5. Specificity detection: Similar to ELISA antibody titer detection, the Helicobacter pylori type B urease antibody detection was performed using an orthogonal design. The results are shown in Tables 3 to 12, which indicate that the antibodies obtained by the present invention have good specificity.

[0118] Table 3

[0119]

[0120] Table 4

[0121] Table 5

[0122]

[0123] Table 6

[0124]

[0125] Table 7

[0126]

[0127] Table 8

[0128]

[0129] Table 9

[0130]

[0131] Table 10

[0132]

[0133] Table 11

[0134]

[0135] Table 12

[0136]

[0137] It can be seen that by comparing the experimental data of the antibody pairing experiment, it is shown that except for the detection wells corresponding to the enzyme-labeled antibodies that are the same as the coated antibodies, other enzyme-labeled antibodies can still bind to the Helicobacter pylori type B urease antigen in the presence of the coated antibodies, and the binding effect increases with the increase in the concentration of the added antigen. The reading value, therefore, reflects the specificity of the binding of the 2D7 antibody, and the sensitivity to the Helicobacter pylori antigen is optimal when combined with the 4F8 antibody to form an immune sandwich reaction.

[0138] 6. Monoclonal antibody sequencing

[0139] About 28 mg (batch number V20210426; 3.52 mg / mL) of the monoclonal antibody against Helicobacter pylori type B urease prepared by the purified hybridoma cell 2D7 was taken. The light chain variable region and the heavy chain variable region were sequenced to find that the complementary determining regions of the antibody were as follows:

[0140] CDR-VH1: amino acid sequence is DYPVH;

[0141] CDR-VH2: amino acid sequence IIWAAGSSNYNSWLMS;

[0142] CDR-VH3: amino acid sequence is AYYVSSDWFAY;

[0143] CDR-VL1: amino acid sequence is KSSRSLLTSSNQKNHLA;

[0144] CDR-VL2: amino acid sequence is WTSTRDS;

[0145] CDR-VL3: The amino acid sequence is QQYHNYSWT.

[0146] The amino acid sequences of the light chain variable region and the heavy chain variable region of the antibody are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The light chain constant region sequence of the antibody is shown in SEQ ID NO.3, and the heavy chain constant region sequence of the antibody is shown in SEQ ID NO.4. The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.5, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.6.

[0147] Example 3 Development of a Lateral Flow Immunochromatographic Test Strip Using Helicobacter pylori Type B Urease Monoclonal Antibody

[0148] 1. Preparation of colloidal gold

[0149] The gold was prepared by reduction method, and the gold preparation conditions were 5 mL of 2% chloroauric acid + 10 mL of 0.01% trisodium citrate.

[0150] 2. Colloidal gold labeled mouse anti-Helicobacter pylori type B urease monoclonal antibody

[0151] Physical adsorption method, by adjusting the pH value, colloidal gold and antibody (targeting Helicobacter pylori type B urease antibody 4F8, the amino acid sequences of the light chain and heavy chain are shown in SEQ ID NO.9 and SEQ ID NO.10 respectively).

[0152] The specific labeling conditions are as follows: 10 μL of 0.1 M potassium carbonate solution is added to each milliliter of colloidal gold, and the labeling concentration of mouse anti-Helicobacter pylori type B urease monoclonal antibody 4F8 is 10 μg / mL.

[0153] 3. Testing line and quality control line

[0154] Test line: Take appropriate concentration (1.0 mg / mL) of mouse anti-Helicobacter pylori type B urease monoclonal antibody 2D7 and coat it on nitrocellulose membrane to prepare the test line, dry it at 37°C, and the spray volume is 1.0 μL / cm.

[0155] Quality control line: Take 1.0 mg / mL goat anti-mouse IgG polyclonal antibody to prepare a quality control line on the fiber membrane, dry at 37°C, and spray the spot at a volume of 1.0 μL / cm.

[0156] The cellulose sample pad, the glass fiber colloidal gold binding pad (containing the above colloidal gold labeled mouse anti-Helicobacter pylori type B urease monoclonal antibody), the NC reaction membrane (containing the above detection line and quality control line) and the cellulose water absorbent pad were sequentially pasted on the bottom plate. Figure 1 a. Make a test strip.

[0157] 4. Inspection methods

[0158] Return the test strips, sample diluent and sample to 18-30°C. The test strips are tested as follows:

[0159] a. Take out the test strip from the aluminum foil bag, mark the sample, and place it flat on a horizontal work surface;

[0160] b. Take the treated sample solution (200, 100, 50, 20, 10, 5, 2, 1, 0 ng / mL of Helicobacter pylori type B urease is used in this embodiment) or the quality control product and add it directly to the sample pad;

[0161] c. Add 100 μL (2 to 3 drops) of sample diluent (feces matrix, prepared as follows: prepare 0.01 M Tris-HCl buffer, pH 8.5; add 1% BSA and 10% sucrose);

[0162] d. Read the results within 15 to 20 minutes. Test results after 20 minutes will be invalid.

[0163] 5. Interpretation of test results

[0164] The results are as follows Figure 5 shown.

[0165] a. Positive: Both the test line and the quality control line are colored, indicating that the sample has detected the type B urease antigen of Helicobacter pylori. It may be an early infection or an active infection, and the final confirmation needs to be combined with clinical symptoms.

[0166] b. Negative: Only one red quality control line appears in the test window, indicating that Helicobacter pylori type B urease antigen was not detected in the sample.

[0167] c. Invalid: No red quality control line appears in the detection window.

[0168] Example 4 Performance Test of Helicobacter pylori Colloidal Gold Immunochromatographic Test Strip

[0169] 1. Sensitivity test

[0170] The Helicobacter pylori colloidal gold immunochromatographic test strip prepared in Example 3 was tested using the recombinant Helicobacter pylori type B urease solution. Figure 6 As shown, the recombinant Helicobacter pylori type B urease can be detected at a minimum of 10 ng / mL (a faint detection line can be seen in the 10 ng / mL group with the naked eye).

[0171] 2. Specificity test (clinical sample test)

[0172] The Helicobacter pylori colloidal gold immunochromatographic test strip prepared in Example 3 was used to test stool samples from 20 normal subjects. The test results were as follows: Figure 7 As shown in the figure, it can be seen that the detection results are clear and the background is clean, indicating that the specificity of the product is good.

[0173] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody or a functional fragment thereof targeting Helicobacter pylori type B urease, characterized in that: The antibody or its functional fragment comprises the following complementarity determining region: CDR-VH1: amino acid sequence is DYPVH; CDR-VH2: amino acid sequence IIWAAGSSNYNSWLMS; CDR-VH3: amino acid sequence is AYYVSSDWFAY; CDR-VL1: amino acid sequence is KSSRSLLTSSNQKNHLA; CDR-VL2: amino acid sequence is WTSTRDS; CDR-VL3: The amino acid sequence is QQYHNYSWT.

2. The antibody or functional fragment thereof according to claim 1, characterized in that: The amino acid sequences of the light chain variable region and the heavy chain variable region of the antibody are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

3. The antibody or functional fragment thereof according to claim 1, characterized in that: The antibody also comprises a constant region.

4. The antibody or functional fragment thereof according to claim 3, characterized in that: The constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

5. The antibody or functional fragment thereof according to claim 3, characterized in that: The species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink or human.

6. The antibody or functional fragment thereof according to claim 3, characterized in that: The light chain constant region sequence of the constant region is shown in SEQ ID NO.3, and the heavy chain constant region sequence of the constant region is shown in SEQ ID NO.

4.

7. The antibody or functional fragment thereof according to any one of claims 1 to 6, characterized in that: The amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.5, and the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.

6.

8. The antibody or functional fragment thereof according to claim 1, characterized in that: The functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv, and scFv of the antibody.

9. A nucleic acid encoding the antibody or a functional fragment thereof according to any one of claims 1 to 8.

10. The nucleic acid according to claim 9, characterized in that The nucleic acid sequences are shown in SEQ ID NO.7 and SEQ ID NO.

8.

11. A product, characterized in that The antibody or functional fragment thereof is contained in any one of claims 1 to 8; the product is any one of a conjugate of an antibody and a protein tag, a detection reagent or a kit, and a test strip.

12. The product according to claim 11, characterized in that In the product, the antibody or its functional fragment is labeled with a detectable marker.

13. The product according to claim 11, characterized in that The product is used for detecting Helicobacter pylori.

14. A colloidal gold immunochromatographic test strip for Helicobacter pylori, characterized in that: The invention comprises a sample pad, a colloidal gold binding pad, a reaction membrane, a water absorbent pad and a bottom plate; the reaction membrane is provided with a detection line and a quality control line, and the sample pad, the colloidal gold binding pad, the reaction membrane and the water absorbent pad are sequentially pasted on the bottom plate; the detection line is coated with the antibody described in any one of claims 1 to 8.

15. Use of the antibody or functional fragment thereof according to any one of claims 1 to 8, the product according to any one of claims 11 to 13, or the colloidal gold immunochromatographic test strip for Helicobacter pylori according to claim 14 in at least one of the following aspects: (1) Application in the preparation of products for detecting Helicobacter pylori; (2) Use in the preparation of products for diagnosing diseases caused by Helicobacter pylori infection; (3) Application in the detection of Helicobacter pylori for non-diagnostic and non-therapeutic purposes; (4) Application in detecting the antigen content of Helicobacter pylori vaccine; (5) Application in quality control of Helicobacter pylori vaccines.

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