Helicobacter pylori antibody, reagent and kit for detecting Helicobacter pylori

Abstract

The present invention discloses antibodies against Helicobacter pylori or reagents and kits for detecting Helicobacter pylori, which relates to the field of antibodies. The antibodies against Helicobacter pylori disclosed by the present invention include heavy chain complementarity determining regions and light chain complementarity determining regions. The antibodies provide an important source of raw materials for the detection of Helicobacter pylori and have good affinity or activity.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular, to an anti-Helicobacter pylori antibody, a reagent and a kit for detecting Helicobacter pylori. Background Art

[0002] Helicobacter pylori (HP for short) is a spiral Gram-negative bacterium in the human gastric mucosa. This bacterium is the culprit causing chronic gastritis, gastric ulcer, duodenal ulcer and even gastric cancer.

[0003] Generally, the clinical process of Helicobacter pylori infection in the stomach is as follows: Helicobacter pylori in the stomach reaches the gastric mucosa through the mouth and settles and infects. After several weeks or months, it causes chronic superficial gastritis. After several years or decades, it develops into duodenal ulcer, gastric ulcer, lymphoproliferative gastric lymphoma, chronic atrophic gastritis, etc., and the latter is the most dangerous factor leading to gastric cancer. Therefore, detecting whether HP exists in the blood has very practical clinical significance.

[0004] Currently, the main method for detecting HP is immunochromatography assay, which is an immunological detection method based on the specific reaction of antigen-antibody. Its basic principle is: using colloidal gold to label an antigen or antibody, and the corresponding paired antigen or antibody is coated on the nitrocellulose membrane. When detecting a sample, the colloidal gold-labeled substance combines with the ligand in the sample to form a complex, and then moves upward through chromatography and combines with the coated antigen or antibody to agglomerate and develop color, thereby realizing the determination of the sample detection result. Similar immunoassay methods all require antibodies against HP. Therefore, preparing antibodies against HP is the key to realizing HP immunoassay.

[0005] Therefore, those skilled in the art have a strong demand for anti-Helicobacter pylori antibodies with good performance. Summary of the Invention

[0006] The present application provides an antibody or its antigen-binding fragment, which provides an important raw material source for the detection of Helicobacter pylori and has good activity or affinity.

[0007] To achieve the above object, according to one aspect of the present invention, there is provided an antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment comprises three complementary determining regions of any one of the heavy chain variable regions having the amino acid sequence SEQ ID NO: 17, 18 and three complementary determining regions of any one of the light chain variable regions having the amino acid sequence SEQ ID NO: 19, 20.

[0008] To achieve the above object, according to a second aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises the following complementarity-determining regions:

[0009] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:1, or consists of the same;

[0010] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:2, or consists of the same;

[0011] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:3, or consists of the same;

[0012] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO:4, or consists of the same;

[0013] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO:5, or consists of the same; and

[0014] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO:6, or consists of the same.

[0015] To achieve the above object, according to a third aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO:17 and 18; the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO:19 and 20.

[0016] To achieve the above object, according to a fourth aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO:21 and 22; the amino acid sequence of the light chain is as shown in any one of SEQ ID NO:23 and 24.

[0017] To achieve the above object, according to a fifth aspect of the present invention, there is provided an antibody conjugate, wherein the antibody conjugate comprises the above-mentioned antibody or an antigen-binding fragment thereof.

[0018] To achieve the above object, according to a sixth aspect of the present invention, there is provided a reagent or a kit, wherein the reagent or the kit comprises the above-mentioned antibody or an antigen-binding fragment thereof or the above-mentioned antibody conjugate.

[0019] To achieve the above object, according to the seventh aspect of the present invention, there is provided a method for detecting Helicobacter pylori, comprising: a) contacting the above-mentioned antibody or its antigen-binding fragment, antibody conjugate, or reagent or kit with Helicobacter pylori in a test sample under conditions sufficient to effect an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample.

[0020] To achieve the above object, according to the eighth aspect of the present invention, there is provided a nucleic acid encoding the above-mentioned antibody or its antigen-binding fragment.

[0021] To achieve the above object, according to the ninth aspect of the present invention, there is provided a vector comprising the above-mentioned nucleic acid.

[0022] To achieve the above object, according to the tenth aspect of the present invention, there is provided a cell comprising the above-mentioned nucleic acid, vector or expressing the above-mentioned antibody or its antigen-binding fragment.

[0023] To achieve the above object, according to the eleventh aspect of the present invention, there is provided a method for preparing the above-mentioned antibody or its antigen-binding fragment, the method comprising culturing the above-mentioned cell.

[0024] To achieve the above object, according to the twelfth aspect of the present invention, there is provided the use of the above-mentioned antibody or its antigen-binding fragment, antibody conjugate, reagent or kit in the preparation of a product for detecting Helicobacter pylori. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0026] Figure 1 Results of reducing SDS-PAGE for Anti-HP 10H5Rmb1 to Anti-HP 10H5 Rmb3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] In a first aspect, an embodiment of the present invention provides an antibody or its antigen-binding fragment, the antibody or its antigen-binding fragment comprising three complementarity-determining regions of any one of the heavy chain variable regions having the amino acid sequence SEQ ID NO:17, 18 and three complementarity-determining regions of any one of the light chain variable regions having the amino acid sequence SEQ ID NO:19, 20.

[0028] It should be noted that HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to HCDR1, HCDR2, and HCDR3 of the same heavy chain variable region defined in the antibody or its antigen-binding fragment described in the first aspect, and LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to LCDR1, LCDR2, and LCDR3 of the same light chain variable region defined in the antibody or its antigen-binding fragment described in the first aspect.

[0029] For example, the said HCDR1, HCDR2, and HCDR3 are amino acid sequences identical to HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO:17; the said LCDR1, LCDR2, and LCDR3 are amino acid sequences identical to LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO:19.

[0030] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, provided that they exhibit the desired biological activity.

[0031] In the present invention, the terms "complementary determining region", "CDR", or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions containing one or more or even all of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In the specific embodiments of the present invention, the CDRs refer to the highly variable regions of the heavy and light chains of the said antibody.

[0032] In the present invention, the heavy chain complementary determining regions are denoted by HCDR and include HCDR1, HCDR2, and HCDR3; the light chain complementary determining regions are denoted by LCDR and include LCDR1, LCDR2, and LCDR3.

[0033] The methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). For the "Chothia definition", see Chothia et al., J Mol Biol 196: 901-917 (1987). There are other CDR definition methods that may not strictly follow one of the above-mentioned schemes but will still overlap with at least a part of the CDR region defined by Kabat, although they may be shortened or lengthened according to the prediction or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different literatures are slightly different. Given the amino acid sequence of the variable region of a given antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods not limited to Table 1 also fall within the scope of protection of the present disclosure.

[0034] Table 1: CDR Definitions 1

[0035]

[0036]

[0037] 1 The numbers of all CDR definitions in Table 1 are based on the Kabat numbering system (see below). The amino acid numbers on the heavy chain are represented by "H + number", and the amino acid numbers on the light chain are represented by "L + number". Those of ordinary skill in the art can clearly map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein, the "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0038] 2 As used in Table 1, "AbM" with a lowercase "b" refers to the CDR defined by the "AbM" antibody modeling software of Oxford Molecular.

[0039] 3If neither H35A nor H35B is present, then CDR-H1 ends at position 35; if only H35A is present, then CDR-H1 ends at position 35A; if both H35A and H35B are present, then CDR-H1 ends at position 35B.

[0040] 4 If neither H35A nor H35B is present, then CDR-H1 ends at position 32; if only H35A is present, then CDR-H1 ends at position 33; if both H35A and H35B are present, then CDR-H1 ends at position 34.

[0041] 5 If neither H35A nor H35B is present, then CDR-H1 ends at position 33; if only H35A is present, then CDR-H1 ends at position 34; if both H35A and H35B are present, then CDR-H1 ends at position 35.

[0042] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM or Contact.

[0043] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0044] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0045] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0046] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0047] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Contact system.

[0048] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of the Kabat, Chothia, IMGT, AbM, or Contact systems.

[0049] According to an embodiment of the present invention, the Kabat, Chothia, AbM, or IMGT system-defined amino acid sequence positions of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 corresponding to the Kabat numbers are as follows:

[0050]

[0051]

[0052] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0053] In a second aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, and the antibody or the antigen-binding fragment thereof includes the following complementarity-determining regions:

[0054] HCDR1, which contains the amino acid sequence shown in SEQ ID NO: 1, or consists of the same;

[0055] HCDR2, which contains the amino acid sequence shown in SEQ ID NO: 2, or consists of the same;

[0056] HCDR3, which contains the amino acid sequence shown in SEQ ID NO: 3, or consists of the same;

[0057] LCDR1, which contains the amino acid sequence shown in SEQ ID NO: 4, or consists of the same;

[0058] LCDR2, which contains the amino acid sequence shown in SEQ ID NO: 5, or consists of the same; and

[0059] LCDR3, which contains the amino acid sequence shown in SEQ ID NO: 6, or consists of the same.

[0060] In the present invention, the "framework region" or "FR" region includes the heavy-chain framework region and the light-chain framework region, and refers to the regions other than the CDRs in the heavy-chain variable region and the light-chain variable region of the antibody; wherein, the heavy-chain framework region can be further subdivided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light-chain framework region can be further subdivided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3, and LFR4 framework regions.

[0061] In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0062] In an alternative embodiment, the antibody or its antigen-binding fragment according to the first or second aspect further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4;

[0063] The HFR1 comprises / is as shown in SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto;

[0064] The HFR2 comprises / is as shown in SEQ ID NO:8 or an amino acid sequence having at least 80% identity thereto;

[0065] The HFR3 comprises / is as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto;

[0066] The HFR4 comprises / is as shown in SEQ ID NO:10 or an amino acid sequence having at least 80% identity thereto;

[0067] The LFR1 comprises / is as shown in SEQ ID NO:11 or an amino acid sequence having at least 80% identity thereto;

[0068] The LFR2 comprises / is as shown in SEQ ID NO:12 or an amino acid sequence having at least 80% identity thereto;

[0069] The LFR3 comprises / is as shown in SEQ ID NO:13 or an amino acid sequence having at least 80% identity thereto;

[0070] The LFR4 comprises / is as shown in SEQ ID NO:14 or an amino acid sequence having at least 80% identity thereto.

[0071] It should be noted that in other embodiments, the amino acid sequences of the respective framework regions of the antibody or its antigen-binding fragment provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding framework regions (SEQ ID NO:7, 8, 9, 10, 11, 12, 13, or 14).

[0072] In an alternative embodiment, the HFR3 comprises the amino acid sequence as shown in SEQ ID NO: 9 or 25.

[0073] In an alternative embodiment, the LFR1 comprises the amino acid sequence as shown in SEQ ID NO: 11 or 26.

[0074] In an alternative embodiment, the antibody or its antigen-binding fragment binds Helicobacter pylori with an affinity of KD < 5.81×10 -8 M.

[0075] In an alternative embodiment, the antibody or its antigen-binding fragment binds Helicobacter pylori with an affinity of KD ≤ 10 -8 M, KD ≤ 10 -9 M, KD ≤ 10 -10 M, KD ≤ 10 -11 M or KD ≤ 10 -12 M.

[0076] In an alternative embodiment, the antibody or its antigen-binding fragment binds Helicobacter pylori with an affinity of KD ≤ 9.39×10 -9 M.

[0077] There are many methods for measuring antibody affinity (KD), which can be classified into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods according to the detection principle. Among them, common thermodynamic detection methods include isothermal titration calorimetry (ITC); common kinetic detection methods include surface plasmon resonance (SPR) and biolayer interferometry (BLI); common dynamic equilibrium detection methods include enzyme-linked immunosorbent assay (ELISA), etc.

[0078] In an alternative embodiment, the KD is measured by a kinetic detection method; optionally, surface plasmon resonance, for example, by using a biosensor system such as system.

[0079] In a third aspect, an embodiment of the present invention provides an antibody or its antigen-binding fragment, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 17 and 18, and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 19 and 20.

[0080] In an alternative embodiment, the heavy chain variable region and the light chain variable region described in the first aspect and the third aspect above are selected from any one of the following combinations:

[0081] Combination Heavy chain variable region Light chain variable region 1 SEQ ID NO:17 SEQ ID NO:19 2 SEQ ID NO:18 SEQ ID NO:19 3 SEQ ID NO:17 SEQ ID NO:20 .

[0082] In an alternative embodiment, the antibody or antigen-binding fragment thereof further comprises a constant region.

[0083] In an alternative embodiment, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0084] In an alternative embodiment, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments.

[0085] In an alternative embodiment, the heavy chain constant region comprises CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0086] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0087] In an alternative embodiment, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0088] In an alternative embodiment, the species origin of the constant region is bovine, equine, dairy cattle, porcine, ovine, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting chicken or human.

[0089] In an alternative embodiment, the species origin of the constant region is mouse.

[0090] In an alternative embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:15, and the light chain constant region (CL) sequence is as shown in SEQ ID NO:16.

[0091] It should be noted that in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above constant region (SEQ ID NO:15 or 16).

[0092] In an alternative embodiment, the antigen-binding fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv and scFv of the antibody.

[0093] The antigen-binding fragments of the above-mentioned antibodies generally have the same binding specificity as the antibodies from which they are derived. Those skilled in the art can easily understand from the content described in the present invention that the antigen-binding fragments of the above-mentioned antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to cleave disulfide bonds. Based on the disclosure of the structure of the complete antibody in the present invention, those skilled in the art can easily obtain the above-mentioned antigen-binding fragments.

[0094] The antigen-binding fragments of the above-mentioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems.

[0095] In a fourth aspect, the present invention provides an antibody or its antigen-binding fragment, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO:21 and SEQ ID NO:22, and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO:23 and SEQ ID NO:24.

[0096] In an alternative embodiment, the antibody according to the first, second, third, or fourth aspect above comprises a heavy chain and a light chain in any of the following combinations:

[0097] Combination Heavy chain Light chain 1 SEQ ID NO:21 SEQ ID NO:23 2 SEQ ID NO:22 SEQ ID NO:23 3 SEQ ID NO:21 SEQ ID NO:24 。

[0098] In a fifth aspect, the present invention provides an antibody conjugate, which comprises the above-mentioned antibody or its antigen-binding fragment.

[0099] In an alternative embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or its antigen-binding fragment.

[0100] In an alternative embodiment, the antibody conjugate further comprises a label conjugated to the antibody or its antigen-binding fragment.

[0101] In an alternative embodiment, the above-mentioned label refers to a class of substances having characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or probed by an instrument, and through which qualitative or quantitative detection of the corresponding target can be achieved.

[0102] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0103] In actual use, those skilled in the art can select a suitable label according to the detection conditions or actual needs. Whatever label is used, it falls within the protection scope of the present invention.

[0104] In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (e.g., including but not limited to rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (e.g., including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (e.g., including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (PerCP), etc.).

[0105] In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0106] In alternative embodiments, the radioisotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0107] In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetane and its derivatives, rosamide and its derivatives, and peroxyoxalate and its derivatives.

[0108] In alternative embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0109] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0110] In alternative embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0111] In alternative embodiments, the colloidal metal is colloidal gold.

[0112] In alternative embodiments, the above antibody conjugate further includes a solid-phase carrier conjugated to the antibody or its antigen-binding fragment.

[0113] In alternative embodiments, the solid-phase carrier is selected from microspheres, plates, and membranes.

[0114] In alternative embodiments, the solid-phase carrier includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic particles, microtiter plates, glass, capillary tubes, nylon, and nitrocellulose membranes.

[0115] In a sixth aspect, the present invention provides a reagent or kit, which includes the above antibody or its antigen-binding fragment or the above antibody conjugate.

[0116] As described above, the antibody or its antigen-binding fragment in some embodiments or examples of the present invention can effectively bind to Helicobacter pylori. Therefore, a reagent or kit containing the Helicobacter pylori antibody or its antigen-binding fragment can effectively qualitatively or quantitatively detect Helicobacter pylori. Applying the reagent or kit provided by the present invention, for example, can be used for detections such as immunoblotting and immunoprecipitation that involve the specific binding performance of Helicobacter pylori and its antibody. As described above, the antibody or its antigen-binding fragment in some embodiments or examples of the present invention has a higher binding activity or affinity for Helicobacter pylori. Therefore, a reagent or kit containing the antibody or its antigen-binding fragment has higher detection sensitivity or specificity.

[0117] In a seventh aspect, the present invention provides a method for detecting Helicobacter pylori, including: a) contacting the above antibody or its antigen-binding fragment, antibody conjugate, reagent or kit with Helicobacter pylori in a test sample under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample.

[0118] In alternative embodiments, the immune complex further includes a second antibody that binds to the antibody or its antigen-binding fragment.

[0119] In alternative embodiments, the immune complex further includes a second antibody that binds to Helicobacter pylori.

[0120] In a eighth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or its antigen-binding fragment.

[0121] In a ninth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0122] In a tenth aspect, the present invention provides a cell containing the above-mentioned vector.

[0123] In an eleventh aspect, the present invention provides a method for preparing an antibody or its antigen-binding fragment, which includes: culturing the cell as described above.

[0124] In a twelfth aspect, the present invention provides the use of the above-mentioned antibody or its antigen-binding fragment, antibody conjugate or the above-mentioned reagent or kit in the preparation of a product for detecting Helicobacter pylori.

[0125] Based on the disclosure of the amino acid sequence of the antibody or its antigen-binding fragment in the present invention, those skilled in the art can easily think of using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression) to prepare the antibody or its antigen-binding fragment. For example, the antibody or its antigen-binding fragment can be isolated and purified from the culture product of a recombinant cell capable of recombinantly expressing the antibody or its antigen-binding fragment as described in any one of the above. This is easily achievable for those skilled in the art. Based on this, no matter which technique is used to prepare the antibody or its antigen-binding fragment of the present invention, it falls within the protection scope of the present invention.

[0126] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0127] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques employed or considered herein are standard methods. The materials, methods and examples are illustrative only and not restrictive.

[0128] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (eds. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (eds. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (eds. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (eds. Mullis et al., 1994); and "Current Protocols in Immunology" (eds. J.E. Coligan et al., 1991), each of which is hereby expressly incorporated by reference.

[0129] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0130] Example 1 Preparation of Anti-HP 10H5 Monoclonal Antibody

[0131] In this example, the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara. The MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting the Anti-HP 10H5 monoclonal antibody was the hybridoma cell line prepared in this laboratory and was revived for standby.

[0132] (1) Preparation of antibody genes

[0133] mRNA was extracted from the hybridoma cell line secreting the Anti-HP 10H5 monoclonal antibody, and DNA products were obtained by RT-PCR. After the A-tailing reaction of the products with rTaq DNA polymerase, they were inserted into the pMD-18T vector and transformed into DH5α competent cells. After colonies grew, 4 clones of the Heavy Chain and Light Chain genes were respectively taken for cloning and sent to a gene sequencing company for sequencing.

[0134] (2) Sequence analysis of the variable region genes of Anti-HP 10H5 antibody

[0135] The gene sequences obtained from the above sequencing were analyzed in the kabat antibody database, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence was 336bp, and there was a 57bp leader peptide sequence in front of it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 360bp, belonging to the VH1 gene family, and there was a 57bp leader peptide sequence in front of it.

[0136] (3) Construction of recombinant antibody expression plasmid

[0137] pcDNA TM 3.4 The pcDNA3.4 vector was the eukaryotic expression vector of the recombinant antibody constructed. Multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI had been introduced into this expression vector, and it was named the pcDNA3.4A expression vector, hereinafter referred to as the 3.4A expression vector for short; according to the antibody variable region gene sequencing results in the above pMD-18T, specific primers for the VL and VH genes of this antibody were designed, with HindIII and EcoRI enzyme digestion sites and protective bases at both ends, and 0.71kb Light Chain gene fragments and 1.39kb Heavy Chain gene fragments were amplified by PCR amplification.

[0138] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI double enzymes respectively, and the 3.4A vector was digested with HindIII / EcoRI double enzymes. After purifying and recovering the fragments and the vector, the Heavy Chain gene and the Light Chain gene were respectively ligated into the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain respectively.

[0139] 2. Recombinant antibody production

[0140] Resuscitate HEK293 cells in advance, passage and culture them to a 200 ml system to make the cell density reach 3 - 5×10 6 cells / ml. Select antibodies and cells with a cell density reaching the required concentration, and the cell viability > 95%; centrifuge and wash the cells, resuspend them with the medium, and at the same time adjust the cell density to 2.9×10 6 cells / ml. Wash the cells, resuspend them with the medium, and at the same time, use it as the cell diluent. Prepare plasmid DNA and transfection reagent diluents with the medium respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let it stand at room temperature for 15 min; slowly add this mixture to the cell diluent within 1 min, mix well, sample and count, record and observe the viability of the cells after transfection, and place them in a 35°C constant temperature incubator for culture, with a rotation speed of 120 rmp and a CO2 content of 8%. After 13 days, centrifuge to collect the samples. Purify the centrifuged supernatant with a protein A affinity chromatography column. Take 6 μg of the purified antibody for reducing SDS-PAGE, and the electrophoresis pattern is shown in the figure. Two bands are shown after reducing SDS-PAGE, one with Mr of 50 KD (heavy chain) and the other with Mr of 28 KD (light chain).

[0141] The obtained antibody was named Anti-HP 10H5Rmb1, and the Anti-HP 10H5Rmb1 was mutated to obtain a mutant antibody. The sequences of the heavy chain (H) and light chain (L) of the above antibodies are shown in the following table:

[0142] Table 2 Antibody sequences

[0143] Antibody name Heavy chain Light chain Anti-HP 10H5Rmb1 SEQ ID NO:21 SEQ ID NO:23 Anti-HP 10H5Rmb2 SEQ ID NO:22 SEQ ID NO:23 Anti-HP 10H5Rmb3 SEQ ID NO:21 SEQ ID NO:24

[0144] Example 2 Performance detection of the antibody

[0145] 1. Affinity analysis

[0146] Dilute the purified antibody in advance, and simultaneously perform gradient dilution on Helicobacter pylori antigen (from PhyGenesis). Use the CM5 chip pre-coupled with goat anti-mouse IgG to test the binding and dissociation curves of the antigen and antibody on the Biacore 8K+ device, and the instrument automatically fits to obtain the affinity constant, association rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., the affinity constant; ka represents the association rate; kd represents the dissociation rate)

[0147] Table 3 Affinity Data

[0148] Sample name KD ka kd Control 5.81E-08 3.12E+04 1.81E-03 Anti-HP 10H5Rmb1 8.47E-09 8.03E+04 6.80E-04 Anti-HP 10H5Rmb2 9.39E-09 7.71E+04 7.24E-04 Anti-HP 10H5Rmb3 8.15E-09 8.18E+04 6.67E-04

[0149] 2. Activity Identification

[0150] Dilute Helicobacter pylori antigen (from PhyGenesis) to 3 μg / ml with coating buffer (main component: NaHCO3), 100 μL per well, and incubate overnight at 4°C. The next day, wash 2 times with washing buffer (main components: Na2HPO4 + NaCl), and pat dry. Add blocking buffer (20% BSA + 80% PBS), 120 μL per well, incubate at 37°C for 1 h, and pat dry. Add the diluted purified antibody and control antibody, 100 μL per well, incubate at 37°C for 30 min. Wash 5 times with washing buffer and pat dry. Add goat anti-mouse IgG-HRP, 100 μL per well, incubate at 37°C for 30 min. Wash 5 times with washing buffer and pat dry. Add chromogenic solution A (50 μL per well), then add chromogenic solution B (50 μL per well), and incubate for 10 min. Add stop solution, 50 μL per well. Read the OD value at 450 nm (reference 630 nm) on an ELISA reader.

[0151] Note: Solution A (main components: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main components: citric acid + EDTA·2Na + TMB + concentrated HCl); Stop solution (EDTA·2Na + concentrated H2SO4)

[0152] Table 4 Activity Data

[0153] Concentration (ng / ml) 125.000 62.500 31.250 15.625 7.813 0.000 Control 1.219 0.806 0.321 0.228 0.103 0.011 Anti-HP 10H5Rmb1 1.874 1.611 0.504 0.289 0.189 0.057 Anti-HP 10H5Rmb2 2.247 1.185 0.468 0.181 0.186 0.050 Anti-HP 10H5Rmb3 2.091 1.337 0.603 0.373 0.171 0.057

[0154] 3. Stability Assessment

[0155] Place the above antibody at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days. Take samples at 7 days, 14 days, and 21 days for status observation, and perform activity detection on the 21-day samples. The results show that no obvious protein state changes were observed after the antibody was placed for 21 days under the three assessment conditions, and the activity did not show a downward trend with the increase of the assessment temperature, indicating that the above antibody is stable. The following Table 5 shows the OD results of the enzyme immunoassay activity detection of the antibody Anti-HP 10H5Rmb1 after 21 days of assessment.

[0156] Table 5 Stability Data

[0157] Sample concentration (ng / ml) 62.500 31.250 0 Sample at 4°C for 21 days 1.611 0.523 0.024 Sample at -80°C for 21 days 1.651 0.565 0.037 Sample at 37°C for 21 days 1.635 0.544 0.026

[0158] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0159] Some of the amino acid sequences involved in this application are shown in Table 6 as follows:

[0160]

[0161]

[0162]

Claims

1. An anti-Helicobacter pylori antibody or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment has three complementarity-determining regions of any one of the heavy chain variable regions with amino acid sequences SEQ ID NO: 17 and 18, and three complementarity-determining regions of any one of the light chain variable regions with amino acid sequences SEQ ID NO: 19 and 20, and the complementarity-determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT or AbM systems.

2. An anti-Helicobacter pylori antibody or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment comprises the following complementarity-determining regions: The amino acid sequence of HCDR1 is as shown in SEQ ID NO: 1; The amino acid sequence of HCDR2 is as shown in SEQ ID NO: 2; The amino acid sequence of HCDR3 is as shown in SEQ ID NO: 3; The amino acid sequence of LCDR1 is as shown in SEQ ID NO: 4; The amino acid sequence of LCDR2 is as shown in SEQ ID NO: 5; and The amino acid sequence of LCDR3 is as shown in SEQ ID NO:

6.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

4. The antibody or its antigen-binding fragment according to claim 3, wherein the HFR1 comprises SEQ ID NO: 7 or an amino acid sequence having at least 80% identity therewith; the HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity therewith; the HFR3 comprises SEQ ID NO: 9 or an amino acid sequence having at least 80% identity therewith; the HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity therewith; the LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity therewith; the LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity therewith; the LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity therewith; the LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity therewith.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 and 4, characterized in that, The antibody or its antigen-binding fragment binds to Helicobacter pylori with an affinity of KD < 5.81×10 -8 M.

6. An anti-Helicobacter pylori antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 19; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 19; or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

20.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, 4, and 6, characterized in that, The antibody or its antigen-binding fragment further comprises a constant region.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein, The constant region includes a heavy chain constant region and a light chain constant region.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein, The heavy chain constant region is selected from the heavy chain constant regions of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments.

10. The antibody or antigen-binding fragment thereof according to claim 8, wherein The heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

11. The antibody or antigen-binding fragment thereof according to claim 7, wherein The species origin of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human.

12. The antibody or antigen-binding fragment thereof according to claim 7, wherein The species origin of the constant region is mouse.

13. The antibody or antigen-binding fragment thereof according to claim 8, wherein The heavy chain constant region sequence is as shown in SEQ ID NO:15 or has at least 80% identity therewith; the light chain constant region sequence is as shown in SEQ ID NO:16 or has at least 80% identity therewith.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, 4, and 6, wherein The antigen-binding fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv, and scFv of the antibody.

15. An anti-Helicobacter pylori antibody, comprising a heavy chain and a light chain, characterized in that, The amino acid sequence of the heavy chain is as shown in SEQ ID NO:21, and the amino acid sequence of the light chain is as shown in SEQ ID NO:23; or the amino acid sequence of the heavy chain is as shown in SEQ ID NO:22, and the amino acid sequence of the light chain is as shown in SEQ ID NO:23; or the amino acid sequence of the heavy chain is as shown in SEQ ID NO:21, and the amino acid sequence of the light chain is as shown in SEQ ID NO:

24.

16. An antibody conjugate, characterized in that, The antibody conjugate includes the antibody or its antigen-binding fragment according to any one of claims 1 to 14, or the antibody according to claim 15, and biotin or a biotin derivative, a label, or a solid-phase carrier conjugated to the antibody or its antigen-binding fragment.

17. The antibody conjugate according to claim 16, wherein The label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

18. The antibody conjugate according to claim 16, wherein, The solid-phase carrier is selected from microspheres, plates, and membranes.

19. A reagent or kit, characterized in that, The reagent or kit includes the antibody or its antigen-binding fragment according to any one of claims 1 to 14, or the antibody according to claim 15, or the antibody conjugate according to any one of claims 16 to 18. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the antibody according to claim 15, the antibody conjugate according to any one of claims 16 to 18, or the reagent or kit according to claim 19 in the preparation of a product for detecting Helicobacter pylori, characterized in that, Comprising: a) contacting the antibody or its antigen-binding fragment according to any one of claims 1 to 14, the antibody according to claim 15, the antibody conjugate according to any one of claims 16 to 18, or the reagent or kit according to claim 19 with Helicobacter pylori in a test sample under conditions sufficient to effect an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample.

21. The use according to claim 20, wherein The immune complex further includes a second antibody that binds to the antibody or its antigen-binding fragment.

22. The use according to claim 20, wherein, The immune complex further includes a second antibody that binds to Helicobacter pylori.

23. A nucleic acid, characterized in that, It encodes the antibody or its antigen-binding fragment according to any one of claims 1 to 14, or the antibody according to claim 15.

24. A carrier, characterized in that, It contains the nucleic acid according to claim 23.

25. A cell, characterized in that, It contains the nucleic acid according to claim 23 or the vector according to claim 24.

26. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, characterized in that, It includes: culturing the cell according to claim 25.

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