Anti-helicobacter pylori antibody and application thereof

Abstract

The invention discloses an anti-helicobacter pylori antibody and application thereof, and relates to the field of antibodies. The anti-helicobacter pylori antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for detection of helicobacter pylori, and has good activity and sensitivity.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of a Chinese patent application with the application number 202311643212.7 and the title "An Antibody Against Helicobacter pylori and Its Application" filed with the Chinese Patent Office on December 1, 2023. The entire content of which is incorporated herein by reference. Technical field

[0003] The present invention relates to the technical field of antibodies, and specifically, to an antibody against Helicobacter pylori and its application. Background art

[0004] Helicobacter Pylori (HP for short) is a spiral - shaped Gram - negative bacterium in the human gastric mucosa. This bacterium is the culprit causing chronic gastritis, gastric ulcer, duodenal ulcer and even gastric cancer.

[0005] Generally, the clinical process of Helicobacter pylori infection in the stomach is as follows: Helicobacter pylori reaches the gastric mucosa through the mouth and then 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. The latter is the most dangerous factor leading to gastric cancer. Therefore, detecting whether HP exists in the blood has very practical clinical significance.

[0006] Currently, the main method for detecting HP is the 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 label 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 immunological detection methods all require antibodies against HP. Therefore, preparing antibodies against HP is the key to realizing HP immunological detection.

[0007] Therefore, those skilled in the art have a strong demand for anti - Helicobacter pylori antibodies with good performance. Summary of the invention

[0008] This application provides an antibody against Helicobacter pylori, which provides an important raw material source for the detection of Helicobacter pylori and has good activity or sensitivity.

[0009] To achieve the above object, according to one aspect of the present invention, there is provided an anti-Helicobacter pylori antibody, which antibody comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19 and three complementary determining regions of any one of the light chain variable regions having amino acid sequences SEQ ID NO: 21, 22, and 23.

[0010] To achieve the above object, according to a second aspect of the present invention, there is provided an anti-Helicobacter pylori antibody, which antibody comprises the following complementary determining regions:

[0011] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1 (SYWMH), or consists of the same;

[0012] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2 (YINPSTGNTEYDQRFKG), or consists of the same;

[0013] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3 (DFDTYYDS), or consists of the same;

[0014] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4 (RASSSVLYIH) or 17 (RASSSVIYIH), or consists of the same;

[0015] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5 (ATSNLAS), or consists of the same;

[0016] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6 (QQWSSNPFT), or consists of the same.

[0017] To achieve the above object, according to a third aspect of the present invention, there is provided an anti-Helicobacter pylori antibody, 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 SEQ ID NO: 19; and the amino acid sequence of the light chain variable region is any one of SEQ ID NO: 21, 22, and 23.

[0018] To achieve the above object, according to a fourth aspect of the present invention, there is provided an anti-Helicobacter pylori antibody, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 20; and the amino acid sequence of the light chain is any one of SEQ ID NO: 24, 25, and 26.

[0019] To achieve the above object, according to a fifth aspect of the present invention, there is provided an antibody conjugate, which antibody conjugate comprises the above antibody.

[0020] To achieve the above object, according to the sixth aspect of the present invention, there is provided a reagent or a kit, which comprises the above-mentioned antibody or the above-mentioned antibody conjugate.

[0021] To achieve the above object, according to the seventh aspect of the present invention, there is provided a use of the above-mentioned antibody or antibody conjugate in the preparation of a product for detecting Helicobacter pylori.

[0022] To achieve the above object, the present invention also provides a nucleic acid, a vector, a cell and a method for preparing the above-mentioned antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other relevant drawings can also be obtained based on these drawings.

[0024] Figure 1 Results of reducing SDS-PAGE for Anti-HP 9D16 Rmb1 to Anti-HP 9D16 Rmb3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] In a first aspect, an embodiment of the present invention provides an antibody against Helicobacter pylori, which comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19 and three complementary determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO: 21, 22, and 23.

[0026] 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 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 described in the first aspect.

[0027] For example, 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: 19; 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: 21.

[0028] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies or antigen-binding fragments of antibodies, provided that they exhibit the required antigen-binding activity.

[0029] Antigen-binding fragments of antibodies generally have the same binding specificity as the antibodies from which they are derived and may be selected from any one of F(ab’)2, Fab’, Fab, Fv and scFv. It is readily understood by those skilled in the art from the content described in the present invention that antigen-binding fragments of 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 intact antibody in the present invention, those skilled in the art can readily obtain the above-mentioned antigen-binding fragments of antibodies.

[0030] Antigen-binding fragments of 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 and the like.

[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 the antigen or epitope it recognizes. In the specific embodiments of the present invention, the CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0032] In the present invention, the heavy-chain complementary determining regions are denoted as HCDR and include HCDR1, HCDR2 and HCDR3; the light-chain complementary determining regions are denoted as 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 schemes but will still overlap at least a portion of the CDR regions defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results for specific residues or groups of residues. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different documents may vary slightly. Given the amino acid sequence of the variable region of a given antibody, those skilled in the art can routinely determine which residues comprise a specific CDR. It should be noted that CDRs defined by other methods not limited to those in Table 1 also fall within the scope of protection of the present disclosure.

[0034] Table 1: CDR Definitions 1

[0035] CDR Kabat AbM2 IMGT Chothia HCDR1 <![CDATA[H31~H35 3 > <![CDATA[H26~H35 3 > <![CDATA[H26~H33..5 5 > <![CDATA[H26~H32..34 4 > HCDR2 H50 - H65 H50 - H58 H51 - H57 H52 - H56 HCDR3 H95 - H102 H95 - H102 H93 - H102 H95 - H102 LCDR1 L24 - L34 L24 - L34 L27 - L32 L24 - L34 LCDR2 L50 - L56 L50 - L56 L50 - L51 L50 - L56 LCDR3 L89 - L97 L89 - L97 L89 - L97 L89 - L97

[0036] 1 The numbers for all CDR definitions in Table 1 are based on the Kabat numbering system (see below), and 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 unambiguously map this Kabat numbering system to any variable region sequence without relying on any experimental data outside of 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).

[0037] 2 As used in Table 1, "AbM" with a lowercase "b" refers to the CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

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

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

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

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

[0047] 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.

[0048] In a second aspect, embodiments of the present invention provide an anti-Helicobacter pylori antibody, the antibody comprising the following complementarity determining regions:

[0049] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:1 (SYWMH), or consists of the same;

[0050] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:2 (YINPSTGNTEYDQRFKG), or consists of the same;

[0051] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:3 (DFDTYYDS), or consists of the same;

[0052] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO:4 (RASSSVLYIH) or 17 (RASSSVIYIH), or consists of the same;

[0053] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO:5 (ATSNLAS), or consists of the same;

[0054] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO:6 (QQWSSNPFT), or consists of the same.

[0055] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0056] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, which refers to the regions other than the CDRs in the variable region of the heavy chain and the variable region of the light chain 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.

[0057] In the present invention, the heavy chain variable region is obtained by connecting the CDRs and FRs numbered below in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the CDRs and FRs numbered below in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0058] In an alternative embodiment, the antibody according to the first aspect or the second aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0059] In an alternative embodiment, the HFR1 comprises / is as shown in SEQ ID NO:7 or an amino acid sequence having at least 80% identity therewith;

[0060] The HFR2 comprises / is as shown in SEQ ID NO:8 or an amino acid sequence having at least 80% identity therewith;

[0061] The HFR3 comprises / is as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% identity therewith;

[0062] The HFR4 comprises / is as shown in SEQ ID NO:10 or an amino acid sequence having at least 80% identity therewith;

[0063] The LFR1 comprises / is as shown in SEQ ID NO:11 or an amino acid sequence having at least 80% identity therewith;

[0064] The LFR2 comprises / is as shown in SEQ ID NO:12 or an amino acid sequence having at least 80% identity therewith;

[0065] The LFR3 comprises / is as shown in SEQ ID NO:13 or an amino acid sequence having at least 80% identity therewith; and

[0066] The LFR4 comprises / is as shown in SEQ ID NO:14 or an amino acid sequence having at least 80% identity therewith.

[0067] It should be noted that in other embodiments, the amino acid sequences of the respective framework regions of the antibody against Helicobacter pylori 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) above.

[0068] In an alternative embodiment, the LFR2 comprises the amino acid sequence as shown in SEQ ID NO:18.

[0069] In a third aspect, an embodiment of the present invention provides an antibody against Helicobacter pylori, 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 SEQ ID NO:19, and the amino acid sequence of the light chain variable region is any one of SEQ ID NO:21, 22, and 23.

[0070] In an alternative embodiment, the antibodies described in the first, second, and third aspects above further comprise a constant region.

[0071] In an alternative embodiment, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0072] 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.

[0073] 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.

[0074] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0075] In an alternative embodiment, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0076] In an alternative embodiment, the species origin of the constant region is bovine, equine, dairy cattle, porcine, ovine, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting chicken or human.

[0077] In an alternative embodiment, the species origin of the constant region is mouse.

[0078] In this article, the division of variable region and constant region sequences refers to the IMGT division method, see Lefranc, the international ImMunoGeneTics database. Nucl. Acids Res., 29(1):207 - 209(2001). DOI:10.1093 / nar / 29.1.207. PMID:11125093. and Martinez-Jean C. and Bosc N. or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: housemouse (Mus musculus) IGHC, IMGT Repertoire. the internationalImMunoGenetics information http: / / www.imgt.org .Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. Or Ehrenmann, Patrice Duroux, Chantal Ginestoux, Gene table: house mouse (Mus musculus) IGLC, IMGT Repertoire. theinternational ImMunoGenetics information http: / / www.imgt.org .Created: 16 / 03 / 2011. Version: 17 / 01 / 2020.. There will be some amino acid differences between the variable regions divided by different methods and the C-terminus of the variable region or the N-terminus of the constant region divided by IMGT. The variable regions or constant regions divided by other methods well known in the art are also within the protection scope of the present invention.

[0079] 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.

[0080] 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).

[0081] In an alternative embodiment, the antibody includes any one of F(ab)2, F(ab’)2, Fab’, Fab, Fv and scFv.

[0082] In a fourth aspect, the present invention provides an antibody against Helicobacter pylori, including a heavy chain and / or a light chain. The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 20, and the amino acid sequence of the light chain is any one of SEQ ID NO: 24, 25, 26.

[0083] In a fifth aspect, the present invention provides an antibody conjugate, and the antibody conjugate includes the above-mentioned antibody.

[0084] In an alternative embodiment, the above-mentioned antibody conjugate further includes biotin or a biotin derivative conjugated to the antibody.

[0085] In an alternative embodiment, the antibody conjugate further includes a label conjugated to the antibody.

[0086] 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 detected by an instrument, and qualitative or quantitative detection of the corresponding target can be achieved through these characteristics.

[0087] In an alternative embodiment, the label includes, but is not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0088] In actual use, those skilled in the art can select a suitable label according to the detection conditions or actual needs. No matter which label is used, it falls within the protection scope of the present invention.

[0089] In an alternative embodiment, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (such as, but not limited to, fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, but not limited to, rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as, 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 (such as, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0090] In an alternative embodiment, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate dehydrogenase.

[0091] 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.

[0092] 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 ester and its derivatives, dioxetane and its derivatives, rosalic acid and its derivatives, and peroxyoxalate and its derivatives.

[0093] 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.

[0094] In alternative embodiments, the colloids include, but are not limited to, colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres, and latex.

[0095] In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0096] In alternative embodiments, the colloidal metal is colloidal gold.

[0097] In alternative embodiments, the above antibody conjugate further includes a solid-phase carrier conjugated to the antibody.

[0098] In alternative embodiments, the solid-phase carrier is selected from microspheres, plates, and membranes.

[0099] In alternative embodiments, the solid-phase carrier includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microtiter plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0100] In a sixth aspect, the present invention provides a reagent or a kit, which includes the above-mentioned antibody or the above-mentioned antibody conjugate.

[0101] As described above, the antibodies in some embodiments or examples of the present invention can effectively bind to Helicobacter pylori. Therefore, reagents or kits containing the Helicobacter pylori antibodies can effectively detect Helicobacter pylori qualitatively or quantitatively. Applying the reagents or kits provided by the present invention, for example, can be used in detections such as immunoblotting and immunoprecipitation that involve the specific binding performance of Helicobacter pylori and its antibodies. As described above, the antibodies in some embodiments or examples of the present invention have higher binding activity or affinity with Helicobacter pylori. Therefore, the reagents or kits containing the antibodies have higher detection sensitivity or specificity.

[0102] In a seventh aspect, the present invention provides a method for detecting Helicobacter pylori, comprising: a) contacting the above-mentioned antibody, 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;

[0103] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody.

[0104] In an alternative embodiment, the immune complex further comprises a second antibody that binds to Helicobacter pylori.

[0105] In an eighth aspect, the present invention provides the use of the above-mentioned anti-Helicobacter pylori antibodies and antibody conjugates in the preparation of products for detecting Helicobacter pylori.

[0106] It should be noted that the products of the present invention include but are not limited to reagents, kits, test strips or detection plates.

[0107] In a ninth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0108] In a tenth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0109] In an eleventh aspect, the present invention provides a cell containing the above-mentioned vector.

[0110] In a twelfth aspect, the present invention provides a method for preparing an anti-Helicobacter pylori antibody, which comprises: culturing the cells as described above.

[0111] Based on the disclosure of the amino acid sequence of the anti-Helicobacter pylori antibody in the present invention, it is easy for those skilled in the art to conceive of preparing the anti-Helicobacter pylori antibody by using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression). For example, the antibody can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the antibody described in any one of the above. This is easily achievable for those skilled in the art. Based on this, regardless of the technique used to prepare the anti-Helicobacter pylori antibody of the present invention, it falls within the protection scope of the present invention.

[0112] 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. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not indicated by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0113] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one 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 noted, the techniques employed or considered herein are standard methods. The materials, methods, and examples are illustrative only and not restrictive.

[0114] 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", 2nd Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M. Weir and C.C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller and M.P.Calos, eds., 1987); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction" (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (J.E. Coligan et al., eds., 1991), each of which is hereby expressly incorporated by reference.

[0115] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0116] Example 1 Preparation of Anti-HP 9D16 Monoclonal Antibody

[0117] 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 9D16 monoclonal antibody was the hybridoma cell line prepared in this laboratory and was revived for standby.

[0118] (1) Preparation of antibody genes

[0119] mRNA was extracted from the hybridoma cell line secreting the Anti-HP 9D16 monoclonal antibody, and DNA products were obtained by RT-PCR. After the addition of A reaction to this product with rTaq DNA polymerase, it was inserted into the pMD-18T vector and transformed into DH5α competent cells. After colonies grew out, Heavy Chain and Light Chain gene clones were taken respectively, and 4 clones each were sent to a gene sequencing company for sequencing.

[0120] (2) Sequence analysis of the variable region genes of Anti-HP 9D16 antibody

[0121] 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 318 bp, and there was a 57 bp leader peptide sequence in front of it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 360 bp, belonging to the VH1 gene family, and there was a 57 bp leader peptide sequence in front of it.

[0122] (3) Construction of recombinant antibody expression plasmid

[0123] pcDNA TM 3.4 The pcDNA 3.4 vector was the recombinant antibody eukaryotic expression vector constructed. This expression vector had introduced multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI, and 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 protection bases at both ends, and a 0.70 kb Light Chain gene fragment and a 1.39 kb Heavy Chain gene fragment were amplified by PCR amplification.

[0124] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI double enzymes, 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.

[0125] 2. Recombinant antibody production

[0126] Resuscitate HEK293 cells in advance and passage 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 culture medium, and at the same time adjust the cell density to 2.9×10 6 cells / ml. Wash the cells, resuspend them with the culture medium, and at the same time, use it as the cell diluent. Prepare the plasmid DNA and transfection reagent diluents with the culture 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. Affinity purify the centrifuged supernatant with a protein A affinity chromatography column. Take 6 ug of the purified antibody for reducing SDS-PAGE, and the electrophoresis pattern is shown in the figure. After reducing SDS-PAGE, two bands are shown, one with Mr of 50 KD (heavy chain) and the other with Mr of 28 KD (light chain).

[0127] The obtained antibody was named Anti-HP 9D16Rmb1, and the Anti-HP 9D16Rmb1 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:

[0128] Table 2: Antibody sequences

[0129] Antibody Name Heavy Chain Light Chain Anti-HP 9D16Rmb1 SEQ ID NO:20 SEQ ID NO:24 Anti-HP 9D16Rmb2 SEQ ID NO:20 SEQ ID NO:26 Anti-HP 9D16Rmb3 SEQ ID NO:20 SEQ ID NO:25

[0130] Example 2 Performance detection of the antibody

[0131] 1. Activity identification

[0132] Dilute goat anti-mouse IgG at 1 ug / ml with the coating solution (main component NaHCO 3 ) for microplate coating, 100 uL per well, overnight at 4°C; the next day, wash with the washing solution (main component Na 2 HPO4 Wash twice with NaCl solution, then pat dry; add blocking solution (20% BSA + 80% PBS), 120 μL per well, incubate at 37 °C for 1 h, then pat dry; add diluted purified antibody and control antibody, 100 μL per well, incubate at 37 °C for 60 min; discard the liquid in the plate, pat dry, add 20% mouse negative blood to block, 120 μL per well, incubate at 37 °C for 1 h; discard the liquid in the plate, pat dry, add diluted HP recombinant antigen (from Fapon Biotech), 100 μL per well, incubate at 37 °C for 40 min; wash 5 times with washing solution, then pat dry; add HRP-labeled HP paired antibody (from Fapon Biotech, which can pair with the purified antibody), 100 μL per well, incubate at 37 °C for 30 min; add chromogenic solution A (50 μL per well), add chromogenic solution B (50 μL per well), incubate for 10 min; add stop solution, 50 μL per well; read the OD value at 450 nm (reference 630 nm) on the microplate reader.

[0133] Note: Solution A (main components: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main components: citric acid

[0134] + EDTA·2Na + TMB + concentrated HCl); Stop solution (EDTA·2Na + concentrated H 2 SO 4 )

[0135] Table 3: Activity data

[0136] Concentration (ng / ml) 250 125 62.50 31.25 15.63 0.00 Control 1.203 0.862 0.412 0.184 0.025 0.018 Anti-HP 9D16Rmb1 1.878 1.267 0.551 0.205 0.080 0.045 Anti-HP 9D16Rmb2 1.870 1.561 1.196 0.629 0.341 0.042 Anti-HP 9D16Rmb3 1.896 1.149 0.559 0.370 0.091 0.040

[0137] 2. Stability assessment

[0138] Place the above-mentioned 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 conduct activity detection on the 21-day samples. The results show that no obvious protein status 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-mentioned antibody is stable. Table 4 below shows the OD results of the enzyme immunoassay activity detection of the antibody Anti-HP 9D16Rmb2 after 21 days of assessment.

[0139] Table 4: Stability data

[0140] Sample Concentration (ng / ml) 125 62.5 0 Sample at 4°C for 21 days 1.557 1.021 0.027 Sample at -80°C for 21 days 1.551 1.001 0.019 Sample at 37°C for 21 days 1.549 1.029 0.005

[0141] 3. Performance detection of colloidal gold platform

[0142] 3.1 Antibody labeling process

[0143] (1) Adjust the labeling pH: Take 10 ml of 4 / 10,000 colloidal gold, centrifuge, add 0.2 M K 2 CO3 150 ul, stir and mix evenly for 2 min;

[0144] (2) Couple antibodies: Add 200 ug of antibody Anti-HP-C (from Fapon Biotech) respectively, and stir for reaction for 15 min;

[0145] (3) Block: Add 10% (mass / volume ratio) BSA, and stir for reaction for 15 min;

[0146] (4) Centrifuge and store: 10000 rpm / 7 min / room temperature, remove the supernatant, resuspend with gold label reconstitution solution to 1 ml, and store at 4 °C for later use;

[0147] 3.2 Coating

[0148] (1) Assemble the nitrocellulose membrane and the colloidal gold PVC bottom plate for later use;

[0149] (2) Dilute antibodies Anti-HP 9D16Rmb1, Anti-HP 9D16Rmb2, Anti-HP 9D16Rmb3 and control antibody Anti-HP-D (from Fapon Biotech) to 0.5 mg / ml and 1.5 mg / ml respectively. Use a gold-spraying and membrane-drawing instrument to draw lines evenly on the NC membrane, and then put it into an incubator at 50 °C for drying, for at least 4 h or more. Assemble, cut into strips, and perform sample addition and detection.

[0150] 3.3 Detection

[0151] (1) Samples: HP quality control products with different concentrations

[0152] (2) Detection method: Colloidal gold detection, observe the depth of the detection line with the naked eye to judge the result. According to the depth of the color of the displayed band, the activity of the antigen-antibody binding in the sample can be indicated. Compare the color of the T line strip of the colloidal gold test strip reaction with the standard color card, select the closest color, and mark the activity of the product with the number of the color number corresponding to this color.

[0153] The smaller the number, the stronger the color development and the higher the activity; the higher the number, the weaker the color development and the lower the activity; the number with a "+" indicates slightly stronger, the number with a "-" indicates slightly weaker, and "B" represents negative.

[0154] 3.4 Detection results

[0155] The detection results are shown in Table 5. The results show that the detection sensitivity of antibodies Anti-HP 9D16Rmb1, Anti-HP 9D16Rmb2, and Anti-HP 9D16Rmb3 on the colloidal gold platform is better than that of the control antibody.

[0156] Table 5: Detection results on the colloidal gold platform

[0157] QC Concentration HP Antigen 1 / 50 HP Antigen 1 / 100 HP Antigen 1 / 1K Diluent Anti-HP-D (0.5mg / ml) 9+ 9+ B B Anti-HP 9D16Rmb1 (0.5mg / ml) 5 7+ 9 B Anti-HP 9D16Rmb2 (0.5mg / ml) 5 7 9 B Anti-HP 9D16Rmb3 (0.5mg / ml) 6 7 9 B Anti-HP-D (1.5mg / ml) 8+ 8 B B Anti-HP 9D16Rmb1 (1.5mg / ml) 5 6 9+ B Anti-HP 9D16Rmb2 (1.5mg / ml) 5 6 9+ B Anti-HP 9D16Rmb3 (1.5mg / ml) 5 6 9+ B

[0158] The partial amino acid sequences involved in this application are shown in Table 6 as follows:

[0159] Table 6: Amino Acid Sequence Table

[0160]

[0161]

[0162] The above are only the preferred embodiments of the present invention and are not used 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.

Claims

1. An anti-Helicobacter pylori antibody, comprising three complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 19 and three complementarity determining regions of a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 21, 22, and 23.

2. The antibody according to claim 1, characterized in that The complementarity determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

3. An anti-Helicobacter pylori antibody, characterized in that: The antibody comprises the following complementarity determining regions: HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:1 (SYWMH); HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:2 (YINPSTGNTEYDQRFKG); HCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO:3 (DFDTYYDS); LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 (RASSSVLYIH) or 17 (RASSSVIYIH); LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5 (ATSNLAS); LCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO:6 (QQWSSNPFT); Optionally, the HFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto; The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; The HFR3 comprises SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto; The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto; The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; and The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

4. An anti-Helicobacter pylori antibody comprising a heavy chain variable region and / or a light chain variable region, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 19; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 21, 22, and 23; Optionally, the antibody further comprises a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species of origin of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is mouse; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto; Optionally, the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto.

5. An anti-Helicobacter pylori antibody, comprising a heavy chain and / or a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in SEQ ID NO: 20; the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 24, 25, and 26.

6. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody according to any one of claims 1 to 5; Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody; Optionally, the antibody conjugate further comprises a label coupled to the antibody; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle labels; Optionally, the antibody conjugate further comprises a solid phase carrier coupled to the antibody.

7. A reagent or a kit, characterized in that: The reagent or kit comprises the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6.

8. Use of the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6 in the preparation of a product for detecting Helicobacter pylori; Optionally, the use includes: a) contacting the antibody according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 with Helicobacter pylori in a sample to be detected 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 the complex indicating the presence of the antigen in the test sample; Optionally, the immune complex further comprises a second antibody, which binds to the antibody; Optionally, the immune complex further comprises a second antibody, which binds to Helicobacter pylori.

9. A nucleic acid, a vector, a cell or a method for preparing the antibody according to any one of claims 1 to 5, wherein the nucleic acid encodes the antibody according to any one of claims 1 to 5; the vector contains a nucleic acid encoding the antibody according to any one of claims 1 to 5; the cell contains the above nucleic acid or vector; the method comprises the above cell.

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