Anti-statin B antibody and application thereof

Abstract

The invention discloses an anti-statin B antibody and application thereof, and relates to the field of antibodies. The anti-statin B 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 statin B, and has good affinity or activity.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and specifically, to an antibody against inhibin B and its applications.

[0002] Background Art

[0003] Inhibin B (INHB) is a heterogeneous glycoprotein derived from germ cells, composed of a BB subunit linked to an a subunit, with a predicted molecular weight of approximately 28 kDa, and belongs to the superfamily of transforming growth factor (TGF-β). Inhibin B is mainly produced by testicular Sertoli cells in males and mainly by ovarian cells in females. High levels of INHB in the body regulate the pituitary gland through negative feedback and inhibit the release of FSH; in addition, it also affects the paracrine function of the gonads by regulating the amount of estradiol (E2). The content of INHB is closely related to male spermatogenic ability, testicular volume, total sperm count, and female ovarian reserve function and female infertility caused by ovarian factors. Its content decreases with age, directly reflecting the gonadal reserve status of both sexes.

[0004] Clinically, inhibin B is commonly used for the detection and diagnosis of gonadal functions such as various spermatogenesis disorders and ovarian reserve function. In males, INHB is a direct product of the vas deferens, reflecting the function of the entire testicular tissue, and is commonly used for the diagnosis of diseases such as spermatogenic function, cryptorchidism, azoospermia, primary testicular failure, hypogonadotropic hypogonadism, Kallmann syndrome, Klinefelter syndrome, precocious puberty, and complete or selective loss of hypothalamic or pituitary function. In females, INHB is an important indicator of ovarian reserve and directly participates in the regulation of follicle formation and development, and is of great significance for the diagnosis and treatment of diseases such as decreased ovarian reserve function, premature ovarian failure, polycystic ovary syndrome, and endometriosis.

[0005] Currently, the detection methods for inhibin B are mainly immunological detection methods, such as enzyme-linked immunosorbent assay. The principle is as follows: 1) Bind the antigen or antibody to the surface of a certain solid-phase carrier and maintain its immunological activity; 2) Link the antigen or antibody with a certain enzyme to form an enzyme-labeled antigen or antibody, which retains both its immunological activity and the activity of the enzyme. React the test specimen (detecting the antibody or antigen therein) and the enzyme-labeled antigen or antibody with the antigen or antibody on the surface of the solid-phase carrier in different steps. Use the method of washing to separate the Antigen-antibody complex formed on the solid-phase carrier from other substances, add enzymes reaction substrate, and the substrate is Enzyme catalysis converted into a colored product. The amount of the product is directly related to the amount of the detected substance in the specimen. Therefore, it can be based on Color reactionQualitative or quantitative analysis is carried out according to the depth. Similar immunological detection methods also include radioimmunoassay, fluorescence immunochromatography, chemiluminescence method, etc.

[0006] All of the above immunological detection methods require antibodies against inhibin B. Therefore, there is a strong demand in the art for antibodies against inhibin B with good performance. SUMMARY OF THE INVENTION

[0007] The present application provides an antibody against inhibin B, which provides an important raw material source for the detection of inhibin B and has good activity or affinity.

[0008] To achieve the above object, according to one aspect of the present invention, there is provided an antibody against inhibin B, which comprises three complementary determining regions of any one of the heavy chain variable regions having the amino acid sequence SEQ ID NO: 20 or 21 and three complementary determining regions of any one of the light chain variable regions having the amino acid sequence SEQ ID NO: 24, 25 or 26.

[0009] To achieve the above object, according to the second aspect of the present invention, there is provided an antibody against inhibin B, and the antibody comprises the following complementary determining regions:

[0010] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the same;

[0011] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of the same;

[0012] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3, or consists of the same;

[0013] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4, or consists of the same;

[0014] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5, or consists of the same;

[0015] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6 or 17, or consists of the same.

[0016] To achieve the above object, according to the third aspect of the present invention, there is provided an antibody against inhibin B, which comprises a heavy chain variable region and / or a light chain variable region, and the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 20 or 21; the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 24, 25 or 26.

[0017] To achieve the above object, according to the fourth aspect of the present invention, an anti-inhibin B antibody is provided, which includes a heavy chain and / or a light chain. The amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO:22 and SEQ ID NO:23; the amino acid sequence of the light chain is as shown in any one of SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29.

[0018] To achieve the above object, according to the fifth aspect of the present invention, an antibody conjugate is provided, and the antibody conjugate includes the above-mentioned antibody.

[0019] To achieve the above object, according to the sixth aspect of the present invention, a reagent or a kit is provided, and the reagent or the kit includes the above-mentioned antibody or the above-mentioned antibody conjugate.

[0020] To achieve the above object, according to the seventh aspect of the present invention, uses of the above-mentioned antibody and antibody conjugate in the preparation of a product for detecting inhibin B are provided.

[0021] 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. Detailed Embodiments

[0022] In the first aspect, an embodiment of the present invention provides an anti-inhibin B antibody, which includes three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO:20 and SEQ ID NO:21, 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:24, SEQ ID NO:25, and SEQ ID NO:26.

[0023] 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.

[0024] 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:20; 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:24.

[0025] 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, as long as they exhibit the required biological activity.

[0026] The above antigen-binding fragments generally have the same binding specificity as the antibodies from which they are derived. It is readily understood by those skilled in the art from the content described in the present invention that the above antigen-binding fragments 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 full antibody in the present invention, those skilled in the art can readily obtain the above antigen-binding fragments.

[0027] The above antigen-binding fragments 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.

[0028] 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.

[0029] 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.

[0030] 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 used 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). The "Chothia definition" is described in 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 with at least a portion of the CDR regions defined by Kabat, although they may be shortened or extended 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 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 comprise a particular 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.

[0031] Table 1: CDR Definition 1

[0032] 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

[0033] 1 The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H+number" and amino acid numbers on the light chain represented by "L+number." One of ordinary skill in the art can unambiguously assign this Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., U.S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0034] 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0035] 3 If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.

[0036] 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.

[0037] 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.

[0038] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

[0039] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0040] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Chothia system.

[0041] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT system.

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

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

[0044] 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.

[0045] In a second aspect, an embodiment of the present invention provides an antibody against inhibin B, the antibody comprising the following complementarity-determining regions:

[0046] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:1, or consists of the same.

[0047] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:2, or consists of the same.

[0048] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:3, or consists of the same.

[0049] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO:4, or consists of the same.

[0050] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO:5, or consists of the same.

[0051] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO:6 or 17, or consists of the same.

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

[0053] 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 an 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.

[0054] 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.

[0055] 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.

[0056] 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 thereto;

[0057] The HFR2 comprises / is as shown in SEQ ID NO:8 or an amino acid sequence having at least 80% identity thereto;

[0058] The HFR3 comprises / is as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto;

[0059] The HFR4 comprises / is as shown in SEQ ID NO:10 or an amino acid sequence having at least 80% identity thereto;

[0060] The LFR1 comprises / is as shown in SEQ ID NO:11 or an amino acid sequence having at least 80% identity thereto;

[0061] The LFR2 comprises / is as shown in SEQ ID NO:12 or an amino acid sequence having at least 80% identity thereto;

[0062] The LFR3 comprises / is as shown in SEQ ID NO:13 or an amino acid sequence having at least 80% identity thereto; and

[0063] The LFR4 comprises / has an amino acid sequence as set forth in SEQ ID NO:14 or having at least 80% identity thereto.

[0064] It should be noted that in other embodiments, the amino acid sequences of the respective framework regions of the anti-inhibin B antibody 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).

[0065] In an alternative embodiment, the HFR1 comprises / has the amino acid sequence as set forth in SEQ ID NO:18.

[0066] In an alternative embodiment, the LFR1 comprises / has the amino acid sequence as set forth in SEQ ID NO:19.

[0067] In an alternative embodiment, the antibody binds inhibin B with an affinity of KD < 6.74×10-10 M.

[0068] In an alternative embodiment, the antibody binds inhibin B 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.

[0069] In an alternative embodiment, the antibody binds inhibin B with an affinity of KD ≤ 5.33×10 -11 M.

[0070] 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.

[0071] In an alternative embodiment, the determination of KD uses a kinetic detection method; optionally, surface plasmon resonance, for example, by using a biosensor system such as [[ID=Z37]] system.

[0072] In a third aspect, an embodiment of the present invention provides an antibody against inhibin B, comprising a heavy chain variable region and / or a light chain variable region. The amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 20 and 21, and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 24, 25, and 26.

[0073] In an optional embodiment, the heavy chain variable region and the light chain variable region described in the above first aspect or third aspect are selected from any one of the following combinations:

[0074] combination Heavy chain variable region Light chain variable region 1 SEQ ID NO:20 SEQ ID NO:24 2 SEQ ID NO:21 SEQ ID NO:24 3 SEQ ID NO:20 SEQ ID NO:26 4 SEQ ID NO:20 SEQ ID NO:25 .

[0075] In an optional embodiment, the antibody described in the above first aspect, second aspect, and third aspect further comprises a constant region.

[0076] In an optional embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0077] In an optional 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.

[0078] In an optional embodiment, 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.

[0079] In an optional embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0080] In an optional embodiment, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0081] In an optional embodiment, the species origin of the constant region is bovine, equine, dairy cow, porcine, ovine, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting chicken or human.

[0082] In an optional embodiment, the species origin of the constant region is mouse.

[0083] In this article, the division of variable region and constant region sequences follows 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 international ImMunoGenetics 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. the international ImMunoGenetics information http: / / www.imgt.org . Created: 16 / 03 / 2011. Version: 17 / 01 / 2020.. There will be some amino acid differences in the variable regions divided by different methods and the C-terminal of the variable region or the N-terminal 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.

[0084] In an optional 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.

[0085] 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%, 9,0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above constant regions (SEQ ID NO:15 or 16).

[0086] In an alternative embodiment, the antibody includes any one of F(ab)2, F(ab’)2, Fab’, Fab, Fv, and scFv.

[0087] In a fourth aspect, the present invention provides an antibody against inhibin B, including 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:22 and 23, and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO:27, 28, and 29.

[0088] In an alternative embodiment, the antibody described in the first, second, third, or fourth aspect above includes a heavy chain and a light chain in any of the following combinations:

[0089] combination Heavy chain light chain 1 SEQ ID NO:22 SEQ ID NO:27 2 SEQ ID NO:23 SEQ ID NO:27 3 SEQ ID NO:22 SEQ ID NO:29 4 SEQ ID NO:22 SEQ ID NO:28

[0090] In a fifth aspect, the present invention provides an antibody conjugate, which includes the above-mentioned antibody.

[0091] In an alternative embodiment, the above-mentioned antibody conjugate further includes biotin or a biotin derivative conjugated to the antibody.

[0092] In an alternative embodiment, the antibody conjugate further includes a marker or a purification tag conjugated to the antibody.

[0093] In an alternative embodiment, the above-mentioned marker refers to a class of substances having characteristics such as luminescence, chromogenicity, 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.

[0094] In an alternative embodiment, the marker includes, but is not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based markers.

[0095] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the protection scope of the present invention.

[0096] 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.).

[0097] 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.

[0098] 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.

[0099] 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, rofluorescein and its derivatives, and peroxyoxalate and its derivatives.

[0100] 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.

[0101] In alternative embodiments, the colloids include, but are not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latex.

[0102] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0103] In an alternative embodiment, the colloidal metal is colloidal gold.

[0104] In an alternative embodiment, the above antibody conjugate further includes a solid-phase carrier conjugated to the antibody.

[0105] In an alternative embodiment, the solid-phase carrier is selected from microspheres, plates, and membranes.

[0106] In an alternative embodiment, 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.

[0107] In a sixth aspect, the present invention provides a reagent or kit, which includes the above antibody or the above antibody conjugate.

[0108] As described above, the antibodies in some embodiments or examples of the present invention can effectively bind to inhibin B. Therefore, a reagent or kit containing the inhibin B antibody can effectively qualitatively or quantitatively detect inhibin B. Using the reagent or kit provided by the present invention, for example, it can be used in detections such as immunoblotting and immunoprecipitation that involve the specific binding property of inhibin B and its antibody. As described above, the antibodies in some embodiments or examples of the present invention have a higher binding activity or affinity for inhibin B. Therefore, a reagent or kit containing the antibody has higher detection sensitivity or specificity.

[0109] In a seventh aspect, the present invention provides a method for detecting inhibin B, including: a) contacting the above antibody, antibody conjugate, reagent or kit with inhibin B in a test sample under conditions sufficient to allow 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;

[0110] In an alternative embodiment, the immune complex further includes a second antibody that binds to the antibody.

[0111] In an alternative embodiment, the immune complex further includes a second antibody that binds to inhibin B.

[0112] In an eighth aspect, the present invention provides the use of the above anti-inhibin B antibody and antibody conjugate in the preparation of a product for detecting inhibin B.

[0113] It should be noted that the products of the present invention include, but are not limited to, reagents, reagent kits, test strips or reagent plates.

[0114] In a ninth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0115] In a tenth aspect, the present invention provides a vector containing the above nucleic acid molecule.

[0116] In an eleventh aspect, the present invention provides a cell containing the above vector.

[0117] In a twelfth aspect, the present invention provides a method for preparing an anti-inhibin B antibody, which includes: culturing the cells as described above.

[0118] Based on the disclosure of the amino acid sequence of the anti-inhibin B antibody 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 anti-inhibin B antibody. 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, no matter which technique is used to prepare the anti-inhibin B antibody of the present invention, it falls within the protection scope of the present invention.

[0119] 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 with the manufacturer are all conventional products that can be obtained through commercial purchase.

[0120] 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 contemplated herein are standard methods. The materials, methods and examples are illustrative only and not restrictive.

[0121] 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 well 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.

[0122] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0123] Example 1 Antibody Discovery of Monoclonal Antibodies

[0124] 1. Animal Immunization

[0125] Mix the inhibin B antigen (from PhyGen Biotech) with complete Freund's adjuvant in equal volume to obtain an oily emulsion. Subcutaneously inject the emulsion into BALB / c mice at multiple points at a dose of 0.2 ml per mouse. After 14 days, immunize the mice intraperitoneally with the same antigen and adjuvant. For the fourth immunization, collect tail blood for titer detection, and the titer meets the fusion requirement. Three days before fusion, mix the same dose of antigen with an equal volume of 0.9% sodium chloride injection and inject intraperitoneally for booster immunization.

[0126] 2. Preparation of hybridoma cell line

[0127] On the third day after the mice are booster immunized, take out the spleens under sterile conditions. Mix mouse tumor cells and immunized spleen cells at a cell number ratio of 1:10, fuse and culture. On the sixth day of culture, change the HT culture medium twice. On the seventh day after fusion, collect cell supernatant for antibody detection to screen hybridoma cell strains secreting specific antibodies. A total of 1 strain is obtained and named Anti-INHB6A12.

[0128] Example 2 Preparation of monoclonal antibody

[0129] 1. Antibody sequence acquisition

[0130] (1) Preparation of antibody gene

[0131] Extract mRNA from the Anti-INHB 6A12 hybridoma cell strain, obtain DNA products by RT-PCR method. After adding A reaction to the products with rTaq DNA polymerase, insert them into the pMD-18T vector, transform them into DH5α competent cells. After colonies grow, respectively take Heavy Chain and Light Chain gene clones, and send 4 clones each to a gene sequencing company for sequencing.

[0132] (2) Sequence analysis of antibody variable region genes

[0133] Put the gene sequences obtained by the above sequencing into the kabat antibody database for analysis, and use VNTI11.5 software for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs are correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence is 321bp, and there is a 57bp leader peptide sequence in front of it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence is 345bp, belonging to the VH1 gene family, and there is a 57bp leader peptide sequence in front of it.

[0134] (3) Construction of recombinant antibody expression plasmid

[0135] pcDNA TM 3.4 The vector is the constructed eukaryotic expression vector of the recombinant antibody. Multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI have been introduced into this expression vector, which is named pcDNA3.4A expression vector, hereinafter referred to as 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. A 0.70 kb Light Chain gene fragment and a 1.37 kb Heavy Chain gene fragment were amplified by PCR amplification method.

[0136] The Heavy Chain and Light Chain gene fragments were respectively digested with HindIII / EcoRI double enzymes, and the 3.4A vector was digested with HindIII / EcoRI double enzymes. After the fragments and the vector were purified and recovered, the Heavy Chain gene and the Light Chain gene were respectively ligated into the 3.4A expression vector, and the recombinant expression plasmids of Heavy Chain and Light Chain were obtained respectively.

[0137] 2. Production of recombinant antibody

[0138] Resuscitate HEK293 cells in advance, passage culture to a 200 ml system to make the cell density reach 3 - 5×10 6 cells / ml. Select the antibody concentration and cells with a 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 to 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 it 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 to obtain the purified antibody.

[0139] The obtained antibody was named Anti-INHB 6A12Rmb1, and the mutant antibody was obtained by mutating Anti-INHB 6A12Rmb1. The sequences of the heavy chain (H) and light chain (L) of the above antibodies are shown in the following table:

[0140] Table 2: Antibody sequences

[0141] Antibody name Heavy chain light chain Anti-INHB 6A12Rmb1 SEQ ID NO:22 SEQ ID NO:27 Anti-INHB 6A12Rmb2 SEQ ID NO:23 SEQ ID NO:27 Anti-INHB 6A12Rmb3 SEQ ID NO:22 SEQ ID NO:29 Anti-INHB 6A12Rmb4 SEQ ID NO:22 SEQ ID NO:28

[0142] Performance Detection of the Antibody in Example 2

[0143] 1. Affinity Analysis

[0144] Dilute the purified antibody in advance, and at the same time perform gradient dilution on the inhibin B recombinant antigen (from Fapon Biotech); use the CM5 chip that has been 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, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, that is, the affinity constant; ka represents the binding rate; kd represents the dissociation rate)

[0145] Table 3: Affinity Data

[0146] Sample name KD ka kd comparison 6.74E-10 4.87E+04 3.28E-05 Anti-INHB 6A12Rmb1 5.33E-11 1.56E+05 8.31E-06 Anti-INHB 6A12Rmb2 5.31E-11 1.11E+05 5.89E-06 Anti-INHB 6A12Rmb3 4.99E-11 1.23E+05 6.14E-06 Anti-INHB 6A12Rmb4 5.01E-11 1.61E+05 8.07E-06

[0147] Conclusion: The affinities of the antibodies Anti-INHB 6A12Rmb1 to Anti-INHB 6A12Rmb4 with inhibin B are at 10× -11, , and they have good affinities.

[0148] 2. Activity Identification

[0149] Dilute the inhibin B recombinant antigen (from Fapon Biotech) to 3 μg / ml with the coating solution (main component NaHCO3), 100 μL per well, and incubate overnight at 4°C; the next day, wash twice with the washing solution (main components Na2HPO4 + NaCl), and pat dry; add the blocking solution (20% BSA + 80% PBS), 120 μL per well, at 37°C for 1 h, and pat dry; add the diluted purified antibody and control antibody, 100 μL / well, at 37°C for 30 min; wash 5 times with the washing solution, and pat dry; add goat anti-mouse IgG-HRP, 100 μL per well, at 37°C for 30 min; wash 5 times with the washing solution, and pat dry; add chromogenic solution A (50 μL / well), add chromogenic solution B (50 μL / well), for 10 min; add the stop solution, 50 μL / well; read the OD value at 450 nm (reference 630 nm) on the microplate reader.

[0150] 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)

[0151] Table 4: Activity Data

[0152] Concentration (ng / ml) 25 12.5 6.25 3.13 1.56 0 comparison 1.402 0.813 0.476 0.196 0.103 0.038 Anti-INHB 6A12Rmb1 1.688 1.176 0.786 0.434 0.267 0.02 Anti-INHB 6A12Rmb2 1.602 1.012 0.749 0.415 0.228 0.013 Anti-INHB 6A12Rmb3 1.654 1.072 0.763 0.455 0.254 0.012 Anti-INHB 6A12Rmb4 1.654 1.072 0.763 0.763 0.254 0.012

[0153] Conclusion: Antibodies Anti-INHB 6A12Rmb1 to Anti-INHB 6A12Rmb4 all bind to inhibin B antigen at low concentrations, and the antibodies have good binding activity.

[0154] 3. Stability assessment

[0155] Place the above-mentioned antibodies 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. Table 5 below shows the OD results of the enzyme immunoassay activity detection of antibody Anti-INHB 6A12Rmb3 after 21 days of assessment.

[0156] Table 5: Stability data

[0157] Sample concentration (ng / ml) 25 12.5 0 4℃, 21-day samples 1.667 1.038 0.022 -80℃, 21-day sample 1.632 1.009 0.018 37℃, 21-day samples 1.655 1.047 0.016

[0158] Conclusion: The results show that there are no obvious protein status changes in the antibodies after being placed for 21 days under the three assessment conditions, and the activity does not show a downward trend with the increase of the assessment temperature, indicating that the above-mentioned antibodies are stable.

[0159] 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.

[0160] Some amino acid sequences involved in this application are shown in Table 6:

[0161]

[0162]

Claims

1. An anti-inhibin B antibody comprising three complementarity determining regions of a heavy chain variable region having the amino acid sequence of any one of SEQ ID NOs: 20 and 21 and three complementarity determining regions of a light chain variable region having the amino acid sequence of any one of SEQ ID NOs: 24, 25, and 26.

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-inhibin B antibody, characterized in that The antibody comprises the following complementarity determining regions: HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1; HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2; HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3; LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4; LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 5; LCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 6 or 17; 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 at least 80% identical thereto; and The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto; Optionally, the antibody has a KD of less than 6.74×10 -10 M binds inhibin B with high affinity.

4. An anti-inhibin B 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 any one of SEQ ID NOs: 20 and 21; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 24, 25, and 26; Optionally, the combination of the heavy chain variable region and the light chain variable region is selected from any one of the following combinations: ； 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 origin of the constant region is cow, 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; Optionally, the antibody comprises any one of F(ab')2, Fab', Fab, Fv and scFv.

5. An anti-inhibin B antibody comprising a heavy chain and / or a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 22 and 23; the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 27, 28, and 29.

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 marker or purification tag coupled to the antibody; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels; Optionally, the antibody conjugate further comprises a solid phase carrier coupled to the antibody.

7. A reagent or 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 inhibin B; 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 inhibin B in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur, to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates 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 inhibin B.

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

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