Anti-mu globin antibody and application thereof

Abstract

The invention discloses an anti-mu globin antibody and application thereof, and relates to the field of antibodies. The anti-mu globin 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 mu globin, and has good specificity and activity.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and more particularly, to an antibody against μ-globin and its applications. Background Art

[0002] Thalassemia, formerly known as Mediterranean anemia or Cooley's anemia, is a group of hereditary hemolytic anemia diseases. Due to genetic defects, the synthesis of one or more globin chains in hemoglobin is absent or insufficient, resulting in anemia or pathological conditions. The molecular structure and synthesis of globin chains are determined by genes. The γ, δ, ε, and β globin genes form the "β gene cluster", and the ζ and α globin genes form the "α gene cluster". Normal individuals inherit two α globin genes (αα / αα) from each parent to synthesize sufficient α globin chains; and inherit one β globin gene from each parent to synthesize sufficient β globin chains. Due to the deletion or point mutation of globin genes, peptide chain synthesis disorders lead to the disease.

[0003] Thalassemia can be divided into β-thalassemia and α-thalassemia. α-Thalassemia refers to a genetic defect on human chromosome 16, resulting in abnormal translation of α-globin. According to the types of α gene defects, α-thalassemia can be divided into deletion type and non-deletion type (mainly point mutations). At least 35 deletions have been discovered globally, and the most common α-thalassemia gene deletion type in southern China is the Southeast Asian type. In Africa, α-thalassemia is mainly caused by point mutations.

[0004] In proteomic analysis of hemoglobin samples from α-thalassemia patients, carriers, and normal populations, it was found that the expression levels of α, β, γ, θ, and δ in normal populations are 2-3 orders of magnitude higher than those of the ζ and μ subunits. However, it was also found that the expression of hemoglobin ζ and μ subunits is significantly up-regulated in α-thalassemia patients and carriers. At the same time, studies have shown that the ratio analysis of the contents of α, ζ, and μ subunits, namely α / μ and α / ζ, can better distinguish normal people, silent α-thalassemia, non-deletion type α-thalassemia, and deletion type α-thalassemia. Detecting μ-globin in adults has positive significance for screening α-thalassemia gene carriers.

[0005] Currently, the main method for detecting μ-globin subunits is immunoassay, which is mainly based on the principle of antigen-antibody binding and requires an antibody against μ-globin subunits. Therefore, those skilled in the art have a strong demand for anti-μ-globin subunit antibodies with good performance. Summary of the Invention

[0006] The present application provides an antibody against μ-globin, which provides an important raw material source for the detection of μ-globin and has good specificity and activity.

[0007] To achieve the above object, according to one aspect of the present invention, there is provided an anti-μ-globin antibody, which antibody comprises three complementary determining regions of any one of the heavy chain variable regions having the amino acid sequences SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 52, 53, 75, 76, 77, 78 and three complementary determining regions of any one of the light chain variable regions having the amino acid sequences SEQ ID NO: 25, 26, 54, 79.

[0008] To achieve the above object, according to the second aspect of the present invention, there is provided an anti-μ-globin antibody, which antibody comprises CDR sequences of any one of the following numbered combinations:

[0009] Combination HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 1 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 2 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:86 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 3 SEQ ID NO:37 SEQ ID NO:38 SEQ ID NO:39 SEQ ID NO:40 SEQ ID NO:41 SEQ ID NO:42 4 SEQ ID NO:58 SEQ ID NO:59 SEQ ID NO:60 SEQ ID NO:61 SEQ ID NO:62 SEQ ID NO:63 5 SEQ ID NO:58 SEQ ID NO:59 SEQ ID NO:72 SEQ ID NO:61 SEQ ID NO:62 SEQ ID NO:63 6 SEQ ID NO:58 SEQ ID NO:59 SEQ ID NO:73 SEQ ID NO:61 SEQ ID NO:62 SEQ ID NO:63 7 SEQ ID NO:58 SEQ ID NO:59 SEQ ID NO:74 SEQ ID NO:61 SEQ ID NO:62 SEQ ID NO:63 。

[0010] To achieve the above object, according to the third aspect of the present invention, there is provided an anti-μ-globin 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 any one of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 52, 53, 75, 76, 77, 78; and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 25, 26, 54, 79.

[0011] To achieve the above object, according to the fourth aspect of the present invention, there is provided an anti-μ-globin antibody, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 80, 81, 82, 83; and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 35, 36, 57, 84.

[0012] To achieve the above object, according to the fifth aspect of the present invention, there is provided an antibody conjugate, which antibody conjugate comprises the above-mentioned antibody.

[0013] To achieve the above object, according to the sixth aspect of the present invention, there is provided a reagent or a kit, which reagent or kit comprises the above-mentioned antibody or the above-mentioned antibody conjugate.

[0014] To achieve the above object, according to the seventh aspect of the present invention, there is provided the use of the above-mentioned antibody and antibody conjugate in the preparation of a product for detecting μ-globin.

[0015] 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

[0016] In a first aspect, embodiments of the present invention provide an anti-μ globin antibody, which comprises three complementarity-determining regions of any one of the heavy chain variable regions having the amino acid sequences SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 52, 53, 75, 76, 77, 78 and three complementarity-determining regions of any one of the light chain variable regions having the amino acid sequences SEQ ID NO: 25, 26, 54, 79.

[0017] 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.

[0018] 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: 17; 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: 25.

[0019] 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 desired biological activity.

[0020] The above antigen-binding fragments generally have the same binding specificity as the antibody from which they are derived. Those skilled in the art can easily understand 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 chemically reducing and cleaving disulfide bonds. Based on the structure of the full antibody disclosed in the present invention, those skilled in the art can easily obtain the above antigen-binding fragments.

[0021] The above antigen-binding fragments can also be obtained by recombinant genetic techniques well 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.

[0022] In the present invention, the term "complementary determining region", "CDR" or "CDRs" refers to the hypervariable regions of the heavy and light chains of an immunoglobulin, and refers to a region 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 hypervariable regions of the heavy and light chains of the antibody.

[0023] In the present invention, the heavy chain complementary determining regions are denoted as HCDRs, which include HCDR1, HCDR2, and HCDR3; the light chain complementary determining regions are denoted as LCDRs, which include LCDR1, LCDR2, and LCDR3.

[0024] 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 lengthened depending on the prediction or experimental results of specific residues or groups of residues. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different literatures are slightly different. Given the amino acid sequence of the variable region of a given antibody, those skilled in the art can routinely determine which residues comprise a particular CDR. It should be noted that CDRs defined by other methods not limited to Table 1 also fall within the scope of protection of the present disclosure.

[0025] Table 1: CDR Definitions 1

[0026] 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

[0027] 1The numbers of all CDR definitions in Table 1 are based on the Kabat numbering system (see below). The amino acid numbers on the heavy chain are represented by "H + number", and the amino acid numbers on the light chain are represented by "L + number". A person of ordinary skill in the art can clearly map the Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein, "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).

[0028] 2 As used in Table 1, "AbM" with a lowercase "b" refers to the CDR defined by the "AbM" antibody modeling software of Oxford Molecular.

[0029] 3 If 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0034] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0035] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0036] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0037] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Contact system.

[0038] 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.

[0039] According to an embodiment of the present invention, the antibody comprises a CDR sequence of any one of the following numbered combinations:

[0040]

[0041]

[0042] In a second aspect, an embodiment of the present invention provides an antibody against μ-globin, the antibody comprising a CDR sequence of any one of the following numbered combinations:

[0043] Combination HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 1 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 2 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:86 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 3 SEQ ID NO:37 SEQ ID NO:38 SEQ ID NO:39 SEQ ID NO:40 SEQ ID NO:41 SEQ ID NO:42 4 SEQ ID NO:58 SEQ ID NO:59 SEQ ID NO:60 SEQ ID NO:61 SEQ ID NO:62 SEQ ID NO:63 5 SEQ ID NO:58 SEQ ID NO:59 SEQ ID NO:72 SEQ ID NO:61 SEQ ID NO:62 SEQ ID NO:63 6 SEQ ID NO:58 SEQ ID NO:59 SEQ ID NO:73 SEQ ID NO:61 SEQ ID NO:62 SEQ ID NO:63 7 SEQ ID NO:58 SEQ ID NO:59 SEQ ID NO:74 SEQ ID NO:61 SEQ ID NO:62 SEQ ID NO:63 .

[0044] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0045] 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 variable regions of the heavy chain and 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.

[0046] 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.

[0047] In an alternative embodiment, the antibody according to the first or second aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0048] In an alternative embodiment, the antibody comprises an FR sequence of any of the following numbered combinations or has at least 80% identity with the FR of any of the following numbered combinations:

[0049]

[0050]

[0051] It should be noted that in other embodiments, the amino acid sequences of the respective framework regions of the anti-μ globin 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).

[0052] In an alternative embodiment, the antibody comprises 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 SEQ ID NO:SEQ ID NO:17, 18, 19, 20, 21, 22, 23, 24, 52, 53, 75, 76, 77, 78, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:25, 26, 54, 79.

[0053] In a third aspect, an embodiment of the present invention provides an anti-μ globin antibody comprising a heavy chain variable region and / or a light chain variable region, the amino acid sequence of the heavy chain variable region is any one of SEQ ID NO:17, 18, 19, 20, 21, 22, 23, 24, 52, 53, 75, 76, 77, 78, and the amino acid sequence of the light chain variable region is any one of SEQ ID NO:25, 26, 54, 79.

[0054] In an alternative embodiment, the heavy chain variable region and the light chain variable region according to the first or third aspect above are selected from any of the following combinations:

[0055] Combination Heavy chain variable region Light chain variable region 1 SEQ ID NO:17 SEQ ID NO:25 2 SEQ ID NO:18 SEQ ID NO:25 3 SEQ ID NO:19 SEQ ID NO:25 4 SEQ ID NO:20 SEQ ID NO:25 5 SEQ ID NO:21 SEQ ID NO:25 6 SEQ ID NO:22 SEQ ID NO:25 7 SEQ ID NO:23 SEQ ID NO:25 8 SEQ ID NO:24 SEQ ID NO:25 9 SEQ ID NO:17 SEQ ID NO:26 10 SEQ ID NO:52 SEQ ID NO:54 11 SEQ ID NO:53 SEQ ID NO:54 12 SEQ ID NO:75 SEQ ID NO:79 13 SEQ ID NO:76 SEQ ID NO:79 14 SEQ ID NO:77 SEQ ID NO:79 15 SEQ ID NO:78 SEQ ID NO:79 。

[0056] In an alternative embodiment, the antibody described in the first, second, or third aspect above comprises a heavy chain variable region and a light chain variable region in any of the following combinations:

[0057]

[0058]

[0059] In an alternative embodiment, the antibody described in the first, second, or third aspect above further comprises a constant region.

[0060] In an alternative embodiment, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0061] 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.

[0062] 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.

[0063] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3, or IgG4.

[0064] In an alternative embodiment, the light chain constant region is selected from the kappa or lambda light chain constant region.

[0065] In an alternative 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 cock, or human.

[0066] In an alternative embodiment, the species origin of the constant region is mouse.

[0067] In this document, 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: house mouse (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 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.

[0068] 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.

[0069] 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 regions (SEQ ID NO: 15 or 16).

[0070] In an alternative embodiment, the antibody includes any one of F(ab)2, F(ab’)2, Fab’, Fab, Fv and scFv.

[0071] In an alternative embodiment, the antibody comprises a heavy chain and / or a light chain, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 80, 81, 82, 83, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 35, 36, 57, 84.

[0072] In a fourth aspect, the present invention provides an anti-μ globin antibody, comprising a heavy chain and / or a light chain, and the amino acid sequence of the heavy chain is any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 80, 81, 82, 83, and the amino acid sequence of the light chain is any one of SEQ ID NO: 35, 36, 57, 84.

[0073] In an alternative embodiment, the antibody according to the first, second, third or fourth aspect above comprises a heavy chain and a light chain in any of the following combinations:

[0074] Combination Heavy chain Light chain 1 SEQ ID NO:27 SEQ ID NO:35 2 SEQ ID NO:28 SEQ ID NO:35 3 SEQ ID NO:29 SEQ ID NO:35 4 SEQ ID NO:30 SEQ ID NO:35 5 SEQ ID NO:31 SEQ ID NO:35 6 SEQ ID NO:32 SEQ ID NO:35 7 SEQ ID NO:33 SEQ ID NO:35 8 SEQ ID NO:34 SEQ ID NO:35 9 SEQ ID NO:27 SEQ ID NO:36 10 SEQ ID NO:55 SEQ ID NO:57 11 SEQ ID NO:56 SEQ ID NO:57 12 SEQ ID NO:80 SEQ ID NO:84 13 SEQ ID NO:81 SEQ ID NO:84 14 SEQ ID NO:82 SEQ ID NO:84 15 SEQ ID NO:83 SEQ ID NO:84 。

[0075] In a fifth aspect, the present invention provides an antibody conjugate, and the antibody conjugate comprises the above antibody.

[0076] In an alternative embodiment, the above antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody.

[0077] In an alternative embodiment, the antibody conjugate further comprises a label or a purification tag conjugated to the antibody.

[0078] In an alternative embodiment, the above label 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.

[0079] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0080] 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.

[0081] In alternative embodiments, 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 (PerCP), etc.).

[0082] In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate dehydrogenase.

[0083] 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.

[0084] 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.

[0085] In alternative embodiments, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0086] In alternative embodiments, the colloids include, but are not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latex.

[0087] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0088] In an alternative embodiment, the colloidal metal is colloidal gold.

[0089] In an alternative embodiment, the above-mentioned antibody conjugate further includes a solid-phase carrier conjugated to the antibody.

[0090] In an alternative embodiment, the solid-phase carrier is selected from microspheres, plates, and membranes.

[0091] 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.

[0092] 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.

[0093] As mentioned above, the antibodies in some embodiments or examples of the present invention can effectively bind to μ-globin. Therefore, the reagent or kit containing the μ-globin antibody can effectively qualitatively or quantitatively detect μ-globin. Using the reagent or kit provided by the present invention, for example, it can be used for detections such as immunoblotting and immunoprecipitation, which involve the specific binding performance of μ-globin and its antibody. As mentioned above, the antibodies in some embodiments or examples of the present invention have higher binding activity and specificity with μ-globin. Therefore, the reagent or kit containing the antibody has higher detection sensitivity or specificity.

[0094] In a seventh aspect, the present invention provides a method for detecting μ-globin, including: a) contacting the above-mentioned antibody, antibody conjugate, reagent, or kit with μ-globin 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;

[0095] In an alternative embodiment, the immune complex further includes a second antibody that binds to the antibody.

[0096] In an alternative embodiment, the immune complex further includes a second antibody that binds to μ-globin.

[0097] In an eighth aspect, the present invention provides the use of the above-mentioned anti-μ-globin antibody and antibody conjugate in the preparation of a product for detecting μ-globin.

[0098] 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.

[0099] In a ninth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0100] In a tenth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0101] In an eleventh aspect, the present invention provides a cell containing the above-mentioned vector.

[0102] In a twelfth aspect, the present invention provides a method for preparing an anti-μ-globin antibody, which includes: culturing the cells as described above.

[0103] In a thirteenth aspect, the present invention provides the use of the above-mentioned antibody, antibody conjugate or the above-mentioned reagent or reagent kit in detecting μ-globin or indicating μ-globin-related diseases.

[0104] In a fourteenth aspect, the present invention provides a method for indicating μ-globin-related diseases in a subject, including:

[0105] a) contacting the above-mentioned antibody, antibody conjugate or the above-mentioned reagent or reagent kit with μ-globin in a sample from the subject under conditions sufficient to cause an antibody / antigen binding reaction to form an immune complex; and

[0106] b) detecting the presence of the immune complex, and the presence of the complex indicates the presence or status of μ-globin-related diseases in the subject.

[0107] In an optional embodiment, the immune complex further includes a second antibody that binds to the antibody.

[0108] In an optional embodiment, the immune complex further includes a second antibody that binds to μ-globin.

[0109] In an optional embodiment, the μ-globin-related diseases described in the thirteenth or fourteenth aspect are selected from thalassemia.

[0110] Based on the disclosure of the amino acid sequence of the anti-μ-globin 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-μ-globin 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 what technique is used to prepare the anti-μ-globin antibody of the present invention, it falls within the protection scope of the present invention.

[0111] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0112] 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 stated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not limiting.

[0113] 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 of ordinary skill in the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (eds. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (eds. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (eds. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (eds. Mullis et al., 1994); and "Current Protocols in Immunology" (eds. J.E. Coligan et al., 1991), each of which is hereby expressly incorporated by reference.

[0114] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0115] Example 1 Antibody Discovery of Monoclonal Antibodies

[0116] (1) Animal Immunization

[0117] Recombinant globin ζ subunit antigen (from Fapon Biotech) at a concentration of 1.2 mg / mL was mixed with an equal volume of Freund's complete adjuvant to obtain an oily emulsion. This emulsion was subcutaneously injected into BALB / c mice at multiple points at a dose of 0.2 mL per mouse. After 14 days, the mice were immunized intraperitoneally with the same antigen and adjuvant. Up to the fourth immunization, tail blood was collected for titer detection, and the titer reached the fusion requirement. Three days before fusion, the same dose of antigen was mixed with an equal volume of 0.9% sodium chloride injection and injected intraperitoneally for booster immunization.

[0118] (2) Preparation of hybridoma cell line

[0119] On the third day after the booster immunization of the mice, the spleens were removed under sterile conditions. Mouse tumor cells and immunized spleen cells were mixed at a cell number ratio of 1:10, fused, and cultured. On the sixth day of culture, the HT culture medium was changed twice. On the seventh day after fusion, cell supernatants were taken for antibody detection to screen 3 hybridoma cell strains secreting specific antibodies, named Anti-DP 3C12, Anti-DP 5C10, and Anti-DP 7H8.

[0120] Example 2 Preparation of monoclonal antibody

[0121] In this example, the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara. The MagExtractor-RNA extraction kit was purchased from TOYOBO. The BD SMART TM 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.

[0122] 1. Antibody sequencing

[0123] (1) Preparation of antibody gene

[0124] mRNA was extracted from the Anti-DP 3C12, Anti-DP 5C10, and Anti-DP 7H8 hybridoma cell strains. DNA products were obtained by RT-PCR. After adding A to the products using rTaq DNA polymerase, they were inserted into the pMD-18T vector and transformed into DH5α competent cells. After colonies grew, 4 clones of the Heavy Chain and Light Chain genes were respectively taken and sent to a gene sequencing company for sequencing.

[0125] (2) Sequence analysis of antibody variable region genes

[0126] The gene sequences obtained from the above sequencing were analyzed in the Kabat antibody database, and the VNTI 11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct.

[0127] (3) Construction of recombinant antibody expression plasmid

[0128] pcDNA TM 3.4 The vector pcDNA 3.4A is the constructed recombinant antibody eukaryotic expression vector. Multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI have been introduced into this expression vector, and it is named the pcDNA 3.4A expression vector, hereinafter simply referred to as the 3.4A expression vector. 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. The Light Chain gene fragment and the Heavy Chain gene fragment were amplified by PCR amplification.

[0129] 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 to obtain the recombinant expression plasmids of Heavy Chain and Light Chain respectively.

[0130] 2. Recombinant antibody production

[0131] Resuscitate HEK293 cells in advance, subculture them to a 200 ml system to make the cell density reach 3 - 5×10 6 cells / ml. When the cell density reaches, select the antibody concentration and cells with 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 6Wash the cells at cells / ml, resuspend them with the culture medium, and at the same time, use it as the cell diluent. Prepare 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 the 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 an incubator at 35 °C for culture, with a rotation speed of 120 rmp and a CO2 content of 8%. After 13 days, collect the samples by centrifugation. Purify the centrifuged supernatant with a protein A affinity chromatography column. The obtained antibodies were named Anti-DP 3C12RMb1, Anti-DP 5C10RMb1, and Anti-DP 7H8RMb1 respectively. Mutate Anti-DP 3C12RMb1, Anti-DP 5C10RMb1, and Anti-DP 7H8RMb1 respectively to obtain mutant antibodies. The sequences of the heavy chain (H) and light chain (L) of the above antibodies are shown in the following table:

[0132] Table 2 Antibody Sequences

[0133] Antibody Name Heavy Chain Light Chain Anti-DP 3C12RMb1 SEQ ID NO:27 SEQ ID NO:35 Anti-DP 3C12RMb2 SEQ ID NO:28 SEQ ID NO:35 Anti-DP 3C12RMb3 SEQ ID NO:29 SEQ ID NO:35 Anti-DP 3C12RMb4 SEQ ID NO:30 SEQ ID NO:35 Anti-DP 3C12RMb5 SEQ ID NO:31 SEQ ID NO:35 Anti-DP 3C12RMb6 SEQ ID NO:32 SEQ ID NO:35 Anti-DP 3C12RMb7 SEQ ID NO:33 SEQ ID NO:35 Anti-DP 3C12RMb8 SEQ ID NO:34 SEQ ID NO:35 Anti-DP 3C12RMb9 SEQ ID NO:27 SEQ ID NO:36 Anti-DP 5C10RMb1 SEQ ID NO:55 SEQ ID NO:57 Anti-DP 5C10RMb2 SEQ ID NO:56 SEQ ID NO:57 Anti-DP 7H8RMb1 SEQ ID NO:80 SEQ ID NO:84 Anti-DP 7H8RMb2 SEQ ID NO:81 SEQ ID NO:84 Anti-DP 7H8RMb3 SEQ ID NO:82 SEQ ID NO:84 Anti-DP 7H8RMb4 SEQ ID NO:83 SEQ ID NO:84

[0134] Example 3 Performance Detection of Antibodies

[0135] 1. Antibody Specificity Identification

[0136] Dilute recombinant ζ-globin antigen, recombinant μ-globin antigen, and recombinant α-globin antigen with the coating solution to 0.5 μg / ml, 100 μl / well, coat the ELISA plate, and incubate overnight at 4 °C; the next day, remove the supernatant, wash once with the washing solution (main components: Na2HPO4 + NaCl), and pat dry; add the blocking solution (20% BSA + 80% PBS), 120 μL / well, incubate at 37 °C for 1 h, and pat dry; add the antibody to be tested diluted to 0.5 μg / ml, 100 μl / well, incubate at 37 °C for 30 min; wash 5 times with the washing solution and pat dry; add goat anti-mouse IgG-HRP, 100 μl / well, incubate 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.

[0137] 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)

[0138] Table 3 Specificity Data

[0139]

[0140] Conclusion: Antibodies Anti-DP 3C12RMb1 to Anti-DP 3C12RMb9, Anti-DP 5C10RMb1 to Anti-DP 5C10RMb2, and Anti-DP 7H8RMb1 to Anti-DP 7H8RMb4 specifically bind to μ-globin antigen and do not bind to ζ-globin antigen and α-globin antigen.

[0141] 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 changes and modifications. 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.

[0142] Some of the amino acid sequences involved in this application are shown in Table 4:

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

Claims

1. An anti-mu globin antibody, the antibody comprising three complementarity determining regions of any one of the heavy chain variable regions having the amino acid sequences SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 52, 53, 75, 76, 77, 78 and three complementarity determining regions of any one of the light chain variable regions having the amino acid sequences SEQ ID NO: 25, 26, 54, 79.

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

3. An anti-mu globin antibody, characterized in that, The antibody comprises the CDR sequences of any one of the following numbered combinations: ； Optionally, the antibody comprises the FR sequences of any one of the following numbered combinations or has at least 80% identity with the FR of any one of the numbered combinations:

4. An anti-μ globin 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 as shown in any one of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 52, 53, 75, 76, 77, 78; the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 25, 26, 54, 79; 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 comprises a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from the heavy chain constant regions of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments; Optionally, 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; Optionally, the species origin of the constant region is bovine, equine, porcine, ovine, caprine, 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 amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 15 or has at least 80% identity therewith; Optionally, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 16 or has at least 80% identity therewith; Optionally, the antibody comprises any one of F(ab’)2, Fab’, Fab, Fv and scFv.

5. An anti-μ globin antibody, comprising a heavy chain and / or a light chain, characterized in that, The amino acid sequence of the heavy chain is as shown in any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 80, 81, 82, 83; the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 35, 36, 57, 84.

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 or a purification tag conjugated to the antibody; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle-based labels; Optionally, the antibody conjugate further comprises a solid-phase carrier conjugated 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 1-5 or the antibody conjugate according to claim 6 in the preparation of a product for detecting μ-globin; Optionally, the use comprises: a) contacting the antibody according to any one of claims 1-5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 with μ-globin in a test sample under conditions sufficient to effect an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of which indicates the presence of the antigen in the test sample; Optionally, the immune complex further comprises a second antibody that binds to the antibody; Optionally, the immune complex further comprises a second antibody that binds to μ-globin.

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