Antibodies against ferritin and uses thereof

Abstract

The application discloses an antibody against ferritin and application thereof, and relates to the field of antibodies. The antibody against ferritin disclosed by the application comprises a heavy chain complementarity determining region and a light chain complementarity determining region, the antibody provides an important raw material source for detection of ferritin, and has good affinity or activity.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically, to an antiferritin antibody and its application. Background Technology

[0002] Ferritin is a widely distributed iron storage protein in living organisms, with a molecular weight of approximately 450 kDa. It has a hydrated iron oxide core and a protein shell surrounding the core. The shell consists of 24 subunits, each containing approximately 163 amino acid residues. As the main form of iron storage in the body, the level of ferritin in the body is positively correlated with the amount of iron stored in the body. Therefore, ferritin has become an important biomarker in medical testing, frequently used to evaluate iron storage status and diagnose iron deficiency anemia.

[0003] Ferritin is present in various cells throughout the body, with the highest concentrations in liver and reticuloendothelial system cells. Its main function is to release excess free iron from cells for the body's use. Therefore, ferritin is a major iron-storing protein in body tissues and an essential protein for maintaining iron balance. Decreased ferritin levels are the most important indicator of iron-deficiency anemia, but generally, levels below 30 ng / ml indicate a tendency towards iron-deficiency anemia, requiring appropriate iron supplementation. Iron-deficiency anemia is a common disease in women and children. When serum ferritin levels exceed 300 ng / ml, it may indicate hepatocyte abnormalities. Normally, serum ferritin is synthesized in large quantities in hepatocytes, while circulating ferritin is absorbed and cleared by hepatocytes. Therefore, hepatocyte abnormalities can cause elevated serum ferritin levels. Additionally, ferritin synthesized by cancer cells can also increase ferritin levels in the body. Therefore, when abnormal serum protein levels are detected, appropriate measures and possible diseases should be considered, and further examinations should be conducted.

[0004] Currently, the main methods for quantitative detection of ferritin include enzyme-linked immunosorbent assay (ELISA), chromatography, chemiluminescence immunoassay, and latex turbidimetry. The principle of ELISA is as follows: 1) the antigen or antibody binds to the surface of a solid-phase carrier while maintaining its immunogenicity; 2) the antigen or antibody is linked to an enzyme to form an enzyme-labeled antigen or antibody, which retains both its immunogenicity and enzyme activity. The test sample (containing the antibody or antigen to be measured) and the enzyme-labeled antigen or antibody react with the antigen or antibody on the surface of the solid-phase carrier in different steps. The antigen-antibody complex formed on the solid-phase carrier is separated from other substances by washing. After adding the substrate for the enzyme reaction, the substrate is catalyzed by the enzyme into a colored product. The amount of product is directly related to the amount of the test substance in the sample; therefore, qualitative or quantitative analysis can be performed based on the intensity of the color reaction.

[0005] All of the above-mentioned immunological detection methods require antibodies against ferritin. Therefore, there is a strong demand in those skilled in the art for antiferritin antibodies with good performance. Summary of the Invention

[0006] This application provides an antibody against ferritin or its antigen-binding fragment, which provides an important source of raw materials for the detection of ferritin and has good activity or affinity.

[0007] To achieve the above objectives, according to one aspect of the present invention, an antibody against ferritin or an antigen-binding fragment thereof is provided, said antibody or antigen-binding fragment comprising three complementary determining regions having a heavy chain variable region as shown in SEQ ID NO:17 and three complementary determining regions having a light chain variable region as shown in SEQ ID NO:19.

[0008] To achieve the above objectives, according to a second aspect of the present invention, an antibody against ferritin or an antigen-binding fragment thereof is provided, said antibody or antigen-binding fragment comprising the following complementarity-determining region:

[0009] HCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:1;

[0010] HCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:2;

[0011] HCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:3;

[0012] LCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:4;

[0013] LCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:5; and

[0014] LCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6.

[0015] To achieve the above objectives, according to a third aspect of the present invention, an antibody against ferritin or an antigen-binding fragment thereof is provided, 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 shown in SEQ ID NO:17; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19.

[0016] To achieve the above objectives, according to a fourth aspect of the present invention, an antibody against ferritin or an antigen-binding fragment thereof is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:18; and the amino acid sequence of the light chain is shown in SEQ ID NO:20.

[0017] To achieve the above objectives, according to a fifth aspect of the present invention, an antibody conjugate is provided, the antibody conjugate comprising the antibody or its antigen-binding fragment described above.

[0018] To achieve the above objectives, according to a sixth aspect of the present invention, a reagent or kit is provided, the reagent or kit comprising the above-described antibody or its antigen-binding fragment or the above-described antibody conjugate.

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

[0020] To achieve the above objectives, according to an eighth aspect of the present invention, the use of the above-described antibody or its antigen-binding fragment, antibody conjugate, reagent or kit in the preparation of a ferritin detection product is provided.

[0021] To achieve the above objectives, the present invention also provides a nucleic acid, a vector, a cell, and a method for preparing the above-mentioned antibody or its antigen-binding fragment. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Results of reductive SDS-PAGE for Anti-FER 7G10 Rmb1. Detailed Implementation

[0024] In a first aspect, embodiments of the present invention provide an antibody against ferritin or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising three complementary determining regions having a heavy chain variable region as shown in SEQ ID NO:17 and three complementary determining regions having a light chain variable region as shown in SEQ ID NO:19.

[0025] In this invention, the term "antibody" is used in the broadest sense, and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, as long as they exhibit the desired biological activity.

[0026] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically 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 specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0027] In this invention, the heavy chain complementarity determination region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determination region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0028] Methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, "Kabat definition" refers to the definition system described in Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" is found in Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below; definitions vary slightly in different literature. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods, not limited to those in Table 1, are also within the scope of this disclosure.

[0029] Table 1: CDR Definition 1

[0030] 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

[0031] 1 The CDRs defined in Table 1 are numbered according to 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". Those skilled in the art can readily map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., USD ept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0032] 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.

[0033] 3 If neither H35A nor H35B exists, then CDR-H1 ends at bit 35; if only H35A exists, then CDR-H1 ends at bit 35A; if both H35A and H35B exist, then CDR-H1 ends at bit 35B.

[0034] 4 If neither H35A nor H35B exists, then CDR-H1 ends at bit 32; if only H35A exists, then CDR-H1 ends at bit 33; if both H35A and H35B exist, then CDR-H1 ends at bit 34.

[0035] 5 If neither H35A nor H35B exists, then CDR-H1 ends at bit 33; if only H35A exists, then CDR-H1 ends at bit 34; if both H35A and H35B exist, then CDR-H1 ends at bit 35.

[0036] According to embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM or Contact.

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

[0038] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Chothia system.

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

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

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

[0042] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM, or Contact systems.

[0043] According to embodiments of the present invention, the Kabat numbering positions corresponding to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 defined by the Kabat, Chothia, AbM, or IMGT systems are as follows:

[0044] CDR Kabat AbM IMGT Chothia HCDR1 H31~H35A H26~H35A H26~H34 H26~H33 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

[0045] Secondly, embodiments of the present invention provide an antibody against ferritin or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof includes the following complementarity-determining region:

[0046] HCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:1;

[0047] HCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:2;

[0048] HCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:3;

[0049] LCDR1, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:4;

[0050] LCDR2, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:5; and

[0051] LCDR3, which contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6.

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

[0053] In this invention, the "frame region" or "FR" region includes the heavy chain frame region and the light chain frame region, referring to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR; wherein, the heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including the HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including the LFR1, LFR2, LFR3 and LFR4 frame regions.

[0054] In this invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: 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 combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0055] In an optional embodiment, the antibody or its antigen-binding fragment described in the first or second aspect further comprises at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4;

[0056] The HFR1 includes / is such as SEQ ID NO:7 or an amino acid sequence having at least 80% identity with it;

[0057] The HFR2 includes / is such as SEQ ID NO:8 or an amino acid sequence having at least 80% identity with it;

[0058] The HFR3 includes / is, for example, SEQ ID NO:9 or an amino acid sequence having at least 80% identity with it;

[0059] The HFR4 includes / is, for example, SEQ ID NO:10 or an amino acid sequence having at least 80% identity with it;

[0060] The LFR1 includes / such as SEQ ID NO:11 or an amino acid sequence having at least 80% identity with it;

[0061] The LFR2 includes / is, for example, SEQ ID NO:12 or an amino acid sequence having at least 80% identity with it;

[0062] The LFR3 includes / is, for example, SEQ ID NO:13 or an amino acid sequence having at least 80% identity with it;

[0063] The LFR4 includes / such as SEQ ID NO:14 or an amino acid sequence having at least 80% identity with it.

[0064] It should be noted that, in other embodiments, the amino acid sequences of each frame region of the antiferritin antibody or its antigen-binding fragment provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding frame regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, or 14) mentioned above.

[0065] In an optional embodiment, the antibody or its antigen-binding fragment has a KD < 2.79 × 10⁻⁶. -9 M binds to ferritin with affinity.

[0066] In an optional embodiment, the antibody or its antigen-binding fragment has a KD ≤ 10. -7 M, KD≤10 -8 M, KD≤10 -9 M, KD≤10 -10 M, KD≤10 -11 M or KD≤10 -12 M binds to ferritin with affinity.

[0067] In an optional embodiment, the antibody or its antigen-binding fragment has a KD ≤ 3.08 × 10⁻⁶. -10 M binds to ferritin with affinity.

[0068] There are many methods for determining antibody affinity (KD), which can be categorized into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods based on their detection principles. Common thermodynamic detection methods include isothermal titration calorimetry (ITC); common kinetic detection methods include surface plasmon resonance (SPR) and biomembrane optical interferometry (BLI); and common dynamic equilibrium detection methods include enzyme-linked immunosorbent assay (ELISA).

[0069] In an optional implementation, KD is measured using a kinetic detection method; alternatively, surface plasmon resonance, for example, by using a method such as The system's biosensor system.

[0070] Thirdly, embodiments of the present invention provide an antibody against ferritin or an antigen-binding fragment thereof, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19.

[0071] In optional embodiments, the antibodies or antigen-binding fragments thereof described in the first, second, and third aspects above further include a constant region.

[0072] In an optional implementation, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0073] In an optional implementation, 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.

[0074] 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.

[0075] In an optional implementation, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0076] In an optional implementation, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0077] In an optional implementation, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans.

[0078] In an optional implementation, the species source of the constant region is mice.

[0079] 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 sequence (CL) is as shown in SEQ ID NO:16.

[0080] It should be noted that, in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the aforementioned constant region (SEQ ID NO: 15 or 16).

[0081] In an optional embodiment, the antigen-binding fragment is selected from any one of the antibody's F(ab)2, F(ab')2, Fab', Fab, Fv, and scFv.

[0082] The antigen-binding fragments of the aforementioned antibodies typically possess the same binding specificity as the source antibody. Those skilled in the art will readily understand, based on the description herein, that the antigen-binding fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned antigen-binding fragments.

[0083] The antigen-binding fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems.

[0084] Fourthly, the present invention provides an antibody against ferritin or an antigen-binding fragment thereof, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:18 and the amino acid sequence of the light chain is shown in SEQ ID NO:20.

[0085] Fifthly, the present invention provides an antibody conjugate comprising the antibody or its antigen-binding fragment described above.

[0086] In an optional embodiment, the antibody conjugate further includes biotin or a biotin derivative conjugated to the antibody or its antigen-binding fragment.

[0087] In an optional embodiment, the antibody conjugate further includes a marker conjugated to the antibody or its antigen-binding fragment.

[0088] In an optional implementation, the aforementioned marker refers to a type of substance that has properties such as luminescence, color development, and radioactivity that can be directly observed by the naked eye or detected or probed by instruments. Through these properties, qualitative or quantitative detection of the corresponding target can be achieved.

[0089] In optional embodiments, the markers include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0090] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0091] In optional 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), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0092] In optional 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.

[0093] In optional embodiments, the radioactive isotopes 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.

[0094] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0095] In optional 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.

[0096] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0097] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0098] In an optional embodiment, the colloidal metal is colloidal gold.

[0099] In an optional embodiment, the antibody conjugate further includes a solid-phase carrier conjugated to the antibody or its antigen-binding fragment.

[0100] In an optional embodiment, the solid support is selected from microspheres, plates, and membranes.

[0101] In optional embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0102] In a sixth aspect, the present invention provides a reagent or kit comprising the antibody or antigen-binding fragment thereof described above or the antibody-conjugate described above.

[0103] As previously mentioned, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present invention can effectively bind to ferritin. Therefore, reagents or kits containing the ferritin antibody or its antigen-binding fragment can effectively perform qualitative or quantitative detection of ferritin. The reagents or kits provided by the present invention can be used, for example, for detections involving the specific binding properties of ferritin and its antibodies, such as immunoblotting and immunoprecipitation. As previously mentioned, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present invention have higher binding activity or affinity for ferritin; therefore, reagents or kits containing the antibody or its antigen-binding fragment have higher detection sensitivity or specificity.

[0104] In a seventh aspect, the present invention provides a method for detecting ferritin, comprising: a) contacting the antibody or its antigen-binding fragment, antibody conjugate, reagent or kit with ferritin in a sample to be tested under conditions sufficient to induce an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample;

[0105] In an optional embodiment, the immune complex further includes a second antibody that binds to the antibody or its antigen-binding fragment.

[0106] In an optional embodiment, the immune complex further includes a second antibody that binds to ferritin.

[0107] Eighthly, the present invention provides the use of the above-described antiferritin antibody or its antigen-binding fragment, antibody conjugate, or the above-described reagent or kit in the preparation of products for detecting ferritin.

[0108] In a ninth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or its antigen-binding fragment.

[0109] In a tenth aspect, the present invention provides a carrier containing the above-mentioned nucleic acid molecules.

[0110] In the eleventh aspect, the present invention provides cells containing the above-described carrier.

[0111] In a twelfth aspect, the present invention provides a method for preparing an antiferritin antibody or an antigen-binding fragment thereof, comprising: culturing cells as described above.

[0112] Based on the amino acid sequence of the antiferritin antibody or its antigen-binding fragment disclosed in this invention, those skilled in the art will readily conceive of preparing the antiferritin antibody or its antigen-binding fragment using genetic engineering or other techniques (chemical synthesis, recombinant expression). For example, the antibody or its antigen-binding fragment can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the antibody or its antigen-binding fragment as described in any of the preceding claims. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the antiferritin antibody or its antigen-binding fragment of this invention, it falls within the protection scope of this invention.

[0113] 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. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0114] 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 pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0115] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0116] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0117] Example 1: Preparation of Anti-FER 7G10 Monoclonal Antibody

[0118] In this embodiment, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The MagExtractor RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen Biotech. Primer synthesis and gene sequencing were performed by Invitrogen. The hybridoma cell line secreting the Anti-FER 7G10 monoclonal antibody was a hybridoma cell line prepared in our laboratory and was revived for later use.

[0119] (1) Antibody gene preparation

[0120] mRNA was extracted from hybridoma cell lines secreting Anti-FER 7G10 monoclonal antibody, and DNA products were obtained by RT-PCR. The product was then inserted into the pMD-18T vector after an A-addition reaction with rTaq DNA polymerase. The vector was then transformed into DH5α competent cells. After bacterial growth, four clones of the Heavy Chain and Light Chain genes were collected and sent to a gene sequencing company for sequencing.

[0121] (2) Sequence analysis of the variable region gene of Anti-FER 7G10 antibody

[0122] The gene sequences obtained from the sequencing were analyzed in the Kabat antibody database and VNTI11.5 software was used to confirm that the genes amplified by both heavy and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain primer pair, the VL gene sequence was 321 bp, with a 57 bp leader peptide sequence preceding it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 354 bp, belonging to the VH1 gene family, with a 57 bp leader peptide sequence preceding it.

[0123] (3) Construction of recombinant antibody expression plasmid

[0124] pcDNA TM 3.4 The vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been introduced with multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the variable region gene of the antibody in pMD-18T, VL and VH gene-specific primers of this antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. The 0.70kb Light Chain gene fragment and the 1.42kb Heavy Chain gene fragment were amplified by PCR.

[0125] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. After purification and recovery of the fragments and vector, the Heavy Chain gene and Light Chain gene were ligated into the 3.4A expression vector to obtain recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0126] 2. Recombinant antibody production

[0127] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cell density reached the required antibody concentration and cell viability >95%; cells were washed by centrifugation, reconstituted with culture medium, and the cell density was adjusted to 2.9 × 10⁻⁶ cells / ml. 6 Cells were washed at a concentration of cells / ml and reconstituted with culture medium, which served as a cell dilution buffer. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. The cells were then incubated at 35°C with a rotation speed of 120 rpm and a CO2 concentration of 8%. After 13 days, the samples were centrifuged and collected. The supernatant was purified using a protein A affinity chromatography column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE, as shown in the figure. The reducing SDS-PAGE showed two bands: one with a Mr of 50 kDa (heavy chain) and the other with a Mr of 28 kDa (light chain).

[0128] The resulting antibody was named Anti-FER 7G10Rmb1. The heavy chain amino acid sequence of the antibody Anti-FER 7G10Rmb1 is shown in SEQ ID NO:18, and the light chain amino acid sequence is shown in SEQ ID NO:20.

[0129] Example 2: Antibody Performance Detection

[0130] 1. Affinity Analysis

[0131] The antibody was pre-diluted and purified, and the recombinant ferritin antigen (from Phytobio) was serially diluted. Using a CM5 chip pre-conjugated with goat anti-mouse IgG, the binding and dissociation curves of the antigen and antibody were tested on a Biacore 8K+ device. The instrument automatically fitted and obtained the affinity constant, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., the affinity constant; ka represents the binding rate; kd represents the dissociation rate)

[0132] Table 2 Affinity Data

[0133] Sample Name KD ka kd Comparison 2.79E-09 5.31E+05 1.48E-03 Anti-FER 7G10Rmb1 3.08E-10 8.21E+06 2.53E-03

[0134] 2. Activity identification

[0135] Dilute goat anti-mouse IgG (1ug / ml) with coating buffer (main component NaHCO3) and coat microplates with 100µL per well at 4°C overnight. The next day, wash twice with washing buffer (main components Na2HPO4 + NaCl) and blot dry. Add blocking buffer (20% BSA + 80% PBS) at 120µL per well and incubate at 37°C for 1 hour, then blot dry. Add diluted purified antibody at 100µL / well and incubate at 37°C for 60 minutes. Discard the liquid in the plate, blot dry, add 20% mouse negative blood for blocking at 120µL per well and incubate at 37°C for 1 hour. Discard the liquid in the plate, blot dry, and add... Add diluted recombinant ferritin antigen (from Fipeng Biotechnology) at 100 μL per well, incubate at 37°C for 40 min; wash 5 times with washing buffer and pat dry; add HRP-labeled ferritin-pairing antibody (from Fipeng Biotechnology, which can pair with the purified antibody) at 100 μL per well, incubate at 37°C for 30 min; add chromogenic solution A (50 μL / well), add chromogenic solution B (50 μL / well), incubate for 10 min; add stop solution at 50 μL / well; read the OD value at 450 nm (630 nm for reference) on the microplate reader.

[0136] Notes: 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)

[0137] Table 3 Activity Data

[0138] Concentration (ng / ml) 15.625 7.813 3.906 1.953 0.977 0.000 Comparison 1.395 0.801 0.463 0.195 0.042 0.016 Anti-FER 7G10Rmb1 2.005 1.216 0.705 0.338 0.169 0.017

[0139] 3. Stability assessment

[0140] The above-mentioned antibodies were placed at 4℃ (refrigerator), -80℃ (refrigerator), and 37℃ (incubator) for 21 days. Samples were taken at 7, 14, and 21 days for observation of their state, and the activity of the 21-day sample was tested. The results showed that no significant changes in protein state were observed under the three testing conditions after 21 days, and the activity did not decrease with increasing testing temperature, indicating that the above-mentioned antibodies were stable. Table 4 below shows the OD results of enzyme immunoassay for antibody Anti-FER 7G10Rmb1 after 21 days of testing.

[0141] Table 4 Stability Data

[0142] Sample concentration (ng / ml) 7.813 3.906 0.000 4℃, 21-day sample 1.225 0.725 0.037 -80℃, 21-day sample 1.237 0.731 0.039 37℃, 21-day sample 1.229 0.717 0.035

[0143] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0144] The partial amino acid sequences involved in this application are shown in Table 5:

[0145]

[0146]

Claims

1. An antibody against ferritin or an antigen-binding fragment thereof, characterized in that, The antibody or antigen-binding fragment of the antiferritin contains three complementary determinant regions of the heavy chain variable region as shown in SEQ ID NO:17 and three complementary determinant regions of the light chain variable region as shown in SEQ ID NO:

19. The complementary determination region of the variable region is defined by any one of the systems Kabat, Chothia, IMGT, AbM, or Contact.

2. An antibody against ferritin or an antigen-binding fragment thereof, characterized in that, The antibody or antigen-binding fragment of the antiferritin includes the following complementary determinant region: HCDR1, whose amino acid sequence is shown in SEQ ID NO:1; HCDR2, the amino acid sequence of which is shown in SEQ ID NO:2; HCDR3, the amino acid sequence of which is shown in SEQ ID NO:3; LCDR1, whose amino acid sequence is shown in SEQ ID NO:4; LCDR2, whose amino acid sequence is shown in SEQ ID NO:5; and LCDR3, whose amino acid sequence is shown in SEQ ID NO:

6.

3. The antibody against the antiferritin or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody or antigen-binding fragment of the antiferritin also has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

4. The antibody or antigen-binding fragment of the antiferritin according to claim 3, characterized in that, The HFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity with it; The HFR2 comprises SEQ ID NO:8 or an amino acid sequence having at least 80% identity with it; The HFR3 comprises SEQ ID NO:9 or an amino acid sequence having at least 80% identity with it; The HFR4 comprises SEQ ID NO:10 or an amino acid sequence having at least 80% identity with it; The LFR1 includes SEQ ID NO:11 or an amino acid sequence having at least 80% identity with it; The LFR2 comprises SEQ ID NO:12 or an amino acid sequence having at least 80% identity with it; The LFR3 includes SEQ ID NO:13 or an amino acid sequence having at least 80% identity with it; The LFR4 includes SEQ ID NO:14 or an amino acid sequence that is at least 80% identical to it.

5. The antibody against antiferritin or its antigen-binding fragment according to any one of claims 1, 2, and 4, characterized in that, The antibody or antigen-binding fragment of the antiferritin has a KD < 2.79 × 10⁻⁶. -9 M binds to ferritin with affinity.

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

19.

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

8. The antibody against the antiferritin or its antigen-binding fragment according to claim 7, characterized in that, The constant region includes the heavy chain constant region and / or the light chain constant region.

9. The antibody against the antiferritin or its antigen-binding fragment according to claim 8, characterized in that, 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.

10. The antibody against the antiferritin according to claim 8, or its antigen-binding fragment, characterized in that, The heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

11. The antibody against the antiferritin according to claim 7, or its antigen-binding fragment, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

12. The antibody against the antiferritin or its antigen-binding fragment according to claim 7, characterized in that, The species source of the constant region is mice.

13. The antibody against the antiferritin or its antigen-binding fragment according to claim 8, characterized in that, The heavy chain constant region sequence is as shown in SEQ ID NO:15 or has at least 80% identity with it.

14. The antibody against the antiferritin or its antigen-binding fragment according to claim 8, characterized in that, The light chain constant region sequence is as shown in SEQ ID NO:16 or has at least 80% identity with it.

15. The antibody against antiferritin or its antigen-binding fragment according to any one of claims 1, 2, 4, and 6, characterized in that, The antigen-binding fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

16. An antibody against ferritin or an antigen-binding fragment thereof, said antibody comprising a heavy chain and a light chain, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID NO:18; the amino acid sequence of the light chain is shown in SEQ ID NO:

20.

17. An antibody conjugate, characterized in that, The antibody-drug conjugate comprises an antibody or antigen-binding fragment of the antiferritin as described in any one of claims 1 to 16 and biotin conjugated to the antibody or antigen-binding fragment of the antiferritin.

18. An antibody conjugate, characterized in that, The antibody-drug conjugate comprises an antibody or antigen-binding fragment of the antiferritin as described in any one of claims 1 to 16, and a label or solid-phase carrier conjugated to the antibody or antigen-binding fragment of the antiferritin.

19. The antibody conjugate according to claim 18, characterized in that, The markers are selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

20. A reagent or kit, characterized in that, The reagent or kit comprises an antibody or antigen-binding fragment of the antiferritin as described in any one of claims 1 to 16, or an antibody-drug conjugate as described in any one of claims 17 to 19.

21. Use of the antibody against ferritin according to any one of claims 1 to 16 or the antigen-binding fragment thereof, or the antibody conjugate according to any one of claims 17 to 19, in the preparation of a product for detecting ferritin.

22. The use according to claim 21, characterized in that, include: a) Under conditions sufficient to induce an antibody / antigen binding reaction, an antibody or antigen-binding fragment of the antiferritin according to any one of claims 1 to 16, an antibody conjugate according to any one of claims 17 to 19, or a reagent or kit according to claim 20 is brought into contact with ferritin in the sample to be tested to form an immune complex. and b) Detect the presence of the immune complex, the presence of which indicates the presence of ferritin in the sample to be tested.

23. The use according to claim 22, characterized in that, The immune complex further includes a second antibody that binds to the antibody or antigen-binding fragment of the antiferritin.

24. The use according to claim 22, characterized in that, The immune complex also includes a second antibody that binds to ferritin.

25. A nucleic acid, characterized in that, It encodes an antibody or antigen-binding fragment of the antiferritin as described in any one of claims 1 to 16.

26. A carrier, characterized in that, It contains the nucleic acid as described in claim 25.

27. A cell characterized in that, It contains the nucleic acid as described in claim 25 or the vector as described in claim 26.

28. A method for preparing an antibody or antigen-binding fragment of the antiferritin according to any one of claims 1 to 16, characterized in that, It includes: Culture the cells as described in claim 27.

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