Antibody combined with E2-E2 antibody compound and application thereof

Abstract

The invention discloses an antibody combined with an E2-E2 antibody compound, and relates to the field of antibodies. The antibody combined with the E2-E2 antibody compound comprises a heavy chain complementarity determining region and a light chain complementarity determining region, the antibody is strongly combined with the E2 and the compound formed by the E2 antibody, is not combined with the free E2 antibody or is not combined with the free E2, the antibody provides an important raw material source for the detection of the E2, and the detection cost is reduced. And the antibody has good sensitivity and activity, good detection linear relationship and relatively high clinical correlation.

Description

[0001] Cross-referencing:

[0002] This application claims priority to Chinese Patent Application No. 202411111212.7, filed on August 14, 2024, entitled "An antibody against E2-E2 antibody complex and its application thereto", the entire contents of which are incorporated herein by reference.

[0003] This application claims priority to Chinese Patent Application No. 202510217161.4, filed on February 25, 2025, entitled "An antibody against E2-E2 antibody complex and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0004] This invention relates to the field of antibody technology, and more specifically, to an antibody that binds to an E2-E2 antibody complex and its application. Background Technology

[0005] Estradiol (E2) is the most potent natural estrogen in the human body. It regulates female reproductive function and, along with progesterone, maintains pregnancy. Estradiol and estrone are interconverted in the body. In normal, non-pregnant women, estradiol synthesized by the ovaries is the primary source of estrone and estriol. Most estradiol in the blood specifically binds to sex hormone-binding globulin, a small portion non-specifically binds to serum albumin, and only a very small amount exists in a free state. Therefore, all serum estradiol measurements must release estradiol from its binding proteins. Although most estradiol is secreted by the ovaries (in non-pregnant women), the testes and adrenal cortex also secrete small amounts. During pregnancy, most of the estradiol in the blood is produced by the placenta. Because most estradiol in normal women is produced by the ovaries, measuring estradiol concentration can be used to assess ovarian function. Furthermore, measuring estradiol levels is also important for assessing amenorrhea, precocious puberty, menopause, infertility, and assisted reproductive technologies.

[0006] In studies of common diseases in postmenopausal women, such as osteoporosis, cognitive impairment, cardiovascular disease, and breast cancer, accurate detection of serum estradiol levels ranging from <1 pg / mL to 30 pg / mL is extremely valuable. Detection of ultra-low estrogen levels is also essential for the study of congenital sex hormone metabolism disorders, puberty disorders, and estrogen deficiency in men.

[0007] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is the standard reference measurement method for estradiol recognized by the Joint Committee on Traceability in Laboratory Medicine (JCTLM). LC-MS / MS technology has high sensitivity, high specificity, and high analytical throughput; however, LC-MS / MS instruments are expensive, have a low degree of automation, and require highly skilled technicians, thus it is not yet widely used in clinical estrogen testing.

[0008] Currently, the mainstream clinical method for detecting E2 is immunoassay. Since E2 is a small molecule hormone compound with a molecular weight of only 272.4 Daltons and a single antigenic determinant, it cannot support sandwich detection with two different antibodies. Therefore, clinical immunoassays generally use competitive methods. Competitive methods lack precision, accuracy, sensitivity, and linearity, especially for low-value samples, and are difficult to achieve good consistency with mass spectrometry results.

[0009] Therefore, it is urgent to address the problems of low sensitivity and insufficient accuracy of existing competitive methods for detecting E2, and to improve the detection rate of low-value samples.

[0010] Currently, the most feasible method is the complex antibody sandwich assay. The specific detection principle is as follows: one anti-E2 antibody (primary antibody) forms an immune complex with the target antigen; another anti-complex antibody (secondary antibody) binds to the aforementioned immune complex, forming an immune sandwich complex. The recognition site is a new epitope formed after the first antibody and the small molecule hapten bind to each other. Furthermore, the complex antibody must not bind to the free first antibody or the small molecule hapten, or must bind only weakly. By using the complex antibody sandwich assay, the sensitivity and accuracy of small molecule antigen detection can be greatly improved. Summary of the Invention

[0011] This application provides an antibody that binds to an E2-E2 antibody complex. This antibody strongly binds to E2 and the complex formed by the E2 antibody, but does not bind to free E2, and does not bind or weakly binds to free E2 antibody. This antibody provides an important source of raw materials for the detection of E2, and has good sensitivity and activity, good linearity in detection, and high clinical relevance.

[0012] To achieve the above objectives, according to one aspect of the present invention, an antibody that binds to an E2-E2 antibody complex is provided, the antibody comprising three complementary determining regions having a heavy chain variable region having an amino acid sequence as shown in any of SEQ ID NO:37, 38, 39, 107, 108, 109, 110 and three complementary determining regions having a light chain variable region having an amino acid sequence as shown in any of SEQ ID NO:40, 41, 42, 111, 112, 113, 114.

[0013] To achieve the above objective, according to a second aspect of the present invention, an antibody that binds to an E2-E2 antibody complex is provided, the antibody comprising the following complementarity-determining region:

[0014] HCDR1, comprising or consisting of any of the amino acid sequences shown in SEQ ID NO:1 (INADG), SEQ ID NO:7 (NYGVG), SEQ ID NO:51 (KNGVD), and SEQ ID NO:69 (SNSVG);

[0015] HCDR2, which comprises, or consists of, any of the amino acid sequences shown in SEQ ID NO:2 (GISGGGSTYYNPALKS), SEQ ID NO:8 (NMDSDGRTYYNPALKS), SEQ ID NO:52 (GIVSVGNTAYNPALRS), and SEQ ID NO:70 (GMGSGGNTDYNPALKS);

[0016] HCDR3, comprising, or consisting of, any of the amino acid sequences shown in SEQ ID NO:3 (IHASGWSDGYNLGLIDF), SEQ ID NO:9 (SGAYGYIFGWDRLMVIDV), SEQ ID NO:13 (SGAYGYIFGWDNLMVIDV), SEQ ID NO:53 (NEGESYGYAYGDERFDY), and SEQ ID NO:71 (NYWGVYYRVPEIDY);

[0017] LCDR1, comprising, or consisting of, any of the amino acid sequences shown in SEQ ID NO:4 (SGSSSNIGADNGVG), SEQ ID NO:10 (SGSSSNVGYGNYVS), SEQ ID NO:14 (TGTSSDIGGYNYIG), SEQ ID NO:54 (SGSYIGGSNVG), SEQ ID NO:60 (SGSNIGSTNVG), SEQ ID NO:66 (TGTSSDIGGYSYVG), and SEQ ID NO:72 (SGSYIGSSGVG);

[0018] LCDR2, comprising, or consisting of, any of the amino acid sequences shown in SEQ ID NO:5 (GTSSRPS), SEQ ID NO:11 (GATSRAS), SEQ ID NO:15 (NVNKRPS), SEQ ID NO:55 (RSTNRPS), SEQ ID NO:61 (GDTRRPS), SEQ ID NO:67 (NVDKRPS), and SEQ ID NO:73 (ANDKRPS);

[0019] LCDR3 comprises, or is composed of, any of the amino acid sequences shown in SEQ ID NO:6 (ASYDSVTRDSI), SEQ ID NO:12 (ASYDSSSSGGV), SEQ ID NO:16 (SSYKSGGTFV), SEQ ID NO:56 (GSYAGSTLRHL), SEQ ID NO:62 (GSWASGIYTV), SEQ ID NO:68 (SSYLGDDDVI), and SEQ ID NO:74 (GNYADSTYNDFV).

[0020] To achieve the above objectives, according to a third aspect of the present invention, an antibody binding an E2-E2 antibody complex is provided, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the aforementioned heavy chain variable region is as shown in any one of SEQ ID NO: 37, 38, 39, 107, 108, 109, 110; and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 40, 41, 42, 111, 112, 113, 114.

[0021] To achieve the above objectives, according to a fourth aspect of the present invention, an antibody that binds to an E2-E2 antibody complex is provided, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain of the aforementioned antibody is as shown in any one of SEQ ID NO: 43, 44, 45, 115, 116, 117, 118; and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 46, 47, 48, 119, 120, 121, 122.

[0022] To achieve the above objectives, according to a fifth aspect of the present invention, an antibody conjugate is provided, the antibody conjugate comprising the antibodies described above.

[0023] 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 antibody or antibody conjugate described above.

[0024] To achieve the above objectives, according to a seventh aspect of the present invention, the use of the above-described antibody, antibody-drug conjugate, reagent, or kit in the preparation of a product for detecting E2 is provided.

[0025] To achieve the above objectives, the present invention also provides a nucleic acid molecule, a vector, a cell, and a method for preparing the above-mentioned antibody. Attached Figure Description

[0026] 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.

[0027] Figure 1 Standard curve for clinical sample detection of antibody Anti-E2-E2-7F11 chemiluminescence platform.

[0028] Figure 2 Standard curves for clinical sample detection of antibody Anti-E2-E2-5J11 and Anti-E2-E2-9C13 fluorescence platform values.

[0029] Figure 3 Standard curves for clinical sample detection of antibody Anti-E2-E2-5H8 and Anti-E2-E2-9C7 chemiluminescence platform values.

[0030] Figure 4 Standard curves for clinical sample detection of antibody Anti-E2-E2-6G12 and Anti-E2-E2-9D9 fluorescence platform values. Detailed Implementation

[0031] In a first aspect, embodiments of the present invention provide an antibody that binds to an E2-E2 antibody complex, wherein the antibody comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in any of SEQ ID NO:37, 38, 39, 107, 108, 109, 110, and three complementary determining regions of a light chain variable region having an amino acid sequence as shown in any of SEQ ID NO:40, 41, 42, 111, 112, 113, 114.

[0032] In an optional implementation, the complementary decision region described in the first aspect above is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

[0033] In this invention, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies, or antigen-binding fragments of antibodies, as long as they exhibit the desired antigen-binding activity. An antigen-binding fragment of an antibody is a substance containing an antibody CDR that lacks some amino acids present in the full-length chain but can still specifically bind to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), and the smallest antibody recognition unit. The antigen-binding fragments of the aforementioned antibodies can bind to the same antigen as the parent antibody.

[0034] Antigen-binding fragments of antibodies typically possess the same binding specificity as the antibody from which they originate. Those skilled in the art will readily understand, based on the description of this invention, that these antigen-binding fragments can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Given the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned antigen-binding fragments.

[0035] Antigen-binding fragments can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, automated peptide synthesizers sold by Applied BioSystems.

[0036] In this invention, the term "E2-E2 antibody complex" refers to a complex formed by the combination of free E2 and free anti-E2 antibody (which can specifically bind to E2), i.e., an antigen-antibody complex.

[0037] The term "antibody that binds to E2-E2 antibody complex" refers to an antibody that binds to the aforementioned antigen-antibody complex and has the following characteristics: (1) it binds to the E2-E2 antibody complex but does not bind to free E2 antigen; nor does it bind to free anti-E2 antibody; and / or (2) it binds strongly to the E2-E2 antibody complex and binds weakly or not to free anti-E2 antibody or free E2 antigen; the binding strength may be at least 1, 1.5, 2, 2.5, 5, 10, 15, 20, 30, 50, 80, or 100 times the binding activity.

[0038] 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 antibodies.

[0039] 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.

[0040] 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.

[0041] Table 1: CDR Definition 1

[0042] CDR Kabat AbM2 IMGT Chothia <![CDATA[Contact 6 ]]> HCDR1 <![CDATA[H31~H35 3 ]]> <![CDATA[H26~H35 3 ]]> <![CDATA[H26~H33..5 5 ]]> <![CDATA[H26~H32..34 4 ]]> H30--H35 HCDR2 H50~H65 H50~H58 H51~H57 H52~H56 H47--H58 HCDR3 H95~H102 H95~H102 H93~H102 H95~H102 H93--H101 LCDR1 L24~L34 L24~L34 L27~L32 L24~L34 L30--L36 LCDR2 L50~L56 L50~L56 L50~L51 L50~L56 L46--L55 LCDR3 L89~L97 L89~L97 L89~L97 L89~L97 L89--L96

[0043] 1In Table 1, except for the Contact definition system where the CDR is based on the Chothia numbering system, the CDRs defined in other definition systems are based on the Kabat numbering system (see below). Amino acid numbers on the heavy chain are represented by "H + number," and amino acid numbers on the light chain are 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., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia numbering" as used herein refers to the numbering system described by Al-Lazikani et al., (1997) JMB 273, 927-948.

[0044] 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.

[0045] 3 If neither H35A nor H35B exists, then HCDR1 ends at bit 35; if only H35A exists, then HCDR1 ends at bit 35A; if both H35A and H35B exist, then HCDR1 ends at bit 35B.

[0046] 4 If neither H35A nor H35B exists, then HCDR1 ends at bit 32; if only H35A exists, then HCDR1 ends at bit 33; if both H35A and H35B exist, then HCDR1 ends at bit 34.

[0047] 5 If neither H35A nor H35B exists, then HCDR1 ends at bit 33; if only H35A exists, then HCDR1 ends at bit 34; if both H35A and H35B exist, then HCDR1 ends at bit 35.

[0048] 6 The CDR numbering shown in Table 1 under the Contact definition system is based on the Chothia numbering system, and the Contact definition system is only applicable to the Chothia or Martin numbering system.

[0049] According to an embodiment of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 are numbered by the Kabat system.

[0050] According to embodiments of the present invention, the above-mentioned 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.

[0051] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0052] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Chothia system.

[0053] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT system.

[0054] In some optional embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0055] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Contact system.

[0056] In some alternative embodiments of the present invention, the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM, or Contact systems.

[0057] Secondly, embodiments of the present invention provide an antibody that binds to an E2-E2 antibody complex, wherein the antibody comprises the following complementarity-determining region:

[0058] HCDR1, comprising or consisting of any of the amino acid sequences shown in SEQ ID NO:1 (INADG), SEQ ID NO:7 (NYGVG), SEQ ID NO:51 (KNGVD), and SEQ ID NO:69 (SNSVG);

[0059] HCDR2, which comprises, or consists of, any of the amino acid sequences shown in SEQ ID NO:2 (GISGGGSTYYNPALKS), SEQ ID NO:8 (NMDSDGRTYYNPALKS), SEQ ID NO:52 (GIVSVGNTAYNPALRS), and SEQ ID NO:70 (GMGSGGNTDYNPALKS);

[0060] HCDR3, comprising, or consisting of, any of the amino acid sequences shown in SEQ ID NO:3 (IHASGWSDGYNLGLIDF), SEQ ID NO:9 (SGAYGYIFGWDRLMVIDV), SEQ ID NO:13 (SGAYGYIFGWDNLMVIDV), SEQ ID NO:53 (NEGESYGYAYGDERFDY), and SEQ ID NO:71 (NYWGVYYRVPEIDY);

[0061] LCDR1, comprising, or consisting of, any of the amino acid sequences shown in SEQ ID NO:4 (SGSSSNIGADNGVG), SEQ ID NO:10 (SGSSSNVGYGNYVS), SEQ ID NO:14 (TGTSSDIGGYNYIG), SEQ ID NO:54 (SGSYIGGSNVG), SEQ ID NO:60 (SGSNIGSTNVG), SEQ ID NO:66 (TGTSSDIGGYSYVG), and SEQ ID NO:72 (SGSYIGSSGVG);

[0062] LCDR2, comprising, or consisting of, any of the amino acid sequences shown in SEQ ID NO:5 (GTSSRPS), SEQ ID NO:11 (GATSRAS), SEQ ID NO:15 (NVNKRPS), SEQ ID NO:55 (RSTNRPS), SEQ ID NO:61 (GDTRRPS), SEQ ID NO:67 (NVDKRPS), and SEQ ID NO:73 (ANDKRPS);

[0063] LCDR3 comprises, or is composed of, any of the amino acid sequences shown in SEQ ID NO:6 (ASYDSVTRDSI), SEQ ID NO:12 (ASYDSSSSGGV), SEQ ID NO:16 (SSYKSGGTFV), SEQ ID NO:56 (GSYAGSTLRHL), SEQ ID NO:62 (GSWASGIYTV), SEQ ID NO:68 (SSYLGDDDVI), and SEQ ID NO:74 (GNYADSTYNDFV).

[0064] According to an embodiment of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0065] In an optional embodiment, the complementarity-determining region of the antibody includes any one of (a) to (g):

[0066] (a) The amino acid sequences are as shown in SEQ ID NO:1 to SEQ ID NO:3, namely HCDR1, HCDR2, and HCDR3, respectively, and the amino acid sequences are as shown in SEQ ID NO:4 to SEQ ID NO:6, namely LCDR1, LCDR2, and LCDR3, respectively.

[0067] (b) The amino acid sequences are as shown in SEQ ID NO:7 to SEQ ID NO:9, namely HCDR1, HCDR2, and HCDR3, and the amino acid sequences are as shown in SEQ ID NO:10 to SEQ ID NO:12, namely LCDR1, LCDR2, and LCDR3;

[0068] (c) The amino acid sequences are HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:8, and HCDR3 as shown in SEQ ID NO:13, and the amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:14 to SEQ ID NO:16 respectively;

[0069] (d) The amino acid sequences are as shown in SEQ ID NO:51 to SEQ ID NO:53, namely HCDR1, HCDR2, and HCDR3, and the amino acid sequences are as shown in SEQ ID NO:54 to SEQ ID NO:56, namely LCDR1, LCDR2, and LCDR3;

[0070] (e) The amino acid sequences are as shown in SEQ ID NO:57 to SEQ ID NO:59, namely HCDR1, HCDR2, and HCDR3, respectively, and the amino acid sequences are as shown in SEQ ID NO:60 to SEQ ID NO:62, namely LCDR1, LCDR2, and LCDR3, respectively.

[0071] (f) The amino acid sequences are as shown in SEQ ID NO:63 to SEQ ID NO:65, namely HCDR1, HCDR2, and HCDR3, and the amino acid sequences are as shown in SEQ ID NO:66 to SEQ ID NO:68, namely LCDR1, LCDR2, and LCDR3; and

[0072] (g) The amino acid sequences are as shown in SEQ ID NO:69 to SEQ ID NO:71, namely HCDR1, HCDR2, and HCDR3, and the amino acid sequences are as shown in SEQ ID NO:72 to SEQ ID NO:74, namely LCDR1, LCDR2, and LCDR3.

[0073] 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.

[0074] 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.

[0075] In optional embodiments, the antibodies described in the first and second aspects further have the framework regions shown in HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0076] In an optional embodiment, the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 of the above-mentioned antibody include any one of (a') to (g'):

[0077] (a') The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:17 to SEQ ID NO:20, and the amino acid sequences are LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:21 to SEQ ID NO:24; or the amino acid sequences that have at least 80% identity with the sequences of each of the frame regions.

[0078] (b') The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:25 to SEQ ID NO:28, and the amino acid sequences are LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:29 to SEQ ID NO:32; or the amino acid sequences that have at least 80% identity with the sequences of each of the frame regions.

[0079] (c') The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:25 to SEQ ID NO:28, and the amino acid sequences are LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:33 to SEQ ID NO:36; or the amino acid sequences that have at least 80% identity with the sequences of each of the frame regions.

[0080] (d') The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:75 to SEQ ID NO:78, and the amino acid sequences are LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:79 to SEQ ID NO:82; or the amino acid sequences that have at least 80% identity with the sequences of each of the frame regions.

[0081] (e') The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:83 to SEQ ID NO:86, and the amino acid sequences are LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:87 to SEQ ID NO:90; or the amino acid sequences that have at least 80% identity with the sequences of each of the frame regions.

[0082] (f') The amino acid sequences are as shown in SEQ ID NO:91 to SEQ ID NO:94, namely HFR1, HFR2, HFR3, and HFR4, and the amino acid sequences are as shown in SEQ ID NO:95 to SEQ ID NO:98, namely LFR1, LFR2, LFR3, and LFR4; or the amino acid sequences having at least 80% identity with the sequences of each of the frame regions; and

[0083] (g') The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:99 to SEQ ID NO:102, and LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:103 to SEQ ID NO:106; or the amino acid sequences have at least 80% identity with the sequences of each of the frame regions.

[0084] In this invention, the term "identity" percentage refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. The amino acid sequence identity percentage alignment can be performed using various methods within the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA.

[0085] In other embodiments, the amino acid sequences of each frame region of the antibody binding the E2-E2 antibody complex 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 described above.

[0086] Thirdly, embodiments of the present invention provide an antibody that binds to an E2-E2 antibody complex, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the aforementioned heavy chain variable region is as shown in any one of SEQ ID NO: 37, 38, 39, 107, 108, 109, 110; and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 40, 41, 42, 111, 112, 113, 114.

[0087] In an optional implementation, the heavy chain variable region and the light chain variable region described in the third aspect above are selected from any combination of (A) to (G):

[0088] (A) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:37, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:40;

[0089] (B) The heavy chain variable region with amino acid sequence as shown in SEQ ID NO:38, and the light chain variable region with amino acid sequence as shown in SEQ ID NO:41;

[0090] (C) The heavy chain variable region with amino acid sequence as shown in SEQ ID NO:39, and the light chain variable region with amino acid sequence as shown in SEQ ID NO:42;

[0091] (D) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:107, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:111;

[0092] (E) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:108, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:112;

[0093] (F) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:109, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:113; and

[0094] (G) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:110, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:114.

[0095] In optional embodiments, the antibodies described in the first, second, or third aspects above further include a constant region.

[0096] In an optional implementation, the aforementioned constant region includes a heavy chain constant region and / or a light chain constant region.

[0097] In an optional embodiment, the aforementioned 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.

[0098] In an optional embodiment, the aforementioned heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, CH4 of IgM, and / or the tail peptide of IgM.

[0099] In this paper, the CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional) are all components of the heavy chain constant region, which is located at the C-terminus of the heavy chain of the antibody molecule. Each heavy chain constant region, from the N-terminus to the C-terminus, includes the CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional). Different types of antibodies (such as IgG, IgA, IgM, etc.) have different amino acid sequences and structures in their heavy chain constant regions, but they all have relatively conserved structural features. These conserved structures enable the heavy chain constant region to perform its biological function. The heavy chain constant region, CH1 region, hinge region (optional), CH2 region, CH3 region, CH4 region (optional), and tail peptide (optional) of different species and subclasses are well known in the art, and their amino acid sequences can be determined based on bioinformatics databases, such as the IMGT database (https: / / www.imgt.org / IMGTrepertoire / Proteins / ). It should be understood that different bioinformatics databases or software may not have completely consistent results in the division and sequence identification of constant regions. However, those skilled in the art have a general and unified understanding of the concept, division and sequence identification of constant regions and their segments. Therefore, the constant regions that those skilled in the art can identify and divide using common knowledge and ordinary methods are all within the scope of protection of this invention.

[0100] For example, the amino acid sequence of the corresponding segment (such as the IgM CH2 region) divided by the IMGT database can be used as the reference sequence. The start or end position of the reference sequence can be moved forward by several amino acid residues (i.e., moved towards the IgM CH1 region) or backward by several amino acid residues (i.e., moved towards the IgM CH3 region) to obtain a sequence of the corresponding segment that is longer or shorter than the reference sequence.

[0101] In this document, the term "hinge region" refers to a polypeptide that links the CH1 and CH2 domains within the constant region of the heavy chain of an antibody. This region is rich in proline, thus allowing for stretching and bending, and typically contains at least one proline (P). Hinge regions are usually dimers, consisting of two polypeptides with the same amino acid sequence. Specific amino acid sequences are not limited and are all within the scope of protection of this application. Hinge regions of different species and subclasses are well known.

[0102] In this paper, the term "tail peptide" refers to a short peptide sequence of about a dozen amino acid residues at the end of the CH3 or CH4 region of an antibody. Tail peptides from different species are well known.

[0103] In this document, the term "IgM tail peptide" refers to a short peptide sequence at the end of the CH4 region of an IgM antibody, located at the C-terminus of the CH4 region. The specific amino acid sequence is not limited and is within the scope of protection of this application. The IgM tail peptide contains cysteine ​​residues that participate in polymer formation and can also bind to the J chain to further stabilize the multimeric structure.

[0104] In an optional implementation, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0105] In an optional implementation, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0106] In an optional implementation, the species source of the aforementioned constant region is cattle, horses, pigs, sheep, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

[0107] In an optional implementation, the species source of the aforementioned constant region is sheep.

[0108] In this paper, the partitioning of the variable and constant regions is based on the IMGT partitioning method, see Lefranc, and Martinez-Jean C. and Bosc N. or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Musmusculus)IGHC,IMGT Repertoire. the international ImMunoGeneticsinformation http: / / www.imgt.org .Created:16 / 03 / 2011.Version:17 / 01 / 2020.or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Musmusculus)IGLC,IMGT Repertoire. the international ImMunoGeneticsinformation http: / / www.imgt.org Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. The variable regions delineated by different methods may differ in some amino acids from the C-terminus of the variable region delineated by IMGT or the N-terminus of the constant region. Variable regions or constant regions delineated by other methods known in the art are also within the scope of protection of this invention.

[0109] In an optional embodiment, the antibody includes any one of the constant regions selected from (V) to (W):

[0110] (V) The amino acid sequence CH as shown in SEQ ID NO:49; and the amino acid sequence CL as shown in SEQ ID NO:50; or an amino acid sequence having at least 80% identity with each of the constant regions; and

[0111] (W) The amino acid sequence CH as shown in SEQ ID NO:123; and the amino acid sequence CL as shown in SEQ ID NO:50; or an amino acid sequence having at least 80% identity with each of the constant regions.

[0112] In other embodiments, the above-described 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 constant region (SEQ ID NO: 49, 50, 123).

[0113] Fourthly, embodiments of the present invention provide an antibody that binds to an E2-E2 antibody complex, comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain of the aforementioned antibody is as shown in any one of SEQ ID NO: 43, 44, 45, 115, 116, 117, 118; and the amino acid sequence of the light chain is as shown in any one of SEQ ID NO: 46, 47, 48, 119, 120, 121, 122.

[0114] In an optional embodiment, the heavy and light chains of the antibodies described in the first, second, third, or fourth aspects above are selected from any combination of (A') to (G'):

[0115] (A') The heavy chain with an amino acid sequence as shown in SEQ ID NO:43, and the light chain with an amino acid sequence as shown in SEQ ID NO:46;

[0116] (B') The heavy chain with the amino acid sequence shown in SEQ ID NO:44, and the light chain with the amino acid sequence shown in SEQ ID NO:47;

[0117] (C') The heavy chain with an amino acid sequence as shown in SEQ ID NO:45, and the light chain with an amino acid sequence as shown in SEQ ID NO:48;

[0118] (D') The heavy chain with an amino acid sequence as shown in SEQ ID NO:115, and the light chain with an amino acid sequence as shown in SEQ ID NO:119;

[0119] (E') The heavy chain with an amino acid sequence as shown in SEQ ID NO:116, and the light chain with an amino acid sequence as shown in SEQ ID NO:120;

[0120] (F') The heavy chain with the amino acid sequence shown in SEQ ID NO:117, and the light chain with the amino acid sequence shown in SEQ ID NO:121; and

[0121] (G') The heavy chain with an amino acid sequence as shown in SEQ ID NO:118, and the light chain with an amino acid sequence as shown in SEQ ID NO:122.

[0122] Fifthly, the present invention provides an antibody conjugate comprising the antibodies described above.

[0123] In an optional embodiment, the antibody conjugate includes biotin or a biotin derivative.

[0124] In an optional embodiment, the antibody conjugate may further include a marker or purification tag.

[0125] 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.

[0126] In an optional embodiment, the conjugation of the antibody with the conjugate does not affect the reaction characteristics of the antibody conjugate with the antibody, nor does it change the activity of the conjugate itself.

[0127] In optional embodiments, the markers include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0128] 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.

[0129] In optional embodiments, the fluorescent dyes mentioned above 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).

[0130] In optional embodiments, the enzymes mentioned above include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0131] In optional embodiments, the aforementioned radioactive isotopes include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、 211 At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、177 Lu、 172 Lu and 18 F.

[0132] In optional embodiments, the chemiluminescent reagents mentioned above 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.

[0133] In optional embodiments, the above-mentioned nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0134] In optional embodiments, the colloids include, but are not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latexes.

[0135] In optional embodiments, the colloidal metals mentioned above include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0136] In an optional embodiment, the antibody conjugate further includes a solid-phase carrier conjugated with the antibody.

[0137] In an optional embodiment, the solid support is selected from microspheres, plates, and membranes.

[0138] 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.

[0139] In this study, the antibody performance was not affected after conjugation with the conjugate.

[0140] In a sixth aspect, embodiments of the present invention provide a reagent or kit, wherein the aforementioned reagent or kit includes the above-described antibody or antibody conjugate.

[0141] As previously stated, the antibodies in some specific embodiments or examples of the present invention can bind to the E2-E2 antibody complex. Therefore, reagents or kits containing the antibodies can effectively detect E2. The reagents or kits provided by the present invention can be used, for example, for detections involving the specific binding properties of the E2-E2 antibody complex and its antibodies, such as immunoblotting and immunoprecipitation. As previously stated, the antibodies of the present invention have improved binding activity, affinity, stability, specificity, and anti-interference ability; therefore, reagents or kits containing the antibodies have improved detection sensitivity or specificity.

[0142] The above kit may contain any one or more of the following: processing solution, anti-E2 antibody, E2-E2 antibody-conjugated complex antibody, E2 quality control, anti-IgG antibody, instructions for use, or literature. E2-E2 antibody-conjugated complex antibodies can be used for different types of diagnostic tests, such as detecting the presence of various diseases, drugs, or other proteins in vitro or in vivo. For example, they can be used to test for related diseases by analyzing the serum or blood of the subject.

[0143] In a seventh aspect, embodiments of the present invention provide a method for detecting E2, comprising: a) under conditions sufficient to induce an antibody / antigen binding reaction, first binding the test sample with an E2 antibody to form complex 1; and

[0144] b) Contact the above-mentioned antibody, antibody conjugate, reagent or kit with complex 1 in a) to form complex 2; and

[0145] c) Detect the presence of the complex 2, the presence of the complex 2 indicating the presence of E2 in the test sample;

[0146] In an optional implementation, the species source of the constant region of the above-mentioned E2 antibody is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

[0147] In an optional implementation, the constant region of the above-mentioned E2 antibody is derived from sheep.

[0148] Eighthly, embodiments of the present invention provide the use of the antibodies, antibody conjugates, reagents or kits that bind the E2-E2 antibody complex described above in the preparation of products for detecting E2.

[0149] It should be noted that the products of this invention include, but are not limited to, reagents, kits, test strips, or reagent plates.

[0150] Ninthly, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0151] In a tenth aspect, the present invention provides a carrier containing the above-mentioned nucleic acid molecules.

[0152] In the eleventh aspect, the present invention provides cells containing the above-described carrier.

[0153] In a twelfth aspect, the present invention provides a method for preparing an antibody that binds to an E2-E2 antibody complex, comprising: culturing cells as described above.

[0154] In this invention, the term "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acid molecules include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. When a nucleic acid molecule encodes a protein or polypeptide, it may optionally encode the sense or antisense strand. Nucleic acid molecules can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably.

[0155] In this invention, the term "vector" refers to a delivery vehicle that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) therein and enable the expression of that genetic element, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. For example, vectors include plasmids, phage particles, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses, etc. Vectors may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein coats. Vectors can be expression vectors or cloning vectors.

[0156] In this invention, the term "recombinant cell" refers to a cell into which exogenous polynucleotides and / or vectors can be introduced, or have already been introduced. The exogenous polynucleotides may or may not be integrated into the genome of the "recombinant cell." When the recombinant cell contains a vector, the vector can be introduced into mammalian cells to construct recombinant cells, which are then used to express the antibodies or antigen-binding fragments provided by this invention. The corresponding antibodies can be obtained by culturing the recombinant cells. Suitable mammalian cells include CHO cells, etc.

[0157] Based on the amino acid sequence of the antibody binding the E2-E2 antibody complex disclosed in this invention, those skilled in the art will readily conceive of preparing the antibody binding the E2-E2 antibody complex using genetic engineering or other techniques (chemical synthesis, recombinant expression). For example, the antibody can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing any of the antibodies described above. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the antibody binding the E2-E2 antibody complex of this invention, it falls within the protection scope of this invention.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] Based on long-term, inventive research on E2, the inventors discovered an antibody that meets the requirements for E2 detection. The preparation, activity identification, and performance testing of the antibody are illustrated in the examples.

[0162] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0163] Example 1: Discovery of monoclonal antibodies against the E2-E2 complex

[0164] The antibody screening in the following embodiments was obtained using phage display technology. The specific steps of this embodiment are as follows:

[0165] 1. Preparation of anti-E2 antibody

[0166] 1.1 Animal Immunization

[0167] Using E2-BSA as an immunogen, emulsified with incomplete Freund's adjuvant (Sigma), sheep were immunized subcutaneously to elicit an immune response. Serum was collected before and after immunization on days 0, 14, 28, 42, and 69. Serum titer was determined, and sheep with the required titer were selected. Whole blood was collected from the sheep, and PBMCs were prepared using a lymphocyte separation kit.

[0168] 1.2 Construction of phage libraries

[0169] RNA was extracted from sheep PBMCs and reverse transcribed into cDNA. Using specifically designed sheep antibody gene amplification primers and cDNA as a template, heavy chain variable region and light chain variable region gene fragments were amplified separately. Then, the heavy chain and light chain variable region gene fragments were sequentially inserted into the phage vector VO2 using enzyme digestion and ligation. Finally, the ligated phage plasmid was electroporated into TG1 competent cells. The next day, single colonies were selected for PCR identification and antibody gene sequencing to assess the quality of the phage library. Once qualified, the phage library was screened.

[0170] 1.3 Screening of phage libraries

[0171] The TG1 phage library was inoculated into a shake flask and cultured until a suitable bacterial concentration (OD600 of 0.8-1.0) was reached. Helper phages were then added for infection for 1 hour, followed by overnight incubation. The bacterial culture was collected the next day, centrifuged, and the supernatant was purified by salting out to obtain the displayed phage library.

[0172] The phage library was panned 3-4 times using magnetic bead panning. Then, monoclonal phage-infected colonies were selected for antibody expression in the supernatant. The monoclonal phage antibody expression supernatant was then screened and identified using ELISA. Finally, one anti-E2 monoclonal phage antibody with superior detection performance was selected and named Anti-E2-Ab1.

[0173] 1.4 Phage antibody gene sequencing

[0174] Anti-E2-Ab1 was sequenced to obtain a unique sheep monoclonal antibody sequence. Sequence analysis removed duplicate and invalid sequences. The heavy chain amino acid sequence of Anti-E2-Ab1 is shown in SEQ ID NO:124, and the light chain amino acid sequence is shown in SEQ ID NO:125. The Anti-E2-Ab1 sequence was then subjected to eukaryotic recombinant expression. The supernatant was collected and purified to obtain a recombinant antibody that specifically binds to E2.

[0175] 2. Preparation of antibodies conjugated to E2-E2 antibody complexes

[0176] 2.1 Animal Immunization

[0177] Both E2 antigen and antibody Anti-E2-Ab1 were dialyzed into 0.01M PBS and incubated at 37°C for 2 hours at a molar ratio of 12:1. Excess antigen was removed by dialyzing into 0.01M PBS and used as the immunogen. This immunogen was emulsified with an equal volume of Freund's complete adjuvant (Sigma) and administered subcutaneously to immunize sheep to induce an immune response. Serum was collected before and after immunization on days 0, 14, 28, 42, and 69. Serum titers were determined, and whole blood was collected from sheep with the required titers to prepare PBMCs using a lymphocyte separation kit.

[0178] 2.2 Following the steps of constructing, screening, and sequencing the Chinese library for "Preparation of Anti-E2 Antibodies," seven antibodies with superior detection performance binding to the E2-E2 antibody complex were finally selected. These seven antibodies were named Anti-E2-E2-5J15, Anti-E2-E2-7F11, Anti-E2-E2-9C13, Anti-E2-E2-5H8, Anti-E2-E2-9C7, Anti-E2-E2-6G12, and Anti-E2-E2-9D9, respectively. The heavy chain amino acid sequence of antibody Anti-E2-E2-5J15 is shown in SEQ ID NO:43, and the light chain amino acid sequence is shown in SEQ ID NO:46; the heavy chain amino acid sequence of antibody Anti-E2-E2-7F11 is shown in SEQ ID NO:44, and the light chain amino acid sequence is shown in SEQ ID NO:47; the heavy chain amino acid sequence of antibody Anti-E2-E2-9C13 is shown in SEQ ID NO:45, and the light chain amino acid sequence is shown in SEQ ID NO:48; the heavy chain amino acid sequence of antibody Anti-E2-E2-5H8 is shown in SEQ ID NO:115, and the light chain amino acid sequence is shown in SEQ ID NO:119; the heavy chain amino acid sequence of antibody Anti-E2-E2-9C7 is shown in SEQ ID NO:116, and the light chain amino acid sequence is shown in SEQ ID NO:120; the heavy chain amino acid sequence of antibody Anti-E2-E2-6G12 is shown in SEQ ID NO:117, and the light chain amino acid sequence is shown in SEQ ID NO:46. As shown in NO:121; the heavy chain amino acid sequence of antibody Anti-E2-E2-9D9 is shown in SEQ ID NO:118, and the light chain amino acid sequence is shown in SEQ ID NO:122.

[0179] Example 2: Preparation of recombinant antibodies binding to E2-E2 antibody complexes

[0180] 1. Construction of recombinant antibody expression plasmid

[0181] pcDNA TM 3.4 The vector is a recombinant antibody eukaryotic expression vector that has been modified to introduce polyclonal restriction enzyme sites, and will be referred to as the 3.4A expression vector. Based on the variable region gene, gene-specific primers for the light chain variable region and the heavy chain variable region were designed, with restriction endonuclease sites and protective bases at both ends, respectively. The light chain gene fragment and the heavy chain gene fragment were amplified by PCR.

[0182] The heavy chain and light chain gene fragments were double-digested with restriction endonucleases, and the 3.4A vector was also double-digested with restriction endonucleases. The purified and recovered heavy chain and light chain genes were then ligated into the 3.4A expression vector to obtain seven heavy chain and light chain recombinant expression plasmids, respectively.

[0183] 2. Recombinant cell preparation

[0184] HEK293 cells were revived early and passaged to a 200 mL 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 3.5 × 10⁶ cells / mL. 6 Cells were washed at a concentration of cells / mL and reconstituted with culture medium, which served as a cell dilution buffer. Diluents for plasmid DNA and transfection reagent were prepared separately using culture medium in step 1 of Example 2. The transfection reagent dilution buffer was added to the three plasmid DNA dilution buffers, 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 recombinant cells were cultured in a 35°C incubator at 120 rpm and a CO2 concentration of 8%.

[0185] 3. Specificity detection of recombinant antibody supernatant

[0186] 3.1 Binding assay of supernatant antibody to E2-E2 antibody complex (detection mode 1)

[0187] (1) ELISA plate coating

[0188] ELISA plates were coated with PK1-SA8 at a concentration of 3 μg / mL, 100 μL / well, and incubated overnight at 4°C; washed twice with 1x PBST; blocked with 1% casein, 120 μL / well, at 37°C for 1 h, patted dry, and ready for use.

[0189] (2) Preparation of E2 and anti-E2 antibody complex

[0190] Anti-E2-Ab1 antibody (1 μg / mL conjugated with BIO) and 0.1 μg / mL free estradiol (from Phypeng Biotechnology) were incubated in centrifuge tubes at 37°C with gentle shaking for 1.5 h to form estradiol and anti-estradiol antibody complexes. The incubated estradiol and anti-estradiol antibody complexes were then added to the ELISA detection plate from step (1), 100 μL / well, and incubated at 37°C for 1 h. The supernatant was discarded, and the plate was neither tapped nor washed; it was ready for use.

[0191] (3) Preparation of supernatant antibodies and controls:

[0192] Take the supernatant from the recombinant cell expression and dilute it with 1% casein by a certain factor.

[0193] Blank control: 1% casein;

[0194] (4) Detection reading

[0195] Add the antibody supernatant and blank control prepared in step (3) to the ELISA in step (2), 100 μL / well, 2 wells in each case. Gently mix and incubate at 37°C for 45 min; wash 3 times with 1x PBST; add rabbit anti-goat FC-HRP (1% casein diluted 15000 times), 100 μL / well, and incubate at 37°C for 45 min; wash 3 times with 1x PBST; add 50 μL each of solutions A and B, display for 10 min, and add 50 μL of stop solution / well for reading. The results are shown in Table 2.

[0196] 3.2 The same experimental steps as detection mode 1 were used, except that the antibody complex preparation in step (2) was replaced with 1 μg / mL Anti-E2-Ab1-Biotin-labeled antibody, and the supernatant antibody was used to perform the binding experiment with anti-estradiol antibody (detection mode 2). The experimental results are shown in Table 2.

[0197] 3.3 Following the same experimental steps as detection mode 1, the antibody complex preparation in step (1) was replaced with 1 μg / mL free estradiol, and the supernatant antibody-estradiol binding experiment was performed (detection mode 3). The experimental results are shown in Table 2. The results show that antibodies Anti-E2-E2-7F11, Anti-E2-E2-9C13, Anti-E2-E2-5H8, Anti-E2-E2-9C7, Anti-E2-E2-6G12, and Anti-E2-E2-9D9 only bind to estradiol and anti-estradiol antibody complex, and do not bind to anti-estradiol antibody or free estradiol; antibody Anti-E2-E2-5J15 strongly binds to estradiol and anti-estradiol antibody complex, weakly binds to anti-estradiol antibody, and does not bind to free estradiol.

[0198] Table 2: Antibody Binding Experiment Data in Supernatant

[0199]

[0200] 4. Purification of recombinant antibodies

[0201] Thirteen days later, the supernatant of the recombinant cells was collected by centrifugation. The supernatant was then purified by affinity chromatography using a protein A column to obtain purified antibodies.

[0202] Example 3: Antibody Performance Detection

[0203] 1. Performance in chemiluminescence detection of magnetic particles

[0204] 1.1 Estradiol antibody coating process

[0205] Wash 10 mg / mL carboxyl magnetic beads three times with MES buffer. Resuspend the beads in MES buffer, add EDC to a final concentration of 1 mg / mL, mix on a shaker at 25°C, and react for 30 min. Resuspend the beads in MES buffer again, add Anti-E2-Ab1 antibody to a final concentration of 0.2 mg / mL, mix on a shaker at 25°C, and react for 120 min. Store in Tris buffer at 2–8°C.

[0206] 1.2 Antibody labeling process

[0207] Antibodies Anti-E2-E2-7F11, Anti-E2-E2-5H8, and Anti-E2-E2-9C7 were replaced with PBS (100 mM PB, 50 mM sodium chloride, pH 8.0) using a Zeba desalting column (10 kM WCO). Acridinium ester was prepared as a 4 mM solution using DMSO. 10 mM acridinium ester was added to each of the antibodies Anti-E2-E2-7F11, Anti-E2-E2-5H8, and Anti-E2-E2-9C7, and the reaction was carried out at 25°C for 2 hours. Excess reagents were removed by desalting, and the acridinium ester-modified Anti-E2-E2-7F11, Anti-E2-E2-5H8, and Anti-E2-E2-9C7 antibodies were stored at 4°C for later use.

[0208] 1.3 Testing Process

[0209] The Anti-E2-Ab1 antibody coated with magnetic beads and the acridil ester-modified Anti-E2-E2-7F11, Anti-E2-E2-5H8, and Anti-E2-E2-9C7 antibodies were diluted using a diluent to prepare magnetic microparticle working solutions and acridil ester working solutions, respectively. Detection was performed on a Shine i2910 fully automated chemiluminescence immunoassay analyzer using a double-antibody sandwich method. Specifically, 30 μL of sample (containing calibrators of different concentrations of E2 and clinical samples of different concentrations) and 70 μL of magnetic microparticle working solution were added sequentially to the instrument, mixed, incubated for 10 minutes, washed, and then 100 μL of acridil ester working solution was added, mixed, and incubated for 10 minutes. After incubation, the reaction mixture was rinsed, pre-excitation and excitation solutions were added, and the relative luminescence intensity (RLU) was measured.

[0210] 1.4 Test Results

[0211] 1.4.1 Calibration Platform Test Results

[0212] The detection data of E2 calibrator on the chemiluminescence platform are shown in Table 3. The results show that the detection linearity of the antibody Anti-E2-E2-7F11 on the chemiluminescence platform is very good in the range of 2-4500 pg / mL.

[0213] Table 3: Test Results of Luminescence Platform Calibrators

[0214]

[0215]

[0216] 1.4.2 Results of chemiluminescence platform detection for clinical samples with mass spectrometry values

[0217] Table 4 shows the detection results of the antibody Anti-E2-E2-7F11 on the chemiluminescence platform using liquid chromatography-mass spectrometry (LC-MS / MS). The clinical relevance standard curve is shown in the figure. Figure 1 As shown in the figure. The results indicate that the detection using the antibody Anti-E2-E2-7F11 on the chemiluminescence platform has high sensitivity and clinical relevance.

[0218] Table 4: Clinical Sample Detection Results of the Chemiluminescence Platform

[0219]

[0220]

[0221]

[0222] 1.4.3 Calibration Platform Test Results - 2

[0223] Table 5 shows the detection data of E2 calibrators on the chemiluminescence platform using antibodies Anti-E2-E2-5H8 and Anti-E2-E2-9C7. The results show that the linearity of detection using antibody Anti-E2-E2-5H8 on the chemiluminescence platform is very good in the range of 5-6000 pg / mL, and the linearity of detection using antibody Anti-E2-E2-9C7 on the chemiluminescence platform is very good in the range of 5-3000 pg / mL.

[0224] Table 5: Test Results of Luminescence Platform Calibrators - 2

[0225]

[0226] 1.4.4 Results of chemiluminescence platform detection for clinical samples with mass spectrometry-based values ​​- 2

[0227] The detection results of Anti-E2-E2-5H8 and Anti-E2-E2-9C7 in LC-MS / MS fixed-value samples on the chemiluminescence platform are shown in Table 6. The clinical correlation standard curve is shown in Table 6. Figure 3 As shown in the figure. The results indicate that, on the chemiluminescence platform, the detection using Anti-E2-E2-5H8 and Anti-E2-E2-9C7 has high sensitivity and clinical relevance.

[0228] Table 6: Clinical Sample Detection Results of the Chemiluminescence Platform - 2

[0229]

[0230]

[0231]

[0232] 2. Performance testing of the fluorescence immunochromatography platform

[0233] 2.1 Antibody labeling

[0234] T-labeled material: Add 100 μL of 1% solids fluorescent microspheres to 900 μL of activation buffer and mix well. After centrifugation and removal of supernatant, add 1 mL of activation buffer and sonicate to mix well. Then add activator, shake and mix in the dark for 20 min, centrifuge and remove supernatant. Add coupling buffer (MES buffer, pH=6.0) of the same volume as the microspheres, sonicate to mix well, add 0.1-0.2 mg Anti-E2-Ab1, shake and mix in the dark for 3 h, and finally add blocking buffer for blocking. Shake and mix in the dark for 45 min to stop labeling, centrifuge and remove supernatant, reconstitute the microspheres with microsphere preservation solution, sonicate to mix well, and store at 4℃ for use.

[0235] C-labeled substance: Add 100 μL of 1% solid content fluorescent microspheres to 900 μL of activation buffer and mix well. After centrifugation and removal of supernatant, add 1 mL of activation buffer and sonicate to mix well. Then add activator, and mix well in the dark by shaking for 20 min. After centrifugation and removal of supernatant, add a coupling buffer (MES buffer, pH=6.5) of the same volume as the microspheres. After sonication and mixing well, add 0.4-0.8 mg of goat anti-chicken IgY labeled antibody, and mix well in the dark by shaking for 3 h. Finally, add blocking buffer for blocking, and stop labeling by shaking for 45 min in the dark. Centrifuge to remove supernatant, reconstitute the microspheres with microsphere preservation solution, sonicate to mix well, and store at 4℃ for use.

[0236] 2.2 Preparation of microsphere working solution

[0237] The anti-estradiol antibody T marker was diluted to 10-20% using microsphere dilution buffer, and the goat anti-chicken IgY marker was diluted to 1-2%. After mixing them together, the markers were sprayed onto glass fibers using a spray pad device to make a fluorescent pad.

[0238] 2.3 Preparation of dried fluorescent pads: Place the sprayed fluorescent pads in a 50℃ oven and dry for more than 2 hours.

[0239] 2.4 Sample pad treatment

[0240] Dilute the blocking agent to 0.4 mg / mL using sample pad diluent, spread it on glass fiber, and dry it in a 50°C oven overnight.

[0241] 2.5NC membrane coating

[0242] T-line: Anti-E2-E2-5J15, Anti-E2-E2-9C13, Anti-E2-E2-6G12, and Anti-E2-E2-9D9 were diluted to 0.5 mg / mL with coating diluent before coating.

[0243] C line: Chicken IgY coated antibody was diluted to 1.0 mg / mL with coating dilution buffer before coating;

[0244] After completing the T and C line scribing, place the film in a 50℃ oven to dry overnight.

[0245] 2.6 Preparation of Fluorescence Chromatography Strips

[0246] Use a strip cutter to cut the fluorescence chromatography strips to the required width, assemble them, add samples, and perform detection.

[0247] 2.7 Testing Process

[0248] Add estradiol samples (calibrators of different concentrations and clinical samples for mass spectrometry determination of different concentrations) to the sample diluent, mix thoroughly, and then load the sample: load 75 μL onto a 3.5 mm wide strip, add the sample to the test card and react for 15 min, then immediately insert the test card into the instrument to read the value and calculate the T / C.

[0249] 2.8 Test Results

[0250] 2.8.1 Detection results of the calibrator fluorescence platform

[0251] The results of E2 calibrators on the fluorescence platform are shown in Table 7. The results show that the use of antibodies Anti-E2-E2-5J15 or Anti-E2-E2-9C13 on the fluorescence platform results in good binding activity and excellent sensitivity.

[0252] Table 7: Test Results of Fluorescence Platform Calibrators

[0253] Sample concentration (pg / mL) Anti-E2-E2-5J15 Anti-E2-E2-9C13 0 0.007 0.15 20 0.45 0.5 50 0.95 0.92 250 3.8 2.5 500 5.8 3.46 1000 7.84 4.5 3000 10.5 7 6000 11.5 8.2

[0254] 2.8.2 Results of fluorescence detection on clinical samples obtained by mass spectrometry

[0255] The clinical detection results of samples coated with antibodies Anti-E2-E2-5J15 and Anti-E2-E2-9C13, respectively, and determined by liquid chromatography-mass spectrometry (LC-MS / MS), are shown in Table 8. The clinical relevance is as follows: Figure 2 As shown in the figure. The results indicate that the detection using antibodies Anti-E2-E2-5J15 or Anti-E2-E2-9C13 on the fluorescence immunochromatographic platform has high sensitivity and clinical relevance.

[0256] Table 8: Clinical Sample Detection Results from Fluorescence Platform

[0257]

[0258]

[0259] 2.8.3 Detection results of the calibrator fluorescence platform 2

[0260] The results of detecting E2 calibrators on the fluorescence platform using antibodies Anti-E2-E2-6G12 and Anti-E2-E2-9D9 are shown in Table 9. The results show that using antibodies Anti-E2-E2-6G12 or Anti-E2-E2-9D9 on the fluorescence platform results in good binding activity and excellent sensitivity.

[0261] Table 9: Detection Results of Fluorescence Platform Calibrators - 2

[0262]

[0263]

[0264] 2.8.4 Results of fluorescence detection on clinical samples obtained by mass spectrometry

[0265] Table 10 shows the clinical detection results of samples coated with antibodies Anti-E2-E2-6G12 and Anti-E2-E2-9D9, respectively, and determined by liquid chromatography-mass spectrometry (LC-MS / MS). The clinical relevance is shown in Table 10. Figure 4 As shown in the figure. The results indicate that the detection using antibodies Anti-E2-E2-6G12 or Anti-E2-E2-9D9 on the fluorescence immunochromatographic platform has high sensitivity and clinical relevance.

[0266] Table 10: Clinical Sample Detection Results from Fluorescence Platform - 2

[0267]

[0268]

[0269]

[0270] The amino acid sequences involved in this application are shown in Table 11:

[0271] Table 11: Amino Acid Sequence List

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279] 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.

Claims

1. An antibody that binds to an E2-E2 antibody complex, characterized in that, The antibody comprises three complementary determinant regions of a heavy chain variable region having an amino acid sequence as shown in any of SEQ ID NO:37, 38, 39, 107, 108, 109, 110, and three complementary determinant regions of a light chain variable region having an amino acid sequence as shown in any of SEQ ID NO:40, 41, 42, 111, 112, 113, 114. Optionally, the complementary determination region of the variable region is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

2. An antibody that binds to an E2-E2 antibody complex, characterized in that, The antibody contains the following complementarity-determining regions: HCDR1, comprising or consisting of any of the amino acid sequences shown in SEQ ID NO:1 (INADG), SEQ ID NO:7 (NYGVG), SEQ ID NO:51 (KNGVD), and SEQ ID NO:69 (SNSVG); HCDR2, comprising, or consisting of, any of the amino acid sequences shown in SEQ ID NO:2 (GISGGGSTYYNPALKS), SEQ ID NO:8 (NMDSDGRTYYNPALKS), SEQ ID NO:52 (GIVSVGNTAYNPALRS), and SEQ ID NO:70 (GMGSGGNTDYNPALKS); HCDR3, comprising, or consisting of, any of the amino acid sequences shown in SEQ ID NO:3 (IHASGWSDGYNLGLIDF), SEQ ID NO:9 (SGAYGYIFGWDRLMVIDV), SEQ ID NO:13 (SGAYGYIFGWDNLMVIDV), SEQ ID NO:53 (NEGESYGYAYGDERFDY), and SEQ ID NO:71 (NYWGVYYRVPEIDY); LCDR1, comprising, or consisting of, any of the amino acid sequences shown in SEQ ID NO:4 (SGSSSNIGADNGVG), SEQ ID NO:10 (SGSSSNVGYGNYVS), SEQ ID NO:14 (TGTSSDIGGYNYIG), SEQ ID NO:54 (SGSYIGGSNVG), SEQ ID NO:60 (SGSNIGSTNVG), SEQ ID NO:66 (TGTSSDIGGYSYVG), and SEQ ID NO:72 (SGSYIGSSGVG); LCDR2, comprising, or consisting of, any of the amino acid sequences shown in SEQ ID NO:5 (GTSSRPS), SEQ ID NO:11 (GATSRAS), SEQ ID NO:15 (NVNKRPS), SEQ ID NO:55 (RSTNRPS), SEQ ID NO:61 (GDTRRPS), SEQ ID NO:67 (NVDKRPS), and SEQ ID NO:73 (ANDKRPS); LCDR3 comprises, or is composed of, any of the amino acid sequences shown in SEQ ID NO:6 (ASYDSVTRDSI), SEQ ID NO:12 (ASYDSSSSGGV), SEQ ID NO:16 (SSYKSGGTFV), SEQ ID NO:56 (GSYAGSTLRHL), SEQ ID NO:62 (GSWASGIYTV), SEQ ID NO:68 (SSYLGDDDVI), and SEQ ID NO:74 (GNYADSTYNDFV).

3. The antibody according to claim 1 or 2, characterized in that, The complementarity-determining region of the antibody includes any one of (a) to (g): (a) The amino acid sequences are as shown in SEQ ID NO:1 to SEQ ID NO:3, namely HCDR1, HCDR2, and HCDR3, respectively, and the amino acid sequences are as shown in SEQ ID NO:4 to SEQ ID NO:6, namely LCDR1, LCDR2, and LCDR3, respectively. (b) The amino acid sequences are as shown in SEQ ID NO:7 to SEQ ID NO:9, namely HCDR1, HCDR2, and HCDR3, and the amino acid sequences are as shown in SEQ ID NO:10 to SEQ ID NO:12, namely LCDR1, LCDR2, and LCDR3; (c) The amino acid sequences are HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:8, and HCDR3 as shown in SEQ ID NO:13, and the amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:14 to SEQ ID NO:16 respectively; (d) The amino acid sequences are as shown in SEQ ID NO:51 to SEQ ID NO:53, namely HCDR1, HCDR2, and HCDR3, and the amino acid sequences are as shown in SEQ ID NO:54 to SEQ ID NO:56, namely LCDR1, LCDR2, and LCDR3; (e) The amino acid sequences are as shown in SEQ ID NO:57 to SEQ ID NO:59, namely HCDR1, HCDR2, and HCDR3, respectively, and the amino acid sequences are as shown in SEQ ID NO:60 to SEQ ID NO:62, namely LCDR1, LCDR2, and LCDR3, respectively. (f) The amino acid sequences are as shown in SEQ ID NO:63 to SEQ ID NO:65, namely HCDR1, HCDR2, and HCDR3, and the amino acid sequences are as shown in SEQ ID NO:66 to SEQ ID NO:68, namely LCDR1, LCDR2, and LCDR3; and (g) The amino acid sequences are as shown in SEQ ID NO:69 to SEQ ID NO:71, namely HCDR1, HCDR2, and HCDR3, and the amino acid sequences are as shown in SEQ ID NO:72 to SEQ ID NO:74, namely LCDR1, LCDR2, and LCDR3.

4. The antibody according to any one of claims 1 to 3, characterized in that, The antibody includes the framework regions shown in HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4; Optionally, HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 are selected from any one of (a') to (g'): (a') The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:17 to SEQ ID NO:20, and the amino acid sequences are LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:21 to SEQ ID NO:24; or the amino acid sequences that have at least 80% identity with the sequences of each of the frame regions. (b') The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:25 to SEQ ID NO:28, and the amino acid sequences are LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:29 to SEQ ID NO:32; or the amino acid sequences that have at least 80% identity with the sequences of each of the frame regions. (c') The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:25 to SEQ ID NO:28, and the amino acid sequences are LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:33 to SEQ ID NO:36; or the amino acid sequences that have at least 80% identity with the sequences of each of the frame regions. (d') The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:75 to SEQ ID NO:78, and the amino acid sequences are LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:79 to SEQ ID NO:82; or the amino acid sequences that have at least 80% identity with the sequences of each of the frame regions. (e') The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:83 to SEQ ID NO:86, and the amino acid sequences are LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:87 to SEQ ID NO:90; or the amino acid sequences that have at least 80% identity with the sequences of each of the frame regions. (f') The amino acid sequences are as shown in SEQ ID NO:91 to SEQ ID NO:94, namely HFR1, HFR2, HFR3, and HFR4, and the amino acid sequences are as shown in SEQ ID NO:95 to SEQ ID NO:98, namely LFR1, LFR2, LFR3, and LFR4; or the amino acid sequences having at least 80% identity with the sequences of each of the frame regions; and (g') The amino acid sequences are HFR1, HFR2, HFR3, and HFR4 as shown in SEQ ID NO:99 to SEQ ID NO:102, and LFR1, LFR2, LFR3, and LFR4 as shown in SEQ ID NO:103 to SEQ ID NO:106; or the amino acid sequences have at least 80% identity with the sequences of each of the frame regions.

5. An antibody that binds to an E2-E2 antibody complex, comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 37, 38, 39, 107, 108, 109, 110; and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 40, 41, 42, 111, 112, 113, 114; Optionally, the combination of the heavy chain variable region and the light chain variable region is selected from any combination of (A) to (G): (A) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:37, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:40; (B) The heavy chain variable region with amino acid sequence as shown in SEQ ID NO:38, and the light chain variable region with amino acid sequence as shown in SEQ ID NO:41; (C) The heavy chain variable region with amino acid sequence as shown in SEQ ID NO:39, and the light chain variable region with amino acid sequence as shown in SEQ ID NO:42; (D) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:107, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:111; (E) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:108, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:112; (F) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:109, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:113; and (G) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:110, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:

114.

6. The antibody according to any one of claims 1 to 5, characterized in that, The antibody also includes a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Optionally, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, CH4 of IgM and / or the tail peptide of IgM. Optionally, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans; Optionally, the species source of the constant region is sheep; Optionally, the antibody includes any one of the constant regions selected from (V) to (W): (V) The amino acid sequence CH as shown in SEQ ID NO:49; and the amino acid sequence CL as shown in SEQ ID NO:50; or an amino acid sequence having at least 80% identity with each of the constant regions; and (W) The amino acid sequence CH as shown in SEQ ID NO:123; and the amino acid sequence CL as shown in SEQ ID NO:50; or an amino acid sequence having at least 80% identity with each of the constant regions.

7. An antibody that binds to an E2-E2 antibody complex, comprising a heavy chain and a light chain, characterized in that, The heavy chain amino acid sequence of the antibody is shown in any one of SEQ ID NO: 43, 44, 45, 115, 116, 117, 118; the light chain amino acid sequence is shown in any one of SEQ ID NO: 46, 47, 48, 119, 120, 121, 122.

8. An antibody conjugate, characterized in that, The antibody conjugate comprises the antibody according to any one of claims 1 to 7; Optionally, the antibody conjugate further includes biotin or a biotin derivative conjugated with the antibody; Optionally, the antibody conjugate further includes a marker conjugated to the antibody; Optionally, the marker is selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers; Optionally, the antibody conjugate further includes a solid-phase support conjugated to the antibody; Optionally, the solid support is selected from microspheres, plates, and membranes.

9. A reagent or kit, characterized in that, The reagent or kit comprises the antibody as described in any one of claims 1 to 7 or the antibody conjugate as described in claim 8.

10. Use of the antibody according to any one of claims 1 to 7, the antibody conjugate according to claim 8, or the reagent or kit according to claim 9 in the preparation of a product for detecting E2; Optionally, the uses include: a) Under conditions sufficient to induce antibody / antigen binding, the test sample is bound to the E2 antibody to form complex 1; and b) Contacting the antibody of any one of claims 1-7, the antibody conjugate of claim 8, or the reagent or kit of claim 9 with complex 1 in a) to form complex 2; and c) Detect the presence of the complex 2, the presence of the complex 2 indicating the presence of E2 in the test sample; Optionally, the species source of the constant region of the E2 antibody is cattle, horses, pigs, sheep, goats, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, or humans; Optionally, the constant region of the E2 antibody is derived from sheep.

11. A nucleic acid molecule, a vector, a cell, or a method for preparing an antibody according to any one of claims 1 to 7, wherein the nucleic acid molecule encodes an antibody according to any one of claims 1 to 7; the vector contains a nucleic acid molecule encoding an antibody according to any one of claims 1 to 7; the cell contains the aforementioned nucleic acid molecule or vector; and the method comprises culturing the aforementioned cell.

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