Anti-digoxigenin antibody or antigen-binding fragment thereof, and use thereof

Abstract

The present invention belongs to the technical field of antibodies. Provided is an anti-digoxigenin antibody or an antigen-binding fragment thereof. The anti-digoxigenin antibody or antigen-binding fragment thereof has high affinity for digoxigenin; and can be used for detection of digoxigenin, sequencing, preparation of a product used for detecting digoxigenin, and preparation of a product used in sequencing.

Description

Anti-digoxin antibodies or their antigen-binding fragments and their applications Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to anti-digoxin antibodies or their antigen-binding fragments and their applications. Background Technology

[0002] Nucleic acid probes are nucleic acid fragments with known sequences that carry labels. They hybridize with complementary nucleic acid sequences to form double strands, allowing for the detection of specific gene sequences in nucleic acid samples. Each pathogen possesses unique nucleic acid fragments; probes can be prepared by isolating and labeling these fragments, and can be used for research such as disease diagnosis.

[0003] Nucleic acid probes are broadly classified into two categories based on their labeling agents: radiolabeled probes and non-radiolabeled probes. Radiolabeled probes use radioactive isotopes as labels and are the earliest and most widely used probe labeling agents. Commonly used isotopes include... 32 P, 3 H, 35 S. The advantages of radiolabeled probes are high sensitivity, capable of detecting at the pg level; the disadvantages are easy radioactive contamination, short isotope half-life, instability, and high cost. Therefore, radiolabeled probes cannot be commercialized. Currently, many laboratories are dedicated to developing non-radiolabeled probes. The most commonly used non-radiolabeled substances are biotin and digoxigenin, both of which are haptens. Non-radiolabeling methods involve linking biotin or digoxigenin to dNTPs, and then incorporating them into nucleic acid chains using enzymatic polymerization, similar to radiolabeling, to prepare labeled probes. Alternatively, biotin and digoxigenin can be directly chemically reacted with nucleic acids to link them to the nucleic acid chain.

[0004] Biotin is a small-molecule, water-soluble vitamin with a unique affinity for avidin, forming a stable complex. Detection is achieved by a chromogenic substance (such as an enzyme or luciferin) attached to avidin or avidin protein. Digoxin is a steroid hapten extracted from digitalis plants and is used medically to treat various acute and chronic heart failures, supraventricular tachycardia, atrial fibrillation, and flutter. Immunoassay using its antibody is similar in principle to biotin detection. However, because digoxigenin (DIG) in digitalis flowers and leaves is the only natural source, anti-DIG antibodies do not bind to other biological substances, thus meeting the requirement for specific labeling. This is precisely where digoxin surpasses biotin—both are small-molecule markers, but biotin is widely present in various tissues, and for highly sensitive labeling experiments, endogenous biotin present in the sample can interfere with the results. Digoxin effectively avoids this problem. The detection sensitivity of digoxigenin-labeled nucleic acid probes is comparable to that of radioactive isotope-labeled probes, while their specificity is superior to that of biotin-labeled probes, leading to their increasingly widespread application. Therefore, it is necessary to develop anti-digoxigenin antibodies or their antigen-binding fragments.

[0005] Summary of the Invention

[0006] In a first aspect, the present invention provides an anti-digoxin antibody or an antigen-binding fragment thereof, comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region comprising CDR-H1 (complementarity-determining region 1), CDR-H2 (complementarity-determining region 2), and CDR-H3 (complementarity-determining region 3) of the heavy chain variable region as shown in SEQ ID NO: 2; the light chain comprising a light chain variable region comprising CDR-L1 (complementarity-determining region 1), CDR-L2 (complementarity-determining region 2), and CDR-L3 (complementarity-determining region 3) of the light chain variable region as shown in SEQ ID NO: 19.

[0007] In some embodiments, the anti-digoxin antibody or its antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3, the light chain comprising a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 being as shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 21, and SEQ ID NO: 22, respectively, the amino acid sequence of CDR-L2 being KVS, and the CDR being defined according to the IMGT definition scheme.

[0008] In some embodiments, the anti-digoxin antibody or its antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3, the light chain comprising a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 being as shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, respectively, and the CDR is defined according to the Kabat definition scheme.

[0009] In some embodiments, the anti-digoxin antibody or its antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3, the light chain comprising a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 being as shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 22, respectively, wherein the CDR is defined according to the Chothia definition scheme.

[0010] In some embodiments, the anti-digoxigenin antibody or its antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3, the light chain comprising a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 being as shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively, wherein the CDR is defined using the Contact definition scheme.

[0011] In some embodiments, the amino acid sequence of the heavy chain variable region comprises: a1) SEQ ID NO: 2; or

[0012] a2) An amino acid sequence of SEQ ID NO: 2 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 2; or

[0013] a3) has an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% or more homology with SEQ ID NO: 2 and has the same function as the protein shown in SEQ ID NO: 2.

[0014] In some embodiments, the amino acid sequence of the light chain variable region comprises: b1) SEQ ID NO: 19; or

[0015] b2) An amino acid sequence of SEQ ID NO: 19 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 19; or

[0016] b3) has an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% or more homology with SEQ ID NO: 19 and has the same function as the protein shown in SEQ ID NO: 19.

[0017] It should be noted that the anti-digoxin antibody or its antigen-binding fragment described in this article includes at least one of the following: full-length antibody, Fab, Fab', F(ab')2, Fd, Fv, scFv, dAb, Fab / c, complementarity-determining region fragment, monoclonal antibody, bispecific antibody, multispecific antibody, and chimeric antibody.

[0018] In some implementations, the heavy chain further includes a heavy chain constant region; and / or the light chain further includes a light chain constant region.

[0019] In some embodiments, the amino acid sequence of the heavy chain constant region comprises: c1) SEQ ID NO: 3; or

[0020] c2) A sequence of amino acids in SEQ ID NO: 3 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 3; or

[0021] c3) has an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 80% or 70% homology to SEQ ID NO: 3 and has the same function as the protein shown in SEQ ID NO: 3.

[0022] In some embodiments, the amino acid sequence of the light chain constant region comprises: d1) SEQ ID NO: 20; or

[0023] d2) An amino acid sequence of SEQ ID NO: 20 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 20; or

[0024] d3) has an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 80% or more homologous to SEQ ID NO: 20 and has the same function as the protein shown in SEQ ID NO: 20.

[0025] In some embodiments, the amino acid sequence of the heavy chain comprises: A1) SEQ ID NO: 1; or

[0026] A2) An amino acid sequence of SEQ ID NO: 1 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 1; or

[0027] A3) has an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 70%, 65%, or 60% or more homology with SEQ ID NO: 1 and has the same function as the protein shown in SEQ ID NO: 1.

[0028] In some embodiments, the amino acid sequence of the light chain comprises: B1) SEQ ID NO: 18; or

[0029] B2) An amino acid sequence of SEQ ID NO: 18 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 18; or

[0030] B3) has an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 70%, 65%, or 60% or more homology to SEQ ID NO: 18 and has the same function as the protein shown in SEQ ID NO: 18.

[0031] In some embodiments, both the heavy and light chains of the anti-digoxin antibody or its antigen-binding fragment contain a signal peptide. In some embodiments, the signal peptide is located at the N-terminus of the heavy and / or light chains.

[0032] In some embodiments, the amino acid sequence of the signal peptide of the heavy chain of the anti-digoxin antibody or its antigen-binding fragment comprises: x1) SEQ ID NO: 38; or

[0033] x2) The amino acid sequence of SEQ ID NO: 38 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 38; or

[0034] x3) ​​has an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94% or 93% homology to SEQ ID NO: 38 and has the same function as the protein shown in SEQ ID NO: 38.

[0035] In some embodiments, the amino acid sequence of the signal peptide of the light chain of the anti-digoxin antibody or its antigen-binding fragment comprises: y1) SEQ ID NO: 40; or

[0036] y2) An amino acid sequence of SEQ ID NO: 40 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 40; or

[0037] y3) has an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94% or 93% homology with SEQ ID NO: 40 and has the same function as the protein shown in SEQ ID NO: 40.

[0038] A second aspect of the invention provides a recombinant protein comprising: an anti-digoxin antibody or an antigen-binding fragment thereof from the first aspect of the invention, and optionally a tag sequence for expression and / or purification.

[0039] In some implementations, the tag sequence is selected from at least one of Poly his, FLAG, Strep-Tag II, Poly arg, C-myc, HA, V5, VSV-G, Trx, SUMO, GST, MBP, and NusA.

[0040] A third aspect of the present invention provides a conjugate comprising: any one of an anti-digoxin antibody or an antigen-binding fragment thereof from the first aspect of the present invention and a recombinant protein from the second aspect of the present invention, and a conjugation portion thereof, said conjugation portion being a detectable marker.

[0041] In some embodiments, the anti-digoxin antibody or its antigen-binding fragment and / or recombinant protein are coupled to the coupling moiety via chemical bonds or linkers.

[0042] In some embodiments, the detectable marker comprises at least one of metal particles, fluorescent markers, luminescent markers, electron-dense markers, radioactive markers, and enzyme markers.

[0043] In some implementations, "fluorescent labeling" refers to any molecule that can be detected by means of the fluorescent properties of a substance. Fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, fluorescent yellow, cascade blue J, Texas red, IAEDANS, EDANS, BODIPY FL, LC red 640, LC red 705, Oregon green, alexa-fluor dyes (alexa-fluor 350, alexa-fluor 430, alexa-fluor 488, alexa-fluor 546, alexa-fluor 568, alexa-fluor 594, alexa-fluor 633, alexa-fluor 647, alexa-fluor 660, alexa-fluor 680), cascade blue, cascade yellow, and R-phycoerythrin (PE), FITC, Cy5, Cy5.5, and Cy7, etc.

[0044] In some embodiments, "luminescent labeling" includes chemiluminescent labels and bioluminescent labels. Chemiluminescent labels include, but are not limited to, luminol, isoluminol, acridine esters, acridine sulfonamides, aromatic acridine esters, imidazoles, acridine salts, and oxalates. Bioluminescent labels include, but are not limited to, luciferase substrates or their derivatives (such as coelenterazine, coelenterazine h, coelenterazine hcp, coelenterazine 400a, coelenterazine f, coelenterazine cp, coelenterazine n, coelenterazine e, and luciferin).

[0045] In some embodiments, the detectable marker comprises at least one of a radioisotope, luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidase, glucose-6-phosphate dehydrogenase, rhodamine, luciferin, glucose oxidase, galactose oxidase, acridinium ester, acridinium sulfonamide, luminol, and isoluminol.

[0046] In some embodiments, the radioactive isotope is selected from at least one of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, P-32, H-3, S-35, Lu-177, and Re-188.

[0047] In some embodiments, the detectable marker is an enzyme marker. In some embodiments, the enzyme marker preferably comprises luciferase. In some embodiments, the luciferase comprises at least one of bacterial luciferase, firefly luciferase, sea firefly luciferase, kidney luciferase, Gaussian luciferase, Nanoluciferase, and spiny shrimp luciferase.

[0048] A fourth aspect of the present invention provides a fusion protein comprising: any one of the anti-digoxin antibody or its antigen-binding fragment from the first aspect of the present invention and the recombinant protein from the second aspect of the present invention, and luciferase.

[0049] In some embodiments, a fusion protein is provided comprising: an anti-digoxin antibody or an antigen-binding fragment thereof from the first aspect of the present invention, and a luciferase.

[0050] In some embodiments, the luciferase comprises at least one selected from bacterial luciferase, firefly luciferase, sea firefly luciferase, kidney luciferase, Gaussian luciferase, Nanoluciferase, and spiny shrimp luciferase. In some embodiments, the luciferase comprises at least one selected from Gaussian luciferase and spiny shrimp luciferase. In some embodiments, the luciferase is either Gaussian luciferase or spiny shrimp luciferase.

[0051] In some embodiments, the luciferase is fused to the heavy chain of the anti-digoxin antibody or its antigen-binding fragment. In some embodiments, the luciferase is fused to the light chain of the anti-digoxin antibody or its antigen-binding fragment. In some embodiments, the luciferase is fused to both the heavy and light chains of the anti-digoxin antibody or its antigen-binding fragment.

[0052] In some embodiments, the fusion protein comprises a third polypeptide and a fourth polypeptide:

[0053] g1) The third polypeptide comprises the heavy chain and luciferase of the anti-digoxin antibody or its antigen-binding fragment, and the fourth polypeptide comprises the light chain of the anti-digoxin antibody or its antigen-binding fragment; or

[0054] g2) The third polypeptide comprises the heavy chain of the anti-digoxin antibody or its antigen-binding fragment, and the fourth polypeptide comprises the light chain of the anti-digoxin antibody or its antigen-binding fragment and luciferase; or

[0055] g3) The third polypeptide comprises the heavy chain and luciferase of the anti-digoxin antibody or its antigen-binding fragment, and the fourth polypeptide comprises the light chain and luciferase of the anti-digoxin antibody or its antigen-binding fragment.

[0056] In some embodiments, h1): the luciferase fuses with the heavy chain of the anti-digoxin antibody or its antigen-binding fragment via a linker; and / or, h2): the luciferase fuses with the light chain of the anti-digoxin antibody or its antigen-binding fragment via a linker.

[0057] In some implementations, the connectors described in h1) and h2) are each independently selected from at least one of linking peptides and flexible peptides.

[0058] In some embodiments, the amino acid sequence of the linker described in h1) and h2) includes at least one of z1) SEQ ID NO: 42 and z2) SEQ ID NO: 45.

[0059] In some embodiments, the luciferase is a Gaussian luciferase, and the amino acid sequence of the linker in h1) and h2) includes z1).

[0060] In some embodiments, the luciferase is spiny shrimp luciferase, and the amino acid sequence of the linker in h1) and h2) includes z2).

[0061] In some embodiments, the amino acid sequence of the Gaussian luciferase comprises: e1) SEQ ID NO: 43; or

[0062] e2) An amino acid sequence of SEQ ID NO: 43 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 43; or

[0063] e3) has an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94% or 93% homology to SEQ ID NO: 43 and has the same function as the protein shown in SEQ ID NO: 43.

[0064] In some embodiments, the amino acid sequence of the spiny shrimp luciferase includes: f1) SEQ ID NO: 46; or

[0065] f2) An amino acid sequence of SEQ ID NO: 46 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 46; or

[0066] f3) has an amino acid sequence that is 99%, 98%, 97%, 96%, 95%, 94% or 93% homology to SEQ ID NO: 46 and has the same function as the protein shown in SEQ ID NO: 46.

[0067] In some embodiments, the luciferase is fused to the light chain and / or heavy chain of the anti-digoxigenin antibody or its antigen-binding fragment via a linkage of N'-R1-L-R2-C', N'-R2-L-R1-C', or N'-R2-L-R1r-C': wherein L is the linker; R1 is the amino acid sequence of the luciferase; R1r is the reverse amino acid sequence of the luciferase; R2 is the light chain and / or heavy chain of the anti-digoxigenin antibody or its antigen-binding fragment; C' represents the carbonyl residue terminus of the fusion protein; and N' represents the amino residue terminus of the fusion protein.

[0068] In some embodiments, the amino acid sequence of the third polypeptide comprises: i11)SEQ ID NO: 1; or

[0069] i12) A sequence of amino acids in SEQ ID NO: 1 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 1; or

[0070] i13) has an amino acid sequence that is 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology to SEQ ID NO: 1 and has the same function as the protein shown in SEQ ID NO: 1; or

[0071] i14)SEQ ID NO: 37; or

[0072] i15) An amino acid sequence of SEQ ID NO: 37 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 37; or

[0073] i16) has 99%, 98%, 97%, 96%, 90%, 80% or 75% homology with SEQ ID NO: 37 and has the same amino acid sequence as the protein shown in i14).

[0074] In some embodiments, the amino acid sequence of the fourth polypeptide comprises: (i17) amino acids 23-429 of SEQ ID NO: 47; or

[0075] i18) An amino acid sequence of i17) with one or more amino acid substitutions and / or deletions and / or additions that has the same function as the protein shown in i17); or

[0076] i19) has 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology with i17) and has the same amino acid sequence as the protein shown in i17); or

[0077] i110)SEQ ID NO: 47; or

[0078] i111) The amino acid sequence of SEQ ID NO: 47 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 47; or

[0079] i112) has an amino acid sequence that is 99%, 98%, 97%, 96%, 90%, 80% or 75% homology to SEQ ID NO: 47 and has the same function as the protein shown in SEQ ID NO: 47.

[0080] In some embodiments, the amino acid sequence of the third polypeptide comprises: i21) amino acids 20-651 of SEQ ID NO: 41; or

[0081] i22) An amino acid sequence of i21) with one or more amino acid substitutions and / or deletions and / or additions that has the same function as the protein shown in i21); or

[0082] i23) has 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology with i21) and has the same amino acid sequence as the protein shown in i21); or

[0083] i24)SEQ ID NO: 41; or

[0084] i25) An amino acid sequence of SEQ ID NO: 41 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 41; or

[0085] i26) has an amino acid sequence that is 99%, 98%, 97%, 96%, 90%, 80% or 75% homology to SEQ ID NO: 41 and has the same function as the protein shown in SEQ ID NO: 41.

[0086] In some embodiments, the amino acid sequence of the fourth polypeptide includes: i27)SEQ ID NO: 18; or

[0087] i28) An amino acid sequence of SEQ ID NO: 18 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 18; or

[0088] i29) has 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology with SEQ ID NO: 18 and has the same amino acid sequence as the protein shown in SEQ ID NO: 18; or

[0089] i210)SEQ ID NO: 39; or

[0090] i211) The amino acid sequence of SEQ ID NO: 39 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 39; or

[0091] i212) has an amino acid sequence that is 99%, 98%, 97%, 96%, 90%, 80% or 75% homology to SEQ ID NO: 39 and has the same function as the protein shown in SEQ ID NO: 39.

[0092] In some embodiments, the amino acid sequence of the third polypeptide comprises: i31) amino acids 20-651 of SEQ ID NO: 41; or

[0093] i32) An amino acid sequence of SEQ ID NO: i31) with one or more amino acid substitutions and / or deletions and / or additions, and having the same function as the protein shown in i31); or

[0094] i33) has 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology with i31) and has the same amino acid sequence as the protein shown in i31); or

[0095] i34)SEQ ID NO: 41; or

[0096] i35) An amino acid sequence of SEQ ID NO: 41 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 41; or

[0097] i36) has an amino acid sequence that is 99%, 98%, 97%, 96%, 90%, 80% or 75% homology to SEQ ID NO: 41 and has the same function as the protein shown in SEQ ID NO: 41.

[0098] In some embodiments, the amino acid sequence of the fourth polypeptide comprises: (i37) amino acids 23-429 of SEQ ID NO: 47; or

[0099] i38) is an amino acid sequence of i37) with one or more amino acid substitutions and / or deletions and / or additions, and which has the same function as the protein shown in i37); or

[0100] i39) shares 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology with i37) and has the same functional amino acid sequence as the protein shown in i37); or

[0101] i310)SEQ ID NO: 47; or

[0102] i311) The amino acid sequence of SEQ ID NO: 47 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 47; or

[0103] i312) has 99%, 98%, 97%, 96%, 90%, 80% or 75% homology with SEQ ID NO: 47 and has the same amino acid sequence as the protein shown in SEQ ID NO: 47.

[0104] In some embodiments, the amino acid sequence of the third polypeptide comprises: i41) SEQ ID NO: 1; or

[0105] i42) A sequence of amino acids in SEQ ID NO: 1 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 1; or

[0106] i43) has an amino acid sequence that is 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology to SEQ ID NO: 1 and has the same function as the protein shown in SEQ ID NO: 1; or

[0107] i44)SEQ ID NO: 37; or

[0108] i45) An amino acid sequence of SEQ ID NO: 37 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 37; or

[0109] i46) has an amino acid sequence that is 99%, 98%, 97%, 96%, 90%, 80% or 75% homology to SEQ ID NO: 37 and has the same function as the protein shown in SEQ ID NO: 37.

[0110] In some embodiments, the amino acid sequence of the fourth polypeptide comprises: (i47) amino acids 23-428 of SEQ ID NO: 48; or

[0111] i48) An amino acid sequence of i47) with one or more amino acid substitutions and / or deletions and / or additions that have the same function as the protein shown in i47); or

[0112] i49) shares 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology with i47) and has the same functional amino acid sequence as the protein shown in i47); or

[0113] i410)SEQ ID NO: 48; or

[0114] i411) An amino acid sequence of SEQ ID NO: 48 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 48; or

[0115] i412) has an amino acid sequence that is 99%, 98%, 97%, 96%, 90%, 80% or 75% homology to SEQ ID NO: 48 and has the same function as the protein shown in SEQ ID NO: 48.

[0116] In some embodiments, the amino acid sequence of the third polypeptide comprises: i51) amino acids 20-650 of SEQ ID NO: 44; or

[0117] i52) An amino acid sequence of i51) with one or more amino acid substitutions and / or deletions and / or additions that has the same function as the protein shown in i51); or

[0118] i53) shares 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology with i51) and has the same functional amino acid sequence as the protein shown in i51); or

[0119] i54)SEQ ID NO: 44; or

[0120] i55) An amino acid sequence of SEQ ID NO: 44 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 44; or

[0121] i56) has an amino acid sequence that is 99%, 98%, 97%, 96%, 90%, 80% or 75% homology to SEQ ID NO: 44 and has the same function as the protein shown in SEQ ID NO: 44.

[0122] In some embodiments, the amino acid sequence of the fourth polypeptide includes: i57)SEQ ID NO: 18; or

[0123] i58) An amino acid sequence of SEQ ID NO: 18 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 18; or

[0124] i59) has 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology with SEQ ID NO: 18 and has the same amino acid sequence as the protein shown in SEQ ID NO: 18; or

[0125] i510)SEQ ID NO: 39; or

[0126] i511) An amino acid sequence of SEQ ID NO: 39 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 39; or

[0127] i512) has an amino acid sequence that is 99%, 98%, 97%, 96%, 90%, 80% or 75% homology to SEQ ID NO: 39 and has the same function as the protein shown in SEQ ID NO: 39.

[0128] In some embodiments, the amino acid sequence of the third polypeptide comprises: i61) amino acids 20-650 of SEQ ID NO: 44; or

[0129] i62) An amino acid sequence of SEQ ID NO: i61) with one or more amino acid substitutions and / or deletions and / or additions, and having the same function as the protein shown in i61); or

[0130] i63) shares 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology with i61) and has the same functional amino acid sequence as the protein shown in i61); or

[0131] i64)SEQ ID NO: 44; or

[0132] i65) An amino acid sequence of SEQ ID NO: 44 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 44; or

[0133] i66) has an amino acid sequence that is 99%, 98%, 97%, 96%, 90%, 80% or 75% homology to SEQ ID NO: 44 and has the same function as the protein shown in SEQ ID NO: 44.

[0134] In some embodiments, the amino acid sequence of the fourth polypeptide comprises: (i67) amino acids 23-428 of SEQ ID NO: 48; or

[0135] i68) is an amino acid sequence of i67) with one or more amino acid substitutions and / or deletions and / or additions, and which has the same function as the protein shown in i67); or

[0136] i69) and i67) have 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology and have the same amino acid sequence as the protein shown in i67); or

[0137] i610)SEQ ID NO: 48; or

[0138] i611) The amino acid sequence of SEQ ID NO: 48 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 48; or

[0139] i612) has 99%, 98%, 97%, 96%, 90%, 80% or 75% homology with SEQ ID NO: 48 and has the same amino acid sequence as the protein shown in SEQ ID NO: 48.

[0140] A fifth aspect of the invention provides biomaterials relating to an anti-digoxin antibody or its antigen-binding fragment of the first aspect of the invention, a recombinant protein of the second aspect of the invention, a conjugate of the third aspect of the invention, or a fusion protein of the fourth aspect of the invention, said biomaterials comprising any one of j1)-j12):

[0141] j1) A nucleic acid molecule encoding an anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the present invention, a recombinant protein of the second aspect of the present invention, a conjugate of the third aspect of the present invention, or a fusion protein of the fourth aspect of the present invention;

[0142] j2) contains an expression cassette containing the nucleic acid molecule described in j1);

[0143] j3) A carrier containing the nucleic acid molecule described in j1);

[0144] j4) A carrier containing the expression box described in j2);

[0145] j5) A transgenic cell line containing the nucleic acid molecules described in j1);

[0146] j6) A transgenic cell line containing the expression cassette described in j2);

[0147] j7) A transgenic cell line containing the vector described in j3);

[0148] j8) A transgenic cell line containing the vector described in j4);

[0149] j9) Recombinant bacteria containing the nucleic acid molecules described in j1);

[0150] j10) contains the recombinant bacteria of the expression cassette described in j2);

[0151] j11) contains recombinant bacteria containing the vector described in j3);

[0152] j12) contains recombinant bacteria containing the vector described in j4).

[0153] In some implementations, the transgenic cell lines described in j5)-j8) do not contain propagation material.

[0154] In some implementations, the vectors in j3) and j4) contain a promoter that is operatively linked to the nucleic acid molecule.

[0155] In some embodiments, the vectors in j3) and j4) are independently selected from non-pathogenic viral vectors and non-viral vectors. In some embodiments, the non-pathogenic viral vectors include adenovirus vectors or retrovirus vectors. In some embodiments, the non-viral vectors include plasmid vectors.

[0156] In some embodiments, the nucleic acid molecule encoding the anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the present invention comprises: a nucleic acid molecule encoding the heavy chain variable region of the anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the present invention and a nucleic acid molecule encoding the light chain variable region of the anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the present invention.

[0157] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the present invention comprises: k1) SEQ ID NO: 16; or

[0158] k2) A nucleotide sequence of SEQ ID NO: 16 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 16; or

[0159] k3) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% or more homology to SEQ ID NO: 16 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 16; or

[0160] k4) Nucleotides 73-432 of SEQ ID NO: 31, SEQ ID NO: 33, or SEQ ID NO: 34; or

[0161] k5) A nucleotide sequence of k4) that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in k4); or

[0162] k6) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% or more homology with k4) and has the same function as the nucleic acid molecule shown in k4).

[0163] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the invention comprises: l1) SEQ ID NO: 29; or

[0164] l2) A nucleotide sequence of SEQ ID NO: 29 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 29; or

[0165] l3) A nucleotide sequence that has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% or more homology to SEQ ID NO: 29 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 29; or

[0166] l4) Nucleotides 82-417 of SEQ ID NO: 32, SEQ ID NO: 35, or SEQ ID NO: 36; or

[0167] l5) A nucleotide sequence of l4) that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in l4); or

[0168] l6) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% or more homology with l4) and has the same function as the nucleic acid molecule shown in l4).

[0169] In some embodiments, the nucleic acid molecule encoding the anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the present invention further comprises: a nucleic acid molecule encoding the heavy chain constant region of the anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the present invention and a nucleic acid molecule encoding the light chain constant region of the anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the present invention.

[0170] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain constant region of the anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the invention comprises: m1) SEQ ID NO: 17; or

[0171] m2) A nucleotide sequence of SEQ ID NO: 17 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 17; or

[0172] m3) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 95%, 80%, or 70% or more homology to SEQ ID NO: 17 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 17; or

[0173] m4) Nucleotides 433-1404 of SEQ ID NO: 31, SEQ ID NO: 33, or SEQ ID NO: 34; or

[0174] m5) is a nucleotide sequence of m4) that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in m4); or

[0175] The nucleotide sequences of m6 and m4 have 99%, 98%, 97%, 96%, 95%, 80% or 70% or more homology and have the same function as the nucleic acid molecule shown in m4).

[0176] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the light chain constant region of the anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the invention comprises: n1) SEQ ID NO: 30; or

[0177] n2) A nucleotide sequence of SEQ ID NO: 30 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 30; or

[0178] n3) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 95%, 80%, or 70% or more homology to SEQ ID NO: 30 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 30; or

[0179] n4) Nucleotides 418-738 of SEQ ID NO: 32, SEQ ID NO: 35, or SEQ ID NO: 36; or

[0180] n5) A nucleotide sequence of n4) that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in n4); or

[0181] n6) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 95%, 80%, or 70% or more homology with n4) and has the same function as the nucleic acid molecule shown in n4).

[0182] In some embodiments, the nucleic acid molecule encoding the anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the present invention comprises: a nucleic acid molecule of a heavy chain encoding the anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the present invention and a nucleic acid molecule of a light chain encoding the anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the present invention.

[0183] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain of the anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the invention comprises: C1) SEQ ID NO: 15; or

[0184] C2) A nucleotide sequence of SEQ ID NO: 15 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 15; or

[0185] C3) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 70%, 65%, or 60% or more homology to SEQ ID NO: 15 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 15; or

[0186] C4) Nucleotides 73-1404 of SEQ ID NO: 31, SEQ ID NO: 33, or SEQ ID NO: 34; or

[0187] C5) A nucleotide sequence of C4) with one or more nucleotide substitutions and / or deletions and / or additions that has the same function as the nucleic acid molecule shown in C4); or

[0188] C6) has a nucleotide sequence that shares 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 70%, 65%, or 60% or more homology with C4) and has the same function as the nucleic acid molecule shown in C4);

[0189] C7) Nucleotides 16-1404 of SEQ ID NO: 31, SEQ ID NO: 33, or SEQ ID NO: 34; or

[0190] C8) A nucleotide sequence of C7) with one or more nucleotide substitutions and / or deletions and / or additions that has the same function as the nucleic acid molecule shown in C7); or

[0191] C9) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 70%, 65%, or 60% or more homology with C7) and has the same function as the nucleic acid molecule shown in C7).

[0192] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the light chain of the anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the invention comprises: D1) SEQ ID NO: 28; or

[0193] D2) A nucleotide sequence of SEQ ID NO: 28 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 28; or

[0194] D3) has a nucleotide sequence that shares 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 70%, 65%, or 60% or more homology with SEQ ID NO: 28 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 28; or

[0195] D4) Nucleotides 82-738 of SEQ ID NO: 32, SEQ ID NO: 35, or SEQ ID NO: 36; or

[0196] D5) A nucleotide sequence of D4) with one or more nucleotide substitutions and / or deletions and / or additions that has the same function as the nucleic acid molecule shown in D4); or

[0197] D6) has a nucleotide sequence that shares 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 70%, 65%, or 60% or more homology with D4) and has the same function as the nucleic acid molecule shown in D4);

[0198] D7) Nucleotides 16-738 of SEQ ID NO: 32, SEQ ID NO: 35, or SEQ ID NO: 36; or

[0199] D8) A nucleotide sequence of D7) with one or more nucleotide substitutions and / or deletions and / or additions that has the same function as the nucleic acid molecule shown in D7); or

[0200] D9) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 70%, 65%, or 60% or more homology with D7) and has the same function as the nucleic acid molecule shown in D7).

[0201] In some embodiments, the nucleic acid molecule encoding the fusion protein of the fourth aspect of the present invention comprises: a nucleic acid molecule encoding a third polypeptide of the fusion protein of the fourth aspect of the present invention and a nucleic acid molecule encoding a fourth polypeptide of the fusion protein of the fourth aspect of the present invention.

[0202] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the third polypeptide of the fusion protein of the fourth aspect of the present invention comprises: nucleotides 73-1404 of SEQ ID NO: 31, or nucleotides 16-1404 of SEQ ID NO: 31; or

[0203] q12) A nucleotide sequence of q11) that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in q11); or

[0204] q13) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology to q11) and has the same function as the nucleic acid molecule shown in q11); or

[0205] q14)SEQ ID NO: 31; or

[0206] q15) A nucleotide sequence of SEQ ID NO: 31 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 31; or

[0207] q16) is a nucleotide sequence that has 99%, 98%, 97%, 96%, 90%, 80% or 75% or more homology with SEQ ID NO: 31 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 31.

[0208] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the fourth polypeptide of the fusion protein of the fourth aspect of the present invention comprises: nucleotides 82-1302 of SEQ ID NO: 35 (q17), or nucleotides 16-1302 of SEQ ID NO: 35; or

[0209] q18) A nucleotide sequence of q17) with one or more nucleotide substitutions and / or deletions and / or additions that has the same function as the nucleic acid molecule shown in q17); or

[0210] q19) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology to q17) and has the same function as the nucleic acid molecule shown in q17); or

[0211] q110)SEQ ID NO: 35; or

[0212] q111) A nucleotide sequence of SEQ ID NO: 35 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 35; or

[0213] q112) is a nucleotide sequence that has 99%, 98%, 97%, 96%, 90%, 80% or 75% homology with SEQ ID NO: 35 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 35.

[0214] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the third polypeptide of the fusion protein of the fourth aspect of the present invention comprises: nucleotides 73-1968 of SEQ ID NO: 33 (q21), or nucleotides 16-1968 of SEQ ID NO: 33; or

[0215] q22) A nucleotide sequence of q21) that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in q21); or

[0216] q23) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology to q21) and has the same function as the nucleic acid molecule shown in q21); or

[0217] q24)SEQ ID NO: 33; or

[0218] q25) A nucleotide sequence of SEQ ID NO: 33 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 33; or

[0219] q26) is a nucleotide sequence that has 99%, 98%, 97%, 96%, 90%, 80% or 75% or more homology with SEQ ID NO: 33 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 33.

[0220] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the fourth polypeptide of the fusion protein of the fourth aspect of the present invention comprises: nucleotides 82-738 of SEQ ID NO: 32 (q27), or nucleotides 16-738 of SEQ ID NO: 32; or

[0221] q28) A nucleotide sequence of q27) with one or more nucleotide substitutions and / or deletions and / or additions that has the same function as the nucleic acid molecule shown in q27); or

[0222] q29) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 90%, 80%, or 75% or higher homology to q27) and has the same function as the nucleic acid molecule shown in q27); or

[0223] q210)SEQ ID NO: 32; or

[0224] q211) A nucleotide sequence of SEQ ID NO: 32 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 32; or

[0225] q212) is a nucleotide sequence that has 99%, 98%, 97%, 96%, 90%, 80% or 75% or more homology with SEQ ID NO: 32 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 32.

[0226] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the third polypeptide of the fusion protein of the fourth aspect of the present invention comprises: q31) nucleotides 73-1968 of SEQ ID NO: 33, or nucleotides 16-1968 of SEQ ID NO: 33; or

[0227] q32) A nucleotide sequence of q31) that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in q31); or

[0228] q33) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology to q31) and has the same function as the nucleic acid molecule shown in q31); or

[0229] q34)SEQ ID NO: 33; or

[0230] q35) A nucleotide sequence of SEQ ID NO: 33 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 33; or

[0231] q36) is a nucleotide sequence that has 99%, 98%, 97%, 96%, 90%, 80% or 75% or more homology with SEQ ID NO: 33 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 33.

[0232] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the fourth polypeptide of the fusion protein of the fourth aspect of the present invention comprises: nucleotides 82-1302 of SEQ ID NO: 35 (q37), or nucleotides 16-1302 of SEQ ID NO: 35; or

[0233] q38) A nucleotide sequence of q37) with one or more nucleotide substitutions and / or deletions and / or additions that have the same function as the nucleic acid molecule shown in q37); or

[0234] q39) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 90%, 80%, or 75% or higher homology to q37) and has the same function as the nucleic acid molecule shown in q37); or

[0235] q310)SEQ ID NO: 35; or

[0236] q311) A nucleotide sequence of SEQ ID NO: 35 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 35; or

[0237] q312) is a nucleotide sequence that has 99%, 98%, 97%, 96%, 90%, 80% or 75% homology with SEQ ID NO: 35 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 35.

[0238] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the third polypeptide of the fusion protein of the fourth aspect of the present invention comprises: nucleotides 73-1404 of SEQ ID NO: 31, or nucleotides 16-1404 of SEQ ID NO: 31; or

[0239] q42) A nucleotide sequence of q41) that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in q41); or

[0240] q43) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology to q41) and has the same function as the nucleic acid molecule shown in q41); or

[0241] q44)SEQ ID NO: 31; or

[0242] q45) A nucleotide sequence of SEQ ID NO: 31 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 31; or

[0243] q46) is a nucleotide sequence that has 99%, 98%, 97%, 96%, 90%, 80% or 75% or more homology with SEQ ID NO: 31 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 31.

[0244] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the fourth polypeptide of the fusion protein of the fourth aspect of the present invention comprises: nucleotides 82-1299 of SEQ ID NO: 36 (q47), or nucleotides 16-1299 of SEQ ID NO: 36; or

[0245] q48) A nucleotide sequence of q47) with one or more nucleotide substitutions and / or deletions and / or additions that has the same function as the nucleic acid molecule shown in q47); or

[0246] q49) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 90%, 80%, or 75% or higher homology to q47) and has the same function as the nucleic acid molecule shown in q47); or

[0247] q410)SEQ ID NO: 36; or

[0248] q411) A nucleotide sequence of SEQ ID NO: 36 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 36; or

[0249] q412) is a nucleotide sequence that has 99%, 98%, 97%, 96%, 90%, 80% or 75% or more homology with SEQ ID NO: 36 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 36.

[0250] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the third polypeptide of the fusion protein of the fourth aspect of the present invention comprises: q51) nucleotides 73-1965 of SEQ ID NO: 34, or nucleotides 16-1965 of SEQ ID NO: 34; or

[0251] q52) A nucleotide sequence of q51) that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in q51); or

[0252] q53) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology to q51) and has the same function as the nucleic acid molecule shown in q51); or

[0253] q54)SEQ ID NO: 34; or

[0254] q55) A nucleotide sequence of SEQ ID NO: 34 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 34; or

[0255] q56) is a nucleotide sequence that has 99%, 98%, 97%, 96%, 90%, 80% or 75% or more homology with SEQ ID NO: 34 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 34.

[0256] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the fourth polypeptide of the fusion protein of the fourth aspect of the present invention comprises: nucleotides 82-738 of SEQ ID NO: 32 (q57), or nucleotides 16-738 of SEQ ID NO: 32; or

[0257] q58) A nucleotide sequence of q57) with one or more nucleotide substitutions and / or deletions and / or additions that has the same function as the nucleic acid molecule shown in q57); or

[0258] q59) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 90%, 80%, or 75% or higher homology to q57) and has the same function as the nucleic acid molecule shown in q57); or

[0259] q510)SEQ ID NO: 32; or

[0260] q511) A nucleotide sequence of SEQ ID NO: 32 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 32; or

[0261] q512) is a nucleotide sequence that has 99%, 98%, 97%, 96%, 90%, 80% or 75% or more homology with SEQ ID NO: 32 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 32.

[0262] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the third polypeptide of the fusion protein of the fourth aspect of the present invention comprises: nucleotides 73-1965 of SEQ ID NO: 34 (q61), or nucleotides 16-1965 of SEQ ID NO: 34; or

[0263] q62) A nucleotide sequence of q61) that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in q61); or

[0264] q63) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 90%, 80%, or 75% or more homology to q61) and has the same function as the nucleic acid molecule shown in q61); or

[0265] q64)SEQ ID NO: 34; or

[0266] q65) A nucleotide sequence of SEQ ID NO: 34 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 34; or

[0267] q66) is a nucleotide sequence that has 99%, 98%, 97%, 96%, 90%, 80% or 75% or more homology with SEQ ID NO: 34 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 34.

[0268] In some embodiments, the nucleotide sequence of the nucleic acid molecule encoding the fourth polypeptide of the fusion protein of the fourth aspect of the present invention comprises: nucleotides 82-1299 of SEQ ID NO: 36 (q67), or nucleotides 16-1299 of SEQ ID NO: 36; or

[0269] q68) A nucleotide sequence of q67) with one or more nucleotide substitutions and / or deletions and / or additions that have the same function as the nucleic acid molecule shown in q67); or

[0270] q69) has a nucleotide sequence that is 99%, 98%, 97%, 96%, 90%, 80%, or 75% or higher homology to q67) and has the same function as the nucleic acid molecule shown in q67); or

[0271] q610)SEQ ID NO: 36; or

[0272] q611) A nucleotide sequence of SEQ ID NO: 36 that has undergone substitution and / or deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 36; or

[0273] q612) is a nucleotide sequence that has 99%, 98%, 97%, 96%, 90%, 80% or 75% or more homology with SEQ ID NO: 36 and has the same function as the nucleic acid molecule shown in SEQ ID NO: 36.

[0274] A sixth aspect of the invention provides the use of the anti-digoxin antibody or its antigen-binding fragment from the first aspect, the recombinant protein from the second aspect, the conjugate from the third aspect, the fusion protein from the fourth aspect, and / or the biological material from the fifth aspect in any one of r1)-r4):

[0275] r1) Prepare products for detecting digoxin (preferably detection reagents, detection kits, test strips, detection plates, detection chips, or detection systems);

[0276] r2) Detect digoxin (preferably digoxin for non-diagnostic destinations);

[0277] r3) Prepare products for nucleic acid sequencing (preferably reagents, kits, or sequencing systems);

[0278] r4) Nucleic acid sequencing.

[0279] Application of any one of s1)-s4):

[0280] s1) The application of the anti-digoxin antibody or its antigen-binding fragment of the first aspect of the present invention, the recombinant protein of the second aspect, the conjugate of the third aspect, the fusion protein of the fourth aspect, and / or the biomaterial of the fifth aspect in the preparation of products for detecting digoxin (the products are preferably detection reagents, detection kits, test strips, detection plates, detection chips, or detection systems);

[0281] s2) The use of the anti-digoxin antibody or its antigen-binding fragment of the first aspect of the present invention, the recombinant protein of the second aspect, the conjugate of the third aspect of the present invention, and / or the fusion protein of the fourth aspect of the present invention in the detection of digoxin (preferably for non-diagnostic digoxin detection);

[0282] s3) The anti-digoxigenin antibody or its antigen-binding fragment of the first aspect of the present invention, the recombinant protein of the second aspect, the conjugate of the third aspect, the fusion protein of the fourth aspect, and / or the biomaterial of the fifth aspect are used to prepare a product for nucleic acid sequencing (the product is preferably a sequencing reagent, a sequencing kit, or a sequencing system);

[0283] s4) Application of the conjugates of the third aspect of the present invention and / or the fusion proteins of the fourth aspect in nucleic acid sequencing.

[0284] A seventh aspect of the invention provides any one of products t1)-t2):

[0285] t1) A product for detecting digoxin, said product comprising at least one of u1)-u4):

[0286] u1) The anti-digoxin antibody or its antigen-binding fragment of the first aspect of the present invention;

[0287] u2) The recombinant protein of the second aspect of the present invention;

[0288] u3) The coupling of the third aspect of the present invention;

[0289] u4) The fusion protein of the fourth aspect of this invention;

[0290] t2) A product for nucleic acid sequencing, said product comprising at least one of v1)-v2):

[0291] v1) The coupling of the third aspect of the present invention;

[0292] v2) The fusion protein of the fourth aspect of the present invention.

[0293] In some implementations, the product described in t1) is a detection reagent, a detection kit, a test strip, a detection plate, a detection chip, or a detection system.

[0294] In some implementations, the product described in t2) is a sequencing reagent, a sequencing kit, or a sequencing system.

[0295] In some embodiments, the products described in t1) and t2) further comprise a chromogenic substrate (e.g., when the product comprises a fusion protein according to the fourth aspect of the present invention; or when the product comprises a conjugate according to the third aspect of the present invention, wherein the conjugate portion is an enzyme marker (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidase, glucose-6-phosphate dehydrogenase, glucose oxidase, galactose oxidase, etc.).

[0296] The eighth aspect of the present invention is to provide any one of w1)-w3):

[0297] w1) The method for preparing the anti-digoxin antibody or its antigen-binding fragment of the first aspect of the present invention, the recombinant protein of the second aspect, the conjugate of the third aspect, or the fusion protein of the fourth aspect, is obtained by culturing the transgenic cell line or recombinant bacteria of the fifth aspect of the present invention;

[0298] w2) A method for detecting digoxin (preferably a non-diagnostic digoxin detection method) comprising the steps of using an anti-digoxin antibody or its antigen-binding fragment from the first aspect of the present invention, a recombinant protein from the second aspect of the present invention, a conjugate from the third aspect of the present invention, a fusion protein from the fourth aspect of the present invention, and / or a product from the seventh aspect of the present invention.

[0299] w3) A nucleic acid sequencing method comprising the steps of using a conjugate of the third aspect of the present invention, a fusion protein of the fourth aspect of the present invention, and / or a product of the seventh aspect of the present invention.

[0300] In some embodiments, the method for detecting digoxin includes the following steps: contacting at least one of u1)-u4) of the anti-digoxin antibody or its antigen-binding fragment of the first aspect of the present invention, the recombinant protein of the second aspect of the present invention, the conjugate of the third aspect of the present invention, the fusion protein of the fourth aspect of the present invention, and / or the product of the seventh aspect of the present invention with the sample to be tested, and detecting whether or not an antigen-antibody immune complex is formed or its content.

[0301] In some embodiments, the nucleic acid sequencing method includes the following steps: using the nucleic acid to be tested as a template, reacting it in a system containing at least one of the conjugates of the third aspect of the present invention, the fusion protein of the fourth aspect of the present invention, and / or the product of the seventh aspect of the present invention (v1)-v2), and determining the nucleic acid sequence of the nucleic acid to be tested based on the signal generated by the detectable marker.

[0302] The beneficial effects of the present invention include, but are not limited to, providing an anti-digoxin antibody or its antigen-binding fragment, which has a high affinity for digoxin and can be used for detecting digoxin, sequencing, preparing products for detecting digoxin, and preparing sequencing products. Attached Figure Description

[0303] Figure 1 is a protein electrophoresis diagram of the anti-digoxin monoclonal antibody obtained by purifying the supernatant of the positive cell line in Example 2.

[0304] Figure 2 shows the titer of the anti-digoxin monoclonal antibody obtained by purifying the supernatant of the positive cell line in Example 2.

[0305] Figure 3 is a map of the anti-digoxigenin antibody heavy chain fusion with the spiny shrimp luciferase (Nluc) expression plasmid in Example 4.

[0306] Figure 4 is a map of the anti-digoxigenin antibody light chain fusion with the spiny shrimp luciferase (Nluc) expression plasmid in Example 4.

[0307] Figure 5 shows the combination of luciferase and anti-digoxin antibody fusion protein in Example 4.

[0308] Figure 6 is a protein electrophoresis diagram of the fusion protein of luciferase and anti-digoxin antibody in Example 4.

[0309] Figure 7 shows the partial activity detection results of the luciferase fusion protein of luciferase and anti-digoxin antibody in Example 5.

[0310] Figure 8 shows the signal intensity results of the fusion protein of luciferase and anti-digoxigenin antibody in Example 7 when sequenced on a DNBSeqE5 autoluminescent sequencer.

[0311] Figure 9 shows the Q30 results of sequencing the fusion protein of luciferase and anti-digoxigenin antibody in Example 7 on a DNBSeqE5 autoluminescent sequencer.

[0312] Figure 10 shows the results of partial activity detection of the fusion protein antibody. Detailed Implementation

[0313] In this application, unless otherwise stated, the term "amino acid" is represented by a single-letter or three-letter code with the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Aspartic acid (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: Histidine; I: Ile (isoleucine); L: Leu (leucine); K: Lysine (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Proline (proline); S: Serine (serine); T: Threonine (threonine); W: Tryptophan (tryptophan); Y: Tyrosine (tyrosine); V: Valine (valine).

[0314] In this application, unless otherwise stated, the nucleotide sequence is displayed as 5' end → 3' end.

[0315] In this application, unless otherwise stated, the amino acid sequence is displayed as N-terminus → C-terminus.

[0316] In this application, unless otherwise stated, “identity” has the conventional meaning in the art as “homology” between two nucleic acid or amino acid sequences, where the percentage represents the statistically significant percentage of identical nucleotide or amino acid residues between the two sequences to be compared after best alignment, with the differences between the two sequences randomly distributed along their entire length.

[0317] The present invention will be further described in detail below through specific embodiments.

[0318] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0319] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0320] Example 1: Subcloning screening of hybridoma cell lines secreting anti-digoxin antibodies

[0321] We commissioned Beijing Yiqiao Shenzhou Company to construct hybridoma cell lines secreting anti-digoxin antibodies and to screen their subclones: After immunizing mice with Dig antigen, we extracted high-titer mouse spleen cells and fused them with myeloma cells to obtain hybridoma parent clones; then, we cultured the hybridoma parent clones and collected the cell supernatant; we performed ELISA on the obtained cell supernatant to obtain the positive value of the antibody in the supernatant; after screening, we obtained positive cell lines, and continued to perform a second round of limiting dilution subcloning, and finally performed ELISA to screen for positive cell lines.

[0322] The method for identifying and screening positive cell lines using ELISA is as follows:

[0323] (1) Coat the enzyme strip with 100 μL of BSA-Dig antigen (solvent is PBS, concentration is 1 μg / mL based on BSA) overnight; add 200 μL PBST and wash 3 times (10 min each time), then add 200 μL PBS and wash 3 times (10 min each time);

[0324] (2) Add 200 μL of 1% BSA-PBS (solvent is PBS, concentration is 1% (m / v, g / mL) based on BSA) and block at 37℃ for 2 h; add 200 μL of PBST and wash 3 times (10 min each time), then add 200 μL of PBS and wash 3 times (10 min each time).

[0325] (3) Add 100 μL of diluted supernatant and incubate at 37 °C for 2 h; add 200 μL of PBST and wash 3 times (10 min each time), then add 200 μL of PBS and wash 3 times (10 min each time);

[0326] (4) Add 100 μL of diluted commercial horseradish peroxidase-conjugated goat anti-mouse antibody and incubate at 37°C for 1 h; add 200 μL of PBST and wash three times (10 min each time), then add 200 μL of PBS and wash three times (10 min each time).

[0327] (5) Use tetramethylbenzidine microporous peroxidase substrate (TMB) as substrate for color development for 10 min;

[0328] (6) After adding 50 μL of 0.1 mol / L sulfuric acid to terminate the reaction, its absorbance at 450 nm was measured.

[0329] Example 2: Preparation of anti-digoxin monoclonal antibody and its purity and potency identification experiments

[0330] The hybridoma cells obtained after the second round of subcloning in Example 1 were expanded and cultured. After centrifugation at 10,000 rpm for 5 min, the cell supernatant was collected. The collected cell supernatant was filtered through a 0.22 μm filter membrane and purified using a Protein A column or a nickel column. After elution with elution buffer, purified anti-digoxin monoclonal antibody was obtained. The potency of the purified anti-digoxin monoclonal antibody was identified by ELISA, and a positive cell line was finally obtained through screening.

[0331] The purity of the anti-digoxin monoclonal antibody obtained by purifying 5 μg of the supernatant of the positive cell line was determined by protein electrophoresis. The results are shown in Figure 1: the protein band is single, indicating that its purity is high.

[0332] The titer of the anti-digoxin monoclonal antibody purified from the supernatant of the positive cell lines was determined by ELISA (using the same method as in Example 1 for identifying and screening positive cell lines). The results are shown in Figure 2: the titer of the anti-digoxin monoclonal antibody purified from the supernatant of the positive cell lines reached 10. -10 The titer is above that of commercial antibodies.

[0333] Example 3: Sequence determination of anti-digoxin monoclonal antibody

[0334] RNA was extracted from the positive cell lines obtained in Example 2, and a cDNA library of hybridoma cells was obtained through reverse transcription. Next, degenerate primers were designed based on the known constant regions of the heavy and light chains of murine antibodies. PCR was performed on the cDNA using the designed primers to obtain the heavy and light chain sequences of monoclonal antibodies. The sequences were then cloned into a T vector, introduced into *E. coli* DH5α for amplification, and single clones were selected. Finally, the selected single clones were expanded and cultured, and the recombinant vector plasmid was extracted and sequenced to obtain the antibody heavy and light chain sequences.

[0335] Its heavy chain amino acid sequence is as follows: (SEQ ID NO: 1; wherein, the single-underlined portion (i.e., amino acids 1-120 of SEQ ID NO: 1) is the heavy chain variable region, which is numbered SEQ ID NO: 2; the double-underlined portion (i.e., amino acids 121-444 of SEQ ID NO: 1) is the heavy chain constant region, which is numbered SEQ ID NO: 3). The CDR-H1, CDR-H2, and CDR-H3 in this heavy chain variable region, defined by different CDR schemes, are shown in Table 1.

[0336] Table 1. CDR-H1, CDR-H2, and CDR-H3 schemes with different CDR definitions in the variable region of the heavy chain.

[0337] Its heavy chain nucleotide sequence is as follows: (SEQ ID NO: 15; wherein, the single underscore portion (i.e., nucleotides 1-360 of SEQ ID NO: 15) is the variable region of the heavy chain, which is numbered SEQ ID NO: 16; the double underscore portion (i.e., nucleotides 361-1332 of SEQ ID NO: 15) is the constant region of the heavy chain, which is numbered SEQ ID NO: 17).

[0338] Its light chain amino acid sequence is as follows: (SEQ ID NO: 18; wherein, the single-underlined portion (i.e., amino acids 1-112 of SEQ ID NO: 18) is the light chain variable region, which is numbered SEQ ID NO: 19; the double-underlined portion (i.e., amino acids 113-219 of SEQ ID NO: 18) is the light chain constant region, which is numbered SEQ ID NO: 20). Table 2 shows CDR-L1, CDR-L2, and CDR-L3 in this light chain variable region with different CDR definitions.

[0339] Table 2. CDR-L1, CDR-L2, and CDR-L3 with different CDR definitions in the variable region of light chains.

[0340] Its light chain nucleotide sequence is as follows: The single-underlined portion (nucleotides 1-336 of SEQ ID NO: 28) is the variable region of the light chain, which is designated as SEQ ID NO: 29; the double-underlined portion (nucleotides 337-657 of SEQ ID NO: 28) is the constant region of the light chain, which is designated as SEQ ID NO: 30.

[0341] Example 4: Expression and purification of a fusion protein comprising luciferase and anti-digoxigenin antibody

[0342] Based on the sequencing results of the anti-digoxin antibody in Example 3, antibody codon optimization and gene synthesis were performed at Genewiz. The anti-digoxin antibody heavy chain expression fragment was synthesized and inserted between the BamHI and HindIII sites on pCDNA3.4 (the nucleotide sequence of the anti-digoxin antibody heavy chain expression fragment is: In SEQ ID NO: 31, nucleotides 1-6 and 1411-1416 (single underlined portion) are restriction enzyme sites (nucleotides 1-6 are BamHI restriction sites, and nucleotides 1411-1416 are HindIII restriction sites) and are not translated; nucleotides 7-15 (double underlined portion) are kozak sequences and are not translated; nucleotides 16-72 (wavy line portion) are the nucleotide sequence encoding the signal peptide; nucleotides 73-1404 (unlabeled portion) are the nucleotide sequence encoding the anti-digoxigenin antibody heavy chain; nucleotides 1405-1410 (dashed line portion) are two stop codons and are not translated to determine the termination of translation; the amino acid sequence obtained by translating the nucleotide sequence shown in SEQ ID NO: 31 is as follows:

[0343] In this sequence, amino acids 1-19 (underlined with wavy lines) represent the amino acid sequence of the signal peptide, designated SEQ ID NO: 38; amino acids 20-463 (unlabeled) represent the amino acid sequence of the anti-digoxigenin antibody heavy chain (SEQ ID NO: 1), thus obtaining the anti-digoxigenin antibody heavy chain expression plasmid; an anti-digoxigenin antibody light chain expression fragment was synthesized and inserted between the BamHI and HindIII sites on pCDNA3.4 (the nucleotide sequence of the anti-digoxigenin antibody light chain expression fragment is: As shown; in SEQ ID NO: 32, nucleotides 1-6 and 745-750 (single underlined portion) are restriction enzyme sites (nucleotides 1-6 are BamHI restriction sites, and nucleotides 745-750 are HindIII restriction sites), and are not translated; nucleotides 7-15 (double underlined portion) are the kozak sequence, and are not translated; nucleotides 16-81 (wavy line portion) are the nucleotide sequence encoding the signal peptide; nucleotides 82-738 (unlabeled portion) are the nucleotide sequence encoding the anti-digoxigenin antibody light chain; nucleotides 739-744 (dashed line portion) are two stop codons, and are not translated to determine the termination of translation; the amino acid sequence obtained by translating the nucleotide sequence shown in SEQ ID NO: 32 is: The amino acids 1-22 (wavy line portion) represent the amino acid sequence of the signal peptide, designated SEQ ID NO: 40; the amino acids 23-241 (unlabeled portion) represent the amino acid sequence of the anti-digoxigenin antibody light chain (SEQ ID NO: 18), resulting in an anti-digoxigenin antibody light chain expression plasmid. An anti-digoxigenin antibody heavy chain fusion Gaussian luciferase (GLuc) expression fragment was synthesized and inserted between the BamHI and HindIII sites on pCDNA3.4 (the nucleotide sequence of the anti-digoxigenin antibody heavy chain fusion Gaussian luciferase (GLuc) expression fragment is: In SEQ ID NO: 33, nucleotides 1-6 and 1975-1980 (single underlined portion) are restriction enzyme sites (nucleotides 1-6 are BamHI restriction sites, and nucleotides 1975-1980 are HindIII restriction sites) and are not translated; nucleotides 7-15 (double underlined portion) are the kozak sequence and are not translated; nucleotides 16-72 (wavy line portion) are the nucleotide sequence encoding the signal peptide; nucleotides 73-1404 (unlabeled portion) are the nucleotide sequence encoding the anti-digoxigenin antibody heavy chain; nucleotides 1405-1464 (italicized portion) are the nucleotide sequence encoding the linker peptide; nucleotides 1465-1968 (bold portion) are the nucleotide sequence encoding Gaussian luciferase (GLuc); nucleotides 1969-1974 (dashed line portion) are two stop codons and are not translated to determine the termination of translation; SEQ ID The amino acid sequence obtained by translating the nucleotide sequence shown in NO: 33 is: In this sequence, amino acids 1-19 (wavy underline) represent the amino acid sequence of the signal peptide, SEQ ID NO: 38; amino acids 20-463 (unlabeled) represent the amino acid sequence of the anti-digoxigenin antibody heavy chain, SEQ ID NO: 1; amino acids 464-483 (italicized) represent the amino acid sequence of the linker peptide, designated SEQ ID NO: 42; and amino acids 484-651 (bolded) represent the amino acid sequence of Gaussian luciferase (GLuc), designated SEQ ID NO: 43, thus obtaining the anti-digoxigenin antibody heavy chain fusion Gaussian luciferase (GLuc) expression plasmid. An anti-digoxigenin antibody heavy chain fusion spiny shrimp luciferase (Nluc) expression fragment was synthesized and inserted between the BamHI and HindIII sites on pCDNA3.4 (Figure 3, the nucleotide sequence of the anti-digoxigenin antibody heavy chain fusion spiny shrimp luciferase (Nluc) expression fragment is: In SEQ ID NO: 34, nucleotides 1-6 and 1972-1977 (single underlined portion) are restriction enzyme sites (nucleotides 1-6 are BamHI restriction sites, and nucleotides 1972-1977 are HindIII restriction sites) and are not translated; nucleotides 7-15 (double underlined portion) are the kozak sequence and are not translated; nucleotides 16-72 (wavy line portion) are the nucleotide sequence encoding the signal peptide; nucleotides 73-1404 (unlabeled portion) are the nucleotide sequence encoding the anti-digoxigenin antibody heavy chain; nucleotides 1405-1458 (italicized portion) are the nucleotide sequence encoding the linker peptide; nucleotides 1459-1965 (bold portion) are the nucleotide sequence encoding the spiny shrimp luciferase (Nluc); nucleotides 1966-1971 (dashed line portion) are two stop codons and are not translated to determine the termination of translation; SEQ ID The amino acid sequence obtained by translating the nucleotide sequence shown in NO:34 is: In this sequence, amino acids 1-19 (wavy underline) represent the amino acid sequence of the signal peptide, SEQ ID NO: 38; amino acids 20-463 (unlabeled) represent the amino acid sequence of the anti-digoxigenin antibody heavy chain, SEQ ID NO: 1; amino acids 464-481 (italicized) represent the amino acid sequence of the linker peptide, designated SEQ ID NO: 45; and amino acids 482-650 (bolded) represent the amino acid sequence of the spiny shrimp luciferase (Nluc), designated SEQ ID NO: 46. This yields an anti-digoxigenin antibody heavy chain fusion expression plasmid for spiny shrimp luciferase (Nluc). An anti-digoxigenin antibody light chain fusion expression fragment for Gaussian luciferase (GLuc) is synthesized and inserted between the BamHI and HindIII sites on pCDNA3.4. (The nucleotide sequence of the anti-digoxigenin antibody light chain fusion expression fragment for Gaussian luciferase (GLuc) is:) In SEQ ID NO: 35, nucleotides 1-6 and 1309-1314 (single underlined portion) are restriction enzyme sites (nucleotides 1-6 are BamHI restriction sites, and nucleotides 1309-1314 are HindIII restriction sites) and are not translated; nucleotides 7-15 (double underlined portion) are kozak sequences and are not translated; nucleotides 16-81 (wavy line portion) are the nucleotide sequence encoding the signal peptide; nucleotides 82-738 (unlabeled portion) are the nucleotide sequence encoding the anti-digoxigenin antibody light chain; nucleotides 739-798 (italicized portion) are the nucleotide sequence encoding the linker peptide; nucleotides 799-1302 (bold portion) are the nucleotide sequence encoding Gaussian luciferase (GLuc); nucleotides 1303-1308 (dashed line portion) are two stop codons and are not translated to determine the termination of translation; the amino acid sequence obtained by translating the nucleotide sequence shown in SEQ ID NO: 35 is as follows: In this sequence, amino acids 1-22 (wavy lines) represent the amino acid sequence of the signal peptide, i.e., SEQ ID NO: 40; amino acids 23-241 (unlabeled) represent the amino acid sequence of the anti-digoxigenin antibody light chain, i.e., SEQ ID NO: 18; amino acids 242-261 (italicized) represent the amino acid sequence of the linker peptide, i.e., SEQ ID NO: 42; and amino acids 262-429 (bolded) represent the amino acid sequence of Gaussian luciferase (GLuc), i.e., SEQ ID NO: 43), thus obtaining an anti-digoxigenin antibody light chain fusion Gaussian luciferase (GLuc) expression plasmid. An anti-digoxigenin antibody light chain fusion spiny shrimp luciferase (Nluc) expression fragment was synthesized and inserted between the BamHI and HindIII sites on pCDNA3.4 (Figure 4, the nucleotide sequence of the anti-digoxigenin antibody light chain fusion spiny shrimp luciferase (Nluc) expression fragment is: In SEQ ID NO: 36, nucleotides 1-6 and 1306-1311 (single underlined portion) are restriction enzyme sites (nucleotides 1-6 are BamHI restriction sites, and nucleotides 1306-1311 are HindIII restriction sites) and are not translated; nucleotides 7-15 (double underlined portion) are kozak sequences and are not translated; nucleotides 16-81 (wavy line portion) are nucleotide sequences encoding a signal peptide; nucleotides 82-738 (unlabeled portion) are nucleotide sequences encoding the light chain of the anti-digoxigenin antibody; nucleotides 739-792 (italicized portion) are nucleotide sequences encoding a linker peptide; nucleotides 793-1299 (bold portion) are nucleotide sequences encoding Nlucose luciferase (Nlucose); nucleotides 1300-1305 (dashed line portion) are two stop codons and are not translated to determine the termination of translation; the amino acid sequence obtained by translating the nucleotide sequence shown in SEQ ID NO: 36 is as follows: In this sequence, amino acids 1-22 (wavy lines) represent the amino acid sequence of the signal peptide, i.e., SEQ ID NO: 40; amino acids 23-241 (unlabeled) represent the amino acid sequence of the anti-digoxigenin antibody light chain, i.e., SEQ ID NO: 18; amino acids 242-259 (italicized) represent the amino acid sequence of the linker peptide, i.e., SEQ ID NO: 45; and amino acids 260-428 (bolded) represent the amino acid sequence of spiny shrimp luciferase (Nluc), i.e., SEQ ID NO: 46), thus obtaining the anti-digoxigenin antibody light chain fusion expression plasmid of spiny shrimp luciferase (Nluc).

[0344] The fusion protein combinations of luciferase and anti-digoxigenin antibody include the following three types: 1) heavy chain fused luciferase and light chain non-fused luciferase; 2) light chain fused luciferase and heavy chain non-fused luciferase; 3) heavy chain fused luciferase and light chain fused luciferase (Figure 5).

[0345] Protein expression was performed using Hek293F cells. When expressing anti-digoxigenin antibody, the plasmids were the anti-digoxigenin antibody heavy chain expression plasmid and the anti-digoxigenin antibody light chain expression plasmid, respectively. When expressing fusion protein N2, the plasmids were the anti-digoxigenin antibody heavy chain fusion with spiny shrimp luciferase (Nluc) expression plasmid and the anti-digoxigenin antibody light chain expression plasmid, respectively. When expressing fusion protein N3, the plasmids were the anti-digoxigenin antibody heavy chain fusion with spiny shrimp luciferase (Nluc) expression plasmid, respectively. When expressing fusion protein N4, the plasmids were the anti-digoxigenin antibody heavy chain fusion with spiny shrimp luciferase (Nluc) expression plasmid and the anti-digoxigenin antibody light chain expression plasmid, respectively. The following plasmids were used to express digoxin antibody light chain fusion with luciferase (Nluc) from spiny shrimp: For fusion protein G2, the plasmids were an anti-digoxin antibody heavy chain fusion with Gaussian luciferase (GLuc) and an anti-digoxin antibody light chain expression plasmid; for fusion protein G3, the plasmids were an anti-digoxin antibody heavy chain fusion with Gaussian luciferase (GLuc) and an anti-digoxin antibody light chain fusion with Gaussian luciferase (GLuc) expression plasmid; for fusion protein G4, the plasmids were an anti-digoxin antibody heavy chain fusion with Gaussian luciferase (GLuc) and an anti-digoxin antibody light chain fusion with Gaussian luciferase (GLuc) expression plasmid. The culture conditions were: 8% CO2, 37℃, 110 rpm, until the cell density reached 3–4 × 10⁻⁴ cells / year. 6 After achieving a cell count / mL, transfection was performed using PEI (1 mg / mL) (200 mL cell transfection system + 10 mL culture medium + 0.24 mg plasmid (total mass of heavy chain expression plasmid and light chain expression plasmid, with a mass ratio of heavy chain expression plasmid to light chain expression plasmid of 1:1) + 0.64 mL PEI); the cells were cultured until the cell viability was below 90%, and the cell supernatant was collected (8000 rpm, 5 min). After filtration through a 0.22 μm filter membrane, the protein was purified using a Protein A column. 5 μg of the purified antibody was used for protein electrophoresis purity assessment, and the results are shown in Figure 6: the protein band was single, indicating high purity.

[0346] Example 5: Detection of partial luciferase activity of fusion protein

[0347] The chemically conjugated products (N enzyme (i.e., anti-digoxigenin antibody chemically conjugated with spiny shrimp luciferase (Nluc)) and G enzyme (i.e. anti-digoxigenin antibody chemically conjugated with Gaussian luciferase (GLuc)), N enzyme and G enzyme were obtained from DNBSEQ-E25RS high-throughput sequencing kit (FCL PE150), catalog number: 940-000571-00, purchased from BGI Genomics), and the fusion proteins N2, N3, N4, G2, G3, and G4 obtained in Example 4 were diluted to 1 ng / μL with substrate dilution buffer (50 mM Tris, pH 8.0, 100 mM NaCl), and 10 μL was added to a 96-well microplate. Add 90 μL of substrate diluted to 5 μM with the same substrate diluent (wherein, the substrate for spiny shrimp luciferase (Nluc) is fluorocoelin, and the substrate for Gaussian luciferase (GLuc) is coelin), and read the luminescence intensity and luminescence mode using the ELISA reader's autoluminescence module. The results are shown in Figure 7: The fusion proteins N2, N3, N4, G2, G3, and G4 obtained in Example 4 retained the luciferase catalytic activity for substrate luminescence, and their catalytic activity for substrate luminescence was higher than that of the chemically coupled products (N enzyme, G enzyme). Furthermore, the more luciferases in the fusion protein (N4, G4), the higher its catalytic activity for substrate luminescence.

[0348] Example 6: Partial Activity Detection of Fusion Protein Antibody

[0349] The titers of the fusion proteins N2 and G2 obtained in Example 4 were determined using an ELISA assay (the method is the same as the ELISA method for identifying and screening positive cell lines in Example 1). The results are shown in Figure 10: the titers of the fusion proteins N2 and G2 reached 10. -10 The above results are consistent with those of hybridoma antibodies (i.e., anti-digoxin monoclonal antibodies purified from the supernatant of the positive cell line in Example 2), indicating that fusion expression of luciferase does not affect the activity of anti-digoxin antibodies.

[0350] Example 7: Instrumental testing of the fusion protein

[0351] The chemically conjugated products (N enzyme, G enzyme, same as in Example 5) and the fusion proteins N2, N3, N4, G2, G3, and G4 obtained in Example 4 were validated for luminescence signals on a DNBSeqE5 autoluminescent sequencer. The sequencing signal intensity and Q30 were detected, and the results are shown in Figures 8 and 9. The signal intensity and Q30 of the fusion proteins N2, N3, N4, G2, G3, and G4 obtained in Example 4 are superior to those of the chemically conjugated products (N enzyme and G enzyme) and can be used in sequencing.

[0352] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An anti-digoxin antibody or antigen-binding fragment thereof, comprising a heavy chain and a light chain: the heavy chain comprises a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region with the amino acid sequence as shown in SEQ ID NO: 2; the light chain comprises a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region with the amino acid sequence as shown in SEQ ID NO:

19. 2.An anti-digoxin antibody or antigen-binding fragment thereof, comprising a heavy chain and a light chain: the heavy chain comprises a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3; the light chain comprises a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3; the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1 and CDR-L3 are shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 21, SEQ ID NO: 22 in turn, the amino acid sequence of CDR-L2 is: KVS, and the CDR is defined according to the IMGT definition scheme; or the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 22 in turn, and the CDR is defined according to the Kabat definition scheme; or the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 22 in turn, and the CDR is defined according to the Chothia definition scheme; or the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 in turn, and the CDR is defined according to the Contact definition scheme. 3.The anti-digoxin antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region comprises: a1) SEQ ID NO: 2; or ​ a2) an amino acid sequence of SEQ ID NO: 2 having one or several amino acid substitutions and / or deletions and / or additions and having the same function as the protein of SEQ ID NO: 2; or a3) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% or more identity to SEQ ID NO: 2 and having the same function as the protein of SEQ ID NO: 2; and / or, the amino acid sequence of the light chain variable region comprises: b1) SEQ ID NO: 19; or b2) an amino acid sequence of SEQ ID NO: 19 having one or several amino acid substitutions and / or deletions and / or additions and having the same function as the protein of SEQ ID NO: 19; or b3) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% or more identity to SEQ ID NO: 19 and having the same function as the protein of SEQ ID NO: 19; Preferably, the amino acid sequence of the heavy chain comprises: A1) SEQ ID NO: 1; or A2) an amino acid sequence of SEQ ID NO: 1 having one or several amino acid substitutions and / or deletions and / or additions and having the same function as the protein of SEQ ID NO: 1; or A3) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 70%, 65%, or 60% or more identity to SEQ ID NO: 1 and having the same function as the protein of SEQ ID NO: 1; Preferably, the amino acid sequence of the light chain comprises: B1) SEQ ID NO: 18; or B2) an amino acid sequence of SEQ ID NO: 18 having one or several amino acid substitutions and / or deletions and / or additions and having the same function as the protein of SEQ ID NO: 18; or B3) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 70%, 65%, or 60% or more identity to SEQ ID NO: 18 and having the same function as the protein of SEQ ID NO:

18.

4. A recombinant protein comprising the anti-digoxin antibody or antigen binding fragment thereof of any one of claims 1-3, and optionally a tag sequence to assist expression and / or purification. ​ Preferably, the tag sequence is selected from at least one of Poly his, FLAG, Strep-Tag II, Poly arg, C-myc, HA, V5, VSV-G, Trx, SUMO, GST, MBP, and NusA.

5. A conjugate comprising: any one of the anti-digoxin antibody or antigen binding fragment thereof of any one of claims 1-3 and the recombinant protein of claim 4, and a conjugation moiety; the conjugation moiety is a detectable label.

6. The conjugate of claim 5, wherein, The detectable label comprises at least one of a metal particle, a fluorescent label, a luminescent label, an electron-dense label, a radioactive label, an enzymatic label.

7. The conjugate of claim 5 or 6, wherein, The enzymatic label comprises a luciferase; Preferably, the luciferase comprises at least one of a bacterial luciferase, a firefly luciferase, a Cypridina luciferase, a Renilla luciferase, a Gaussia luciferase, a Nanoluc luciferase, and a sea pansy luciferase.

8. A fusion protein comprising: any one of the anti-digoxin antibody or antigen binding fragment thereof of any one of claims 1-3 and the recombinant protein of claim 4, and a luciferase.

9. The fusion protein of claim 8, wherein, The luciferase is fused to a heavy chain in the anti-digoxin antibody or antigen binding fragment thereof, and / or the luciferase is fused to a light chain in the anti-digoxin antibody or antigen binding fragment thereof.

10. A biological material related to the anti-digoxin antibody or antigen binding fragment thereof of any one of claims 1-3, the recombinant protein of claim 4, the conjugate of any one of claims 5-7, or the fusion protein of claim 8 or 9, the biological material comprises j1) - j12) any one of: j1) a nucleic acid molecule encoding the anti-digoxin antibody or antigen binding fragment thereof of any one of claims 1-3, the recombinant protein of claim 4, the conjugate of any one of claims 5-7, or the fusion protein of claim 8 or 9; j2) an expression cassette comprising the nucleic acid molecule of j1); j3) a vector comprising the nucleic acid molecule of j1); j4) a vector comprising the expression cassette of j2); j5) a transgenic cell line comprising the nucleic acid molecule of j1); j6) a transgenic cell line comprising the expression cassette of j2); j7) a transgenic cell line comprising the vector of j3); j8) a transgenic cell line comprising the vector of j4); j9) a recombinant bacterium comprising the nucleic acid molecule of j1); j10) a recombinant bacterium comprising the expression cassette of j2); j11) a recombinant bacterium comprising the vector of j3); j12) a recombinant bacterium comprising the vector of j4); Preferably, the transgenic cell line in j5) - j8) does not comprise reproductive material; Preferably, the vector in j3), j4) comprises a promoter operably linked to the nucleic acid molecule; Preferably, the vector in j3), j4) is independently selected from a non-pathogenic viral vector and a non-viral vector; Preferably, the non-pathogenic viral vector comprises an adenoviral vector or a retroviral vector; Preferably, the non-viral vector comprises a plasmid vector.

11. The anti-digoxin antibody or antigen-binding fragment thereof of any one of claims 1-3, the recombinant protein of claim 4, the conjugate of any one of claims 5-7, the fusion protein of claim 8 or 9, and / or the biomaterial of claim 10 for use in any one of r1)-r4): r1) for the manufacture of a product for detecting digoxin; r2) for detecting digoxin; r3) for the manufacture of a product for nucleic acid sequencing; r4) for nucleic acid sequencing.

12. A product for detecting digoxin, comprising at least one of u1)-u4): u1) the anti-digoxin antibody or antigen-binding fragment thereof of any one of claims 1-3; u2) the recombinant protein of claim 4; u3) the conjugate of any one of claims 5-7; u4) the fusion protein of claim 8 or 9.

13. A product for nucleic acid sequencing, comprising at least one of v1)-v2): v1) the conjugate of any one of claims 5-7; v2) the fusion protein of claim 8 or 9.

14. The product of claim 12 or 13, wherein: the product for detecting digoxin is a detection reagent, a detection kit, a detection test strip, a detection plate, a detection chip, or a detection system; or the product for nucleic acid sequencing is a sequencing reagent, a sequencing kit, or a sequencing system; preferably, the product for detecting digoxin and / or the product for nucleic acid sequencing further comprises a chromogenic substrate.

15. Any one of w1)-w3): w1) a method for preparing the anti-digoxin antibody or antigen-binding fragment thereof of any one of claims 1-3, the recombinant protein of claim 4, the conjugate of any one of claims 5-7, or the fusion protein of claim 8 or 9, by culturing the transgenic cell line or recombinant bacteria described in claim 10; w2) a method for detecting digoxin, comprising the step of using the anti-digoxin antibody or antigen-binding fragment thereof of any one of claims 1-3, the recombinant protein of claim 4, the conjugate of any one of claims 5-7, the fusion protein of claim 8 or 9, and / or the product of claim 12 or 14; w3) a method for nucleic acid sequencing, comprising the step of using the conjugate of any one of claims 5-7, the fusion protein of claim 8 or 9, and / or the product of claim 13 or 14.

16. The method of claim 15, wherein: the method for detecting digoxin comprises the step of contacting the at least one of u1)-u4) described in claim 12 or 14, the anti-digoxin antibody or antigen-binding fragment thereof of any one of claims 1-3, the recombinant protein of claim 4, the conjugate of any one of claims 5-7, and / or the fusion protein of claim 8 or 9 with a sample to be tested, and detecting the formation or content of antigen-antibody immune complexes; or The nucleic acid sequencing method comprises the following steps: taking a nucleic acid to be detected as a template, reacting in a system comprising at least one of v1)-v2) in claim 13 or 14, the conjugate in any one of claims 5-7, and / or the fusion protein in claim 8 or 9, and determining the nucleic acid sequence of the nucleic acid to be detected according to the signal generated by the detectable label.

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