Anti-CA153 antibodies and detection kits

Abstract

The present invention discloses an anti-CA153 antibody and a detection kit, and relates to the field of antibody technology. The anti-CA153 antibody disclosed in the present invention comprises the heavy chain complementary determining region and the light chain complementary determining region, and provides an important raw material source for the detection of CA153, and can be used for the qualitative or quantitative detection of CA153.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application with application number 202211686897.9 filed with the China Patent Office on December 26, 2022, entitled “Anti-CA153 Antibodies or Functional Fragments thereof and Detection Kits,” the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present invention relates to the field of antibody technology, in particular to an anti-CA153 antibody and a detection kit. Background Art

[0004] Breast cancer is one of the most common malignant tumors in women. Approximately 1.2 million women worldwide are diagnosed with breast cancer each year, and approximately 500,000 die from it. In recent years, the incidence of breast cancer has been increasing year by year. Its characteristics include a relatively young age of onset, asymptomatic early stages, relatively late presentation, and metastasis, which shortens survival. Therefore, early diagnosis and treatment are key to improving patient survival.

[0005] Improving the early diagnosis of breast cancer is a common concern among researchers both domestically and internationally. Breast cancer serum markers, when used alone for early diagnosis, have limited their clinical utility due to their low sensitivity and specificity. In recent years, tumor marker testing has become increasingly used in the clinical diagnosis and treatment of malignant tumors, with a variety of tumor markers being identified for breast cancer. CA153 is currently recognized as a highly specific tumor marker for breast cancer. It is overexpressed in breast cancer, independent of pathological type, and correlates with clinical stage, tumor size, axillary lymph node status, and estrogen and progesterone receptors. CA153, a variant of a glycoprotein on the surface of breast epithelial cells, is a key marker for breast cancer. CA153 measurement can aid in treatment monitoring for breast cancer patients. Dynamic measurement aids in the early detection of recurrence after treatment in patients with stage II and III breast cancer, as well as in monitoring treatment response in patients with metastatic disease. It is the most useful marker for detecting recurrence after breast cancer surgery. In 96% of patients with local or systemic recurrence, CA153 levels significantly increase, predating radiological and clinical findings. Increased serum concentrations are also closely associated with tumor severity; conversely, decreased levels indicate significant therapeutic efficacy.

[0006] Clinical methods for detecting CA153 levels include enzyme-linked immunosorbent assay (ELISA), chemiluminescence, colloidal gold, etc. Different methods require antibodies against CA153. Therefore, there is a strong demand in this field for antibodies that can effectively bind to CA153 and detect it. Summary of the Invention

[0007] The purpose of the present invention is to provide an anti-CA153 antibody and a detection kit.

[0008] In a first aspect, an embodiment of the present invention provides an antibody comprising: a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 1 or any one or more of the following mutations are made thereto: F in F29 is mutated to T, S in S30 is mutated to K, D in D31 is mutated to S or N, Y in Y61 is mutated to H, P in P100 is mutated to M or A, and A in A109 is mutated to Y, F, or S;

[0009] The amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 2 or any one or more of the following mutations are made thereto: N in N34 is mutated to S, N in N37 is mutated to S, and Q in Q95 is mutated to P;

[0010] The heavy chain variable region and the light chain variable region have at least one of the above mutations.

[0011] In a second aspect, an embodiment of the present invention provides an antibody comprising CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3, wherein:

[0012] CDR-VH1, CDR-VH2 and CDR-VH3 include or have amino acid sequences identical to those of CDR1, CDR2 and CDR3 in the heavy chain variable region described in the preceding examples;

[0013] CDR-VL1, CDR-VL2 and CDR-VL3 include or have the same amino acid sequences as CDR1, CDR2 and CDR3 in the light chain variable region described in the previous examples.

[0014] In a third aspect, an embodiment of the present invention provides an antibody conjugate comprising the antibody described in the aforementioned embodiment.

[0015] In a fourth aspect, an embodiment of the present invention provides a reagent or a kit, which includes the antibody described in the above embodiment or the antibody conjugate described in the above embodiment.

[0016] In a fifth aspect, an embodiment of the present invention provides a method for detecting CA153, comprising: mixing the antibody described in the preceding embodiment with a sample to be detected, allowing the antibody to contact CA153 in the sample to be detected to form an immune complex.

[0017] In a sixth aspect, an embodiment of the present invention provides an isolated nucleic acid encoding the antibody described in the above embodiment.

[0018] In a seventh aspect, an embodiment of the present invention provides a vector comprising the isolated nucleic acid described in the preceding embodiment.

[0019] In an eighth aspect, an embodiment of the present invention provides a cell containing the isolated nucleic acid described in the preceding embodiment or the vector described in the preceding embodiment.

[0020] In an eighth aspect, an embodiment of the present invention provides a method for preparing the antibody described in the preceding embodiment, comprising: culturing the cells described in the preceding embodiment.

[0021] In a tenth aspect, embodiments of the present invention provide use of the antibody described in the preceding embodiments, the antibody conjugate described in the preceding embodiments, or the reagent or kit described in the preceding embodiments in detecting CA153 or preparing a product for detecting CA153.

[0022] In an eleventh aspect, an embodiment of the present invention provides a method for screening anti-CA153 antibodies, the method comprising:

[0023] a) designing primers for amino acid substitution at least one amino acid position of CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3 described in the preceding embodiments;

[0024] b) constructing a mutation library using the nucleic acid described in the preceding embodiment, the vector described in the preceding embodiment, or the cell described in the preceding embodiment as a template and the primers described in a);

[0025] c) screening anti-CA153 antibodies from the mutant library.

[0026] In a twelfth aspect, an embodiment of the present invention provides a mutant library comprising the antibodies described in the aforementioned embodiments.

[0027] The anti-CA153 antibody disclosed in the present invention comprises the heavy chain complementary determining region and the light chain complementary determining region. The antibody provides an important source of raw materials for the detection of CA153 and has improved activity or affinity. DETAILED DESCRIPTION

[0028] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0029] An embodiment of the present invention provides an antibody comprising: a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 1 or any one or more of the following mutations are made thereto: F in F29 is mutated to T, S in S30 is mutated to K, D in D31 is mutated to S or N, Y in Y61 is mutated to H, P in P100 is mutated to M or A, and A in A109 is mutated to Y, F, or S;

[0030] The amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 2 or any one or more of the following mutations are made thereto: N in N34 is mutated to S, N in N37 is mutated to S, and Q in Q95 is mutated to P;

[0031] The heavy chain variable region and the light chain variable region have at least one of the above mutations.

[0032] It should be noted that the numbering of the mutation sites herein is obtained by numbering the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 from the N-terminus to the C-terminus. For example, position 29 refers to the 29th position from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 1; and "F29T" refers to the replacement of phenylalanine at position 29 of the amino acid sequence set forth in SEQ ID NO: 1 with threonine.

[0033] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies or functional fragments, so long as they exhibit the desired antigen-binding activity.

[0034] As used herein, the terms "full-length antibody," "full-length monoclonal antibody," or "full-length monoclonal antibody" are all composed of at least two identical light chains and at least two identical heavy chains linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE). They typically include a light chain with a lighter molecular weight and a heavy chain with a heavier molecular weight, with the heavy chain (H chain) and light chain (L chain) linked by disulfide bonds to form an antibody molecule. The amino acid sequence at the amino terminus (N-terminus) of the peptide chain varies greatly and is called the variable region (V region); the carboxyl terminus (C-terminus) is relatively stable and varies little, and is called the constant region (C region). The V regions of the L chain and H chain are called VL and VH, respectively.

[0035] In this article, the terms "polyantibody" and "multispecific antibody" are synonymous, both referring to antibodies that can recognize multiple antigenic epitopes, such as antibodies that can recognize two antigenic epitopes (bispecific antibodies, abbreviated as bispecific antibodies), antibodies that recognize three antigenic epitopes, or antibodies that recognize four antigenic epitopes. This is understood in a broad sense and the specific structure is not limited, as long as it can recognize multiple antigenic epitopes. In the present invention, at least one of the multiple antigenic epitopes is derived from CA153.

[0036] As used herein, the term "functional fragment" refers to a fragment comprising part or all of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. For example, the fragment may comprise part or all of an antibody CDR. Such fragments are biologically active because they bind to an antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments include Fv fragments, disulfide-stabilized Fv fragments (dsFv), F(ab')2 fragments, Fab' fragments, Fab fragments, F(ab)2 fragments, scFv fragments, scFv-Fc fusion proteins, scFv-Fv fusion proteins, Fv-Fc fusion proteins, multispecific antibodies formed by functional fragments, single domain antibodies, VHH nanobodies, domain antibodies, bivalent domain antibodies, or at least one of the minimum recognition units. Such fragments can be produced by recombinant nucleic acid technology, or can be produced by enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0037] As used herein, the term "at least 80% homology" refers to at least 80%, and may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology to each reference sequence. The term "at least 90% homology" refers to at least 90%, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% homology to each reference sequence.

[0038] Optionally, the heavy chain variable region and the light chain variable region are selected from any one of the following mutation combinations 1 to 65:

[0039]

[0040]

[0041] The amino acid sequence of the wild-type WT heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the wild-type WT light chain variable region is shown in SEQ ID NO: 2;

[0042] In some embodiments, the heavy chain variable region and the light chain variable region are selected from any one of mutation combinations 1 to 52;

[0043] In some embodiments, the heavy chain variable region and the light chain variable region are selected from any one of mutation combinations 2-3, 6-9, 11, 16-22, 24-29, 31-34, 37-38, 40, 42-45, 47, 49-50, and 52;

[0044] In some embodiments, the heavy chain variable region and the light chain variable region are selected from any one of mutation combinations 3, 7, 9, 19, 25, 29, 34, 38, 40, 42, and 52;

[0045] In some embodiments, the heavy chain variable region and the light chain variable region are selected from any one of mutation combinations 9, 19, 34, and 40.

[0046] On the other hand, an embodiment of the present invention further provides an antibody comprising CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3, wherein:

[0047] CDR-VH1, CDR-VH2 and CDR-VH3 include or have amino acid sequences identical to CDR1, CDR2 and CDR3 in the heavy chain variable region of any of the preceding embodiments;

[0048] CDR-VL1, CDR-VL2 and CDR-VL3 include or have the same amino acid sequences as CDR1, CDR2 and CDR3 in the light chain variable region described in any of the preceding embodiments.

[0049] As used herein, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and include one or more, or even all, of the amino acid residues that contribute substantially to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present invention, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[0050] In the present invention, the heavy chain complementary determining region is represented by CDR-VH, and the three CDRs contained in the heavy chain variable region include CDR-VH1 (HCDR1), CDR-VH2 (HCDR2) and CDR-VH3 (HCDR3); the light chain complementary determining region is represented by CDR-, and the three CDRs contained in the light chain variable region include CDR-VL1 (LCDR1), CDR-VL2 (LCDR2) and CDR-VL3 (LCDR3).

[0051] Commonly used CDR numbering schemes in the art include: Kabat numbering, Chothia numbering, IMGT numbering, Chothia, Martin numbering and AHo, Lesk numbering. CDR definition schemes include: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition. As used herein, "Kabat numbering" and "Kabat definition" refer to the numbering and definition system described in Kabat et al., US Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" refers to Chothia et al., J Mol Biol 196: 901-917 (1987). Exemplary defined CDRs are listed in Table 1 below. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues comprise a particular CDR.

[0052] Table 1: CDR Definition 1

[0053] CDR Kabat <![CDATA[AbM 2 ]]> IMGT HCDR1 31-35 26-35 26-35 HCDR2 50-65 50-58 51-56 HCDR3 95-102 95-102 93-102 LCDR1 24-34 24-34 27-32 LCDR2 50-56 50-56 50-51 LCDR3 89-97 89-97 89-97

[0054] 1 The numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below).

[0055] "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0056] The polypeptide sequences in the sequence listing are not numbered according to the Kabat numbering system. However, one of ordinary skill in the art is fully capable of converting the sequence numbers in the sequence listing into Kabat numbers.

[0057] In some embodiments, CDR1 to CDR3 in the heavy chain variable region and / or the light chain variable region are defined by any one of Kabat, Chothia, AbM, Contact, and IMGT, or a combination of multiple schemes.

[0058] In some embodiments, CDR-VH1, CDR-VH2, and CDR-VH3 include or are, respectively, the amino acid sequences of positions 26-35, 50-65, and 94-102 of the heavy chain variable region under Kabat numbering; and CDR-VL1, CDR-VL2, and CDR-VL3 include or are, respectively, the amino acid sequences of positions 24-34, 50-56, and 89-97 of the light chain variable region under Kabat numbering. It should be noted that CDRs defined by methods other than those in Table 1 also fall within the scope of protection of the present disclosure.

[0059] In some embodiments, the amino acid sequences of CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3 are as follows:

[0060] CDR-VH1: GFT-X1-X2-X3-VWMD;

[0061] CDR-VH2:EIRTKPNNHAT-X4-YAESVKG;

[0062] CDR-VH3: X5-HGNYYVGF-X6-Y;

[0063] CDR-VL1:KSSQSLLNSGX7QKX8YLT;

[0064] CDR-VL2:GASIRES;

[0065] CDR-VL3: X9HNHGSFIPYT;

[0066] Among them, X1 is F or T, X2 is S or K, X3 is D, S or N; X4 is Y or H; X5 is P, M or A, X6 is A, Y, F or S; X7 is N or S, X8 is N or S; X9 is Q or P, and X1 to X9 exclude the combination of X1 / X2 / X3 / X4 / X5 / X6 / X7 / X8 / X9 being F / S / D / Y / P / A / N / N / Q.

[0067] In some embodiments, X1-X9 are selected from any one of the following combinations 1-65:

[0068]

[0069]

[0070] In some embodiments, X1-X9 are selected from any one of mutation combinations 1-52.

[0071] In some embodiments, X1-X9 is selected from any one of mutation combinations 2-3, 6-9, 11, 16-22, 24-29, 31-34, 37-38, 40, 42-45, 47, 49-50 and 52.

[0072] In some embodiments, X1 to X9 are selected from any one of mutation combinations 3, 7, 9, 19, 25, 29, 34, 38, 40, 42, and 52.

[0073] In some embodiments, X1 to X9 are selected from any one of mutation combinations 9, 19, 34, and 40.

[0074] In some embodiments, the heavy chain variable region and light chain variable region of the antibody further include a framework region (FR region).

[0075] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region of the antibody heavy chain variable region and the light chain variable region excluding CDR; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0076] In some embodiments, the antibody further comprises at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4, wherein at least a portion of at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 is derived from any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, and humans.

[0077] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0078] In some embodiments, the HFR1 comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 80% homology thereto;

[0079] The HFR2 comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 80% homology thereto;

[0080] The HFR3 comprises the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having at least 80% homology thereto;

[0081] The HFR4 comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 80% homology thereto;

[0082] The LFR1 comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 80% homology thereto;

[0083] The LFR2 comprises the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 80% homology thereto;

[0084] The LFR3 comprises the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 80% homology thereto;

[0085] The LFR4 comprises the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% homology thereto;

[0086] In some embodiments, the antibody comprises the heavy chain variable region and / or light chain variable region of any of the preceding embodiments.

[0087] In some embodiments, the antibody further comprises a constant region, wherein the constant region comprises at least one of a heavy chain constant region and a light chain constant region.

[0088] In some embodiments, the species origin of the constant region is any one of cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose and human.

[0089] In some embodiments, the species origin of the constant region is mouse.

[0090] In some embodiments, the heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

[0091] In some embodiments, the light chain constant region comprises a light chain constant region selected from a kappa-type or a lambda-type light chain constant region.

[0092] In some embodiments, the heavy chain constant region sequence is as shown in SEQ ID NO: 11 or is at least 80% identical thereto.

[0093] In some embodiments, the light chain constant region sequence is as shown in SEQ ID NO: 12 or is at least 80% identical thereto.

[0094] On the other hand, an embodiment of the present invention further provides an antibody conjugate, which includes the antibody described in any of the aforementioned embodiments.

[0095] In some embodiments, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody.

[0096] In some embodiments, the antibody conjugate further comprises a solid phase support coupled to the antibody. In the antibody conjugate, the antibody is coupled to the solid phase support.

[0097] In an alternative embodiment, the solid support is selected from microspheres, plates, and membranes.

[0098] In an optional embodiment, the solid phase includes but is not limited to magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0099] In an optional embodiment, the solid phase carrier is a nitrocellulose membrane.

[0100] In some embodiments, the antibody conjugate further comprises a label conjugated to the antibody.

[0101] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0102] In some embodiments, the label is selected from at least one of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle label.

[0103] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the scope of protection of the present invention.

[0104] In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0105] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxidase.

[0106] In an optional embodiment, the radioactive isotopes include but are not limited to 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.

[0107] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

[0108] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0109] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal selenium, disperse dyes, dye-labeled microspheres, and latex.

[0110] In an optional embodiment, the colloidal metal includes but is not limited to colloidal gold or colloidal silver.

[0111] In an optional embodiment, the colloidal metal is colloidal gold.

[0112] On the other hand, an embodiment of the present invention further provides a reagent or kit, which includes the antibody described in any of the foregoing embodiments or the antibody conjugate described in any of the foregoing embodiments.

[0113] As previously described, the antibodies in some embodiments or examples of the present invention are capable of effectively binding to CA153. Therefore, reagents or kits containing such antibodies are capable of effectively performing qualitative or quantitative detection of CA153. The reagents or kits provided by the present invention can be used, for example, in immunoblotting, immunoprecipitation, and other assays that utilize the specific binding properties of CA153 and its antibodies. As previously described, the antibodies in some embodiments or examples of the present invention have higher binding activity or affinity for CA153. Therefore, reagents or kits containing such antibodies have higher detection sensitivity or specificity.

[0114] On the other hand, an embodiment of the present invention further provides a method for detecting CA153, comprising:

[0115] The antibody described in any of the above embodiments is mixed with a sample to be tested, so that the antibody contacts CA153 in the sample to be tested to form an immune complex.

[0116] In some embodiments, whether the sample to be tested contains CA153 or the content of CA153 is determined based on the signal of the immune complex.

[0117] In some embodiments, the immune complex further comprises a second antibody that binds to the antibody.

[0118] In some embodiments, the immune complex further comprises a second antibody that binds to CA153.

[0119] On the other hand, an embodiment of the present invention further provides an isolated nucleic acid encoding the antibody described in any of the above embodiments.

[0120] On the other hand, an embodiment of the present invention further provides a vector comprising the isolated nucleic acid described in any of the aforementioned embodiments.

[0121] On the other hand, an embodiment of the present invention further provides a cell comprising the isolated nucleic acid described in any of the foregoing embodiments or the vector described in any of the foregoing embodiments.

[0122] On the other hand, an embodiment of the present invention further provides a method for preparing the antibody described in any of the aforementioned embodiments, comprising: culturing the cell described in any of the aforementioned embodiments.

[0123] On the other hand, embodiments of the present invention further provide use of the antibody as described in any of the foregoing embodiments, the antibody conjugate as described in any of the foregoing embodiments, or the reagent or kit as described in any of the foregoing embodiments in detecting CA153 or preparing a product for detecting CA153.

[0124] On the other hand, an embodiment of the present invention further provides the use of the antibody described in any of the preceding embodiments, or the antibody conjugate described in any of the preceding embodiments, or the reagent or kit described in any of the preceding embodiments in the preparation of a product having at least one of the following uses, wherein the uses include: diagnosing or assisting in diagnosing CA153 metabolic-related diseases, and predicting or assisting in predicting at least one of the prognosis and efficacy of CA153 metabolic-related diseases.

[0125] In an optional embodiment, the CA153 metabolism-related disease includes at least one of breast cancer, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, cervical cancer, primary liver cancer, and metastatic cancer. The metastatic cancer includes at least one of metastatic ovarian cancer, metastatic colon cancer, metastatic liver cancer, metastatic bile duct cancer, metastatic pancreatic cancer, metastatic lung cancer, and metastatic bronchogenic carcinoma.

[0126] In an alternative embodiment, the product comprises a reagent or a kit.

[0127] Based on the amino acid sequence of the antibody disclosed in the present invention, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody. For example, the antibody can be isolated and purified from the culture product of recombinant cells that can recombinantly express the antibody as described in any of the above items. This is easy for those skilled in the art to achieve. Based on this, regardless of the technology used to prepare the antibody of the present invention, it falls within the scope of protection of the present invention.

[0128] On the other hand, an embodiment of the present invention further provides a method for screening anti-CA153 antibodies, the method comprising:

[0129] a) designing primers for amino acid substitution at least one amino acid position of CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3 described in any of the preceding embodiments;

[0130] b) constructing a mutation library using the nucleic acid described in any of the preceding embodiments, the vector described in any of the preceding embodiments, or the cell described in any of the preceding embodiments as a template and the primers described in a);

[0131] c) screening anti-CA153 antibodies from the mutant library.

[0132] Optionally, the primers are used to perform amino acid substitutions at 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the F29, S30, D31, Y61, P100, A109, N34, N37 and Q95 sites described in any of the preceding embodiments.

[0133] In some embodiments, the mutation library is a single-site saturation mutation library.

[0134] In addition, an embodiment of the present invention further provides a mutation library comprising the antibody described in any of the aforementioned embodiments.

[0135] In some embodiments, the mutation library is constructed by the steps of constructing a mutation library described in any of the preceding embodiments.

[0136] The amino acid sequences or nucleotide sequences involved herein are shown in the following table, wherein the mutation sites of the heavy chain variable region or HCDRs are based on the amino acid sequence shown in SEQ ID NO: 1 or the HCDRs in SEQ ID NO: 1, and the mutation sites of the light chain variable region or LCDRs are based on the amino acid sequence shown in SEQ ID NO: 2 or the LCDRs in SEQ ID NO: 2. WT indicates no mutation:

[0137] Amino acid sequence listing

[0138]

[0139]

[0140] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0141] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only.

[0142] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0143] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0144] In this example, restriction endonucleases, T4 DNA ligase, and DNA polymerase were purchased from New England Biolabs, Taq DNA polymerase was purchased from TaKaRa, the V01 expression vector was constructed in our laboratory, gel recovery kits and plasmid extraction kits were commercially available, and primer synthesis and gene sequencing were performed by a contract company. The sequence of the CA153 monoclonal antibody (hereinafter referred to as WT antibody) was derived from mouse hybridoma cell sequencing.

[0145] Example 1: Construction and screening of affinity maturation mutation library

[0146] 1. Construction of WT template plasmid

[0147] (1) Antibody gene synthesis

[0148] The VH and VL sequences of the WT antibody sequence were codon-optimized in E. coli, and the antibody gene sequence was then outsourced to a company for gene synthesis. The VH and VL amino acid sequences of the WT antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0149] (2) WT antibody gene fragment amplification

[0150] The synthesized antibody sequence is amplified by PCR using DNA polymerase, and then the antibody bands are separated by agarose gel electrophoresis, and then purified using a gel recovery kit to obtain the antibody gene fragment.

[0151] (3) Enzymatic digestion and ligation of WT antibody gene fragments

[0152] The antibody gene fragment and V01 vector plasmid were simultaneously double-digested with restriction endonucleases, and then purified using a gel recovery kit to obtain the antibody gene fragment with sticky ends and the V01 vector. Subsequently, the antibody gene fragment and the V01 vector were ligated with T4 DNA ligase at 22°C for 4 hours. The ligation reaction product was recovered and purified, and the DNA concentration was determined. Finally, 100 ng of the plasmid was transformed into 100 μl of TG1 competent Escherichia coli, and the entire bacterial liquid was spread on a plate containing ampicillin resistance and cultured at 37°C overnight.

[0153] (4) Extraction and sequencing verification of WT template plasmid

[0154] The next day, 10 monoclonal colonies were selected and tested by colony PCR and gel electrophoresis using Taq DNA polymerase. The bacteria with the correct insertion of the antibody gene sequence were selected for culture and amplification. The WT template plasmid was obtained using a plasmid extraction kit and sent to a sequencing company for gene sequencing verification.

[0155] 2. Construction of single-point mutation library

[0156] (1) Primer design and synthesis

[0157] By using the degenerate base codons, 75 pairs of upstream and downstream primers for single-point saturation mutagenesis of the entire CDR region of VH and VL (75 amino acid sites) were designed and handed over to an outsourcing company for primer synthesis.

[0158] (2) Single-point saturation mutation plasmid PCR amplification

[0159] The PCR method was used, and the reaction system was configured according to Table 2. The PCR reaction conditions in Table 3 were then used to amplify and prepare the single-point saturation mutation library plasmid. Finally, the WT template plasmid was digested with restriction endonucleases at 37°C for 1 hour to obtain 75 mutation library plasmids.

[0160] Table 2: PCR system

[0161] WT template plasmid 50ng DNA polymerase 1 μl DNA polymerase buffer 10 μl dNTP (2.5 mM) 4 μl Upstream primer (10uM) 1 μl Downstream primer (10uM) 1 μl <![CDATA[ddH2O]]> Make up to 50 μl

[0162] Table 3: PCR reaction conditions

[0163] Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 temperature 95℃ 95℃ 55-60℃ 72℃ 72℃ 4℃ time 5min 30s 30s 2min 5min ∞

[0164] Step2 to Step4: 22 cycles.

[0165] (3) Single-site saturation mutagenesis plasmid transformation

[0166] Take 10 μl of the reaction product and transform it into 100 μl of TG1 competent E. coli. Spread the entire bacterial solution on a plate containing ampicillin resistance and culture at 37°C overnight.

[0167] 3. Screening of single-point mutation libraries

[0168] (1) Antibody expression from mutant library

[0169] The next day, 500 μl of culture medium was added to a 96-well culture plate in advance. 92 monoclonal colonies were selected for each single-point mutation library, and WT, negative, and blank control colonies were set up. After culturing at 37°C for 5-6 hours, the bacterial solution was transferred to a new 96-well culture plate. Then, the culture was carried out at 37°C for 1-2 hours. Finally, induction medium was added and the antibodies were expressed at 37°C overnight to obtain the antibody expression supernatant of 75 mutation libraries.

[0170] (2) Mutation library screening and sequencing

[0171] Using the double antibody sandwich method, CA153 antibody was added to 75 ELISA plates at 1 μg / ml and 100 μl / well, and the plates were coated overnight at 4°C. The next day, the plates were blocked with 1% to 2% skim milk powder, and the antibody expression supernatants of 75 mutation libraries were added to the ELISA plate wells at 100 μl / well. WT, negative, and blank controls were set up. After incubation at room temperature for 1 hour, ascites containing CA153 was added to the ELISA plate wells at a dilution of 1:800 and 100 μl / well, and incubated at room temperature for 1 hour. Then, HRP-labeled paired antibodies were added, and conventional ELISA detection methods were used for subsequent plate washing, color development, and reading. Subsequently, the data results were sorted and analyzed, and the improved clones were sent for sequencing. Finally, the sequencing results were analyzed, and the mutation sites of 13 unique mutation candidate clones (see Table 4) were selected for combinatorial mutation library construction. (Ratio value explanation: The Ratio value represents the degree of affinity improvement. When the Ratio value is equal to 1, it means that the affinity of the mutant clone is the same as that of the WT; when the Ratio value is greater than 1, it means that the affinity has improved).

[0172] Table 4: Screening results and mutation sites of candidate clones

[0173]

[0174]

[0175] 4. Construction of combinatorial mutation library

[0176] (1) Library primer design and synthesis

[0177] Based on the mutation sites on VH and VL, amplification primers for the combinatorial mutation library are designed and handed over to an outsourcing company for primer synthesis.

[0178] (2) Fragment amplification and ligation

[0179] The antibody mutant fragments were amplified according to the PCR system in Table 2 and the PCR reaction conditions in Table 3. The antibody mutant fragments were then recovered from the gel. Overlap PCR was used to splice the antibody mutant fragments into complete antibody fragments.

[0180] Finally, the antibody fragment was inserted into the V01 vector using enzyme digestion and ligation to form a complete antibody expression plasmid. The method is the same as for "WT Antibody Gene Fragment Enzyme Digestion and Ligation." 100 ng of plasmid was transformed into 100 μl of competent TG1 E. coli. The entire suspension was plated onto an ampicillin-resistant plate and cultured overnight at 37°C.

[0181] 5. Screening of combinatorial mutation libraries

[0182] 52 combined mutant antibodies were randomly selected for supernatant expression, ELISA screening, and positive clone sequencing analysis. The Ratio values ​​are shown in Table 5.

[0183] Table 5: Information of candidate clones for combined mutation

[0184]

[0185]

[0186]

[0187] Example 2: Verification of eukaryotic recombinant expression of antibodies

[0188] 1. Construction of eukaryotic recombinant expression plasmid

[0189] pcDNA TM 3.4 The vector is a recombinant antibody eukaryotic expression vector constructed using a vector having multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the variable region gene sequences of the 52 candidate clones obtained by screening the combinatorial mutation library (see Table 5), VL and VH gene-specific amplification primers and constant region overlap primers (the amino acid sequences of the heavy chain constant region and the light chain constant region are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively, wherein the N-terminus of the heavy chain constant region is connected to the C-terminus of VH, representing the heavy chain, and the N-terminus of the light chain constant region is connected to the C-terminus of VL, representing the light chain) of the corresponding antibody sequences were designed. The primers at both ends contained HindIII and EcoRI restriction sites and protective bases, respectively. A 0.73 KB light chain gene fragment and a 1.40 kb heavy chain gene fragment were amplified by PCR.

[0190] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was digested with HindIII / EcoRI. After purification and recovery of the fragments and vector, the Heavy Chain and Light Chain genes were ligated into the 3.4A expression vector and transformed into DH5α competent cells. After colonies grew, single colonies were picked for PCR identification of positive clones. Positive clones were sequenced to confirm sequence accuracy. Correctly sequenced clones were selected for plasmid extraction and set aside.

[0191] 2. Sample preparation of recombinant antibodies

[0192] Resuscitate HEK293 cells in advance and subculture them into 200 ml system to make the cell density reach 3-5×10 6 cells / ml, cell viability>95%; wash the cells by centrifugation, resolubilize with culture medium, and adjust the cell density to 2.9×10 6 cells / ml as the cell diluent. Prepare plasmid DNA and transfection reagent diluents separately using culture medium. Add the transfection reagent diluent to the plasmid DNA diluent, mix thoroughly, and let stand at room temperature for 15 minutes. Slowly add this mixture to the cell diluent over 1 minute, mix thoroughly, and then sample and count. Record and observe the viability of the cells after transfection. Place the cells in a 35°C incubator at 120 rpm and 8% CO2. After 13 days, collect the samples by centrifugation. Affinity purification of the antibodies was performed using a protein A affinity chromatography column.

[0193] 3. Affinity analysis

[0194] The wild-type antibody (the amino acid sequence of the heavy chain is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain is shown in SEQ ID NO: 14), the 13 single-site mutated candidate cloned antibodies obtained in step 3 of Example 1, and the 52 mutant antibodies obtained in step 2 of Example 2 were subjected to affinity detection analysis. The specific steps are: the binding and dissociation curves of the antigen and antibody are tested on the Biacore8K+ device, and the instrument automatically fits to obtain the affinity constant, association rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., the affinity constant. The smaller the KD value, the higher the affinity; ka represents the association rate; kd represents the dissociation rate). The results show that the mutant antibodies have an improved affinity for CA153 than the wild type. The affinity test results of the exemplary displayed antibodies are shown in Table 6 below:

[0195] Table 6 Antibody affinity test results

[0196]

[0197]

[0198] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An anti-CA153 antibody, characterized in that It includes: A heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 1 or any one or more of the following mutations are added thereto: F in F29 is mutated to T, S in S30 is mutated to K, D in D31 is mutated to S or N, Y in Y61 is mutated to H, P in P100 is mutated to M or A, and A in A109 is mutated to Y, F, or S; The amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 2 or any one or more of the following mutations are made thereto: N in N34 is mutated to S, N in N37 is mutated to S, and Q in Q95 is mutated to P; The heavy chain variable region and the light chain variable region are selected from any one of the following mutation combinations 1 to 65: The amino acid sequence of the wild-type WT heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the wild-type WT light chain variable region is shown in SEQ ID NO:

2. The mutation sites are numbered by numbering the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 from N-terminus to C-terminus.

2. The antibody according to claim 1, characterized in that The heavy chain variable region and the light chain variable region are selected from any one of mutation combinations 1 to 52.

3. The antibody according to claim 1, characterized in that The heavy chain variable region and the light chain variable region are selected from any one of mutation combinations 2-3, 6-9, 11, 16-22, 24-29, 31-34, 37-38, 40, 42-45, 47, 49-50 and 52.

4. The antibody according to claim 1, characterized in that The heavy chain variable region and the light chain variable region are selected from any one of mutation combinations 3, 7, 9, 19, 25, 29, 34, 38, 40, 42 and 52.

5. The antibody according to claim 1, characterized in that The heavy chain variable region and the light chain variable region are selected from any one of mutation combinations 9, 19, 34 and 40.

6. An anti-CA153 antibody, characterized in that It includes CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3, wherein: CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3 are, in order, the amino acid sequences identical to those of CDR1, CDR2 and CDR3 in the heavy chain variable region and CDR1, CDR2 and CDR3 in the light chain variable region in the same mutation combination of claim 1; The CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3 are defined by any one of the schemes of Kabat, Chothia, AbM, Contact and IMGT.

7. An anti-CA153 antibody, characterized in that It includes CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3. The amino acid sequences of CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3 are as follows: CDR-VH1: GFT-X1-X2-X3-VWMD; CDR-VH2:EIRTKPNNHAT-X4-YAESVKG; CDR-VH3: X5-HGNYYVGF-X6-Y; CDR-VL1:KSSQSLLNSGX7QKX8YLT; CDR-VL2:GASIRES; CDR-VL3: X9HNHGSFIPYT; X1~X9 are selected from any one of the following combinations 1~65:

8. The antibody according to claim 7, characterized in that The X1-X9 are selected from any one of mutation combinations 1-52.

9. The antibody according to claim 7, characterized in that The X1-X9 are selected from any one of mutation combinations 2-3, 6-9, 11, 16-22, 24-29, 31-34, 37-38, 40, 42-45, 47, 49-50 and 52.

10. The antibody according to claim 7, characterized in that The X1-X9 are selected from any one of mutation combinations 3, 7, 9, 19, 25, 29, 34, 38, 40, 42 and 52.

11. The antibody according to claim 7, characterized in that The X1-X9 are selected from any one of mutation combinations 9, 19, 34 and 40.

12. The antibody according to any one of claims 1 to 11, characterized in that The antibodies also include HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4; Wherein, at least a portion of at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 comes from any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese and humans.

13. The antibody according to claim 12, characterized in that The HFR1 comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 80% homology thereto; The HFR2 comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 80% homology thereto; The HFR3 comprises the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having at least 80% homology thereto; The HFR4 comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 80% homology thereto; The LFR1 comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 80% homology thereto; The LFR2 comprises the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 80% homology thereto; The LFR3 comprises the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 80% homology thereto; The LFR4 comprises the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% homology thereto.

14. The antibody according to any one of claims 1 to 11, characterized in that The antibody further comprises a constant region; wherein the constant region comprises at least one of a heavy chain constant region and a light chain constant region.

15. The antibody according to claim 14, characterized in that The species origin of the constant region is any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese and humans.

16. The antibody according to claim 15, characterized in that The species origin of the constant region is mouse.

17. The antibody according to claim 14, characterized in that The heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

18. The antibody according to claim 14, characterized in that The light chain constant region comprises a light chain constant region selected from a kappa type or a lambda type.

19. The antibody according to claim 14, characterized in that The heavy chain constant region sequence is as shown in SEQ ID NO: 11 or has at least 80% identity thereto; the light chain constant region sequence is as shown in SEQ ID NO: 12 or has at least 80% identity thereto.

20. An antibody conjugate, characterized in that It comprises the antibody according to any one of claims 1 to 19; the antibody conjugate further comprises biotin, a solid phase carrier or a label coupled to the antibody.

21. The antibody conjugate according to claim 20, characterized in that The label is selected from at least one of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

22. The antibody conjugate according to claim 20, characterized in that The label is colloidal gold.

23. A reagent or kit, characterized in that It comprises the antibody according to any one of claims 1 to 19 or the antibody conjugate according to any one of claims 20 to 22.

24. Use of the antibody according to any one of claims 1 to 19, or the antibody conjugate according to any one of claims 20 to 22, or the reagent or kit according to claim 23 in the preparation of a product for detecting CA153, characterized in that: It includes: The antibody according to any one of claims 1 to 19 is mixed with a sample to be tested, so that the antibody contacts CA153 in the sample to be tested to form an immune complex.

25. The use according to claim 24, characterized in that Based on the signal of the immune complex, it is determined whether the sample to be tested contains CA153 or the content of CA153.

26. The use according to claim 24, characterized in that The immune complex further includes a second antibody that binds to the antibody.

27. The use according to claim 24, characterized in that The immune complex further comprises a second antibody that binds to CA153.

28. An isolated nucleic acid, characterized in that The nucleic acid encodes the antibody according to any one of claims 1 to 19.

29. A carrier, characterized in that The vector contains the isolated nucleic acid of claim 28.

30. A cell, characterized in that The cell contains the isolated nucleic acid of claim 28 or the vector of claim 29.

31. A method for preparing the antibody according to any one of claims 1 to 19, characterized in that: The method comprises: culturing the cell of claim 30.

32. Use of the antibody according to any one of claims 1 to 19, the antibody conjugate according to any one of claims 20 to 22, or the reagent or kit according to claim 23 in preparing a product for detecting CA153 or detecting CA153 for non-disease diagnosis purposes.

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