Abnormal prothrombin antibody or functional fragment thereof and application thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody technology, in particular to an antibody against abnormal prothrombin and its application. BACKGROUND

[0002] Abnormal prothrombin (PIVKA-II protein, full name Protein Induced by Vitamin K Absence or Antagonist-II) is an abnormal protein produced in the presence of vitamin K deficiency or antagonists. It is also known as des-gamma-carboxy prothrombin (DCP), which is a variant form of prothrombin. I will introduce its structure, in vivo effect, and the significance and method of clinical detection in detail.

[0003] PIVKA-II is a variant form of prothrombin (factor II). Under normal circumstances, when factor II is synthesized in the liver, a specific glutamic acid residue in its amino acid sequence will be carboxylated into gamma-carboxyglutamic acid (gamma-carboxyglutamic acid, gamma-carboxy-Glu or Gla). This process requires vitamin K as a cofactor. In the absence of vitamin K or in the presence of antagonists, part of the glutamic acid residues of factor II cannot be carboxylated, resulting in a protein that loses its normal biological function and is called PIVKA-II.

[0004] Under normal physiological conditions, prothrombin participates in the blood clotting process and is activated into active thrombin, which promotes the formation of fibrin, thereby achieving hemostatic function. PIVKA-II loses its normal coagulation activity due to partial or complete carboxylation of glutamic acid residues.

[0005] The detection of PIVKA-II is mainly used in the screening and monitoring of hepatocellular carcinoma (HCC) in clinical practice. Studies have shown that the level of PIVKA-II in HCC patients is significantly higher than that in non-HCC liver disease patients and other healthy individuals. PIVKA-II has a high correlation with the development of hepatocellular carcinoma and can be used as a valuable serological marker. PIVKA-II has higher specificity than traditional liver cancer marker alpha-fetoprotein (AFP) in some cases, and combined detection with AFP can improve the diagnostic accuracy of HCC.

[0006] The detection of PIVKA-II is usually carried out by immunological methods, including but not limited to:

[0007] · Radioimmunoassay (RIA)

[0008] · Enzyme-linked immunosorbent assay (ELISA)

[0009] Chemiluminescent immunoassay (CLIA)

[0010] These detection methods primarily work by identifying and quantifying the PIVKA-II protein in blood samples. A specific threshold is typically used as a diagnostic criterion, such as 40 mAU / mL, to determine the risk of hepatocellular carcinoma (HCC). In summary, PIVKA-II is an abnormal prothrombin, and due to its high expression in HCC patients, it has become an important auxiliary biomarker for diagnosing HCC. Detecting PIVKA-II levels in the blood can improve the accuracy of early diagnosis and is also significant for monitoring disease progression and evaluating treatment effectiveness. This patent mainly describes an antibody that specifically recognizes PIVKA-II for its clinical detection. Specific Implementation

[0011] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0012] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. The methods and materials described herein, and any similar or equivalent methods and materials, may be used in the practice or testing of formulations or unit doses described herein. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0014] As used herein, the terms “comprising,” “including,” “having,” “may,” and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional actions or structures.

[0015] In a first aspect, embodiments of the present invention provide an antibody or antigen-binding fragment thereof against an abnormal prothrombin, wherein the antibody or antigen-binding fragment thereof comprises a complementarity-determining region of any one of the following groups:

[0016] (1) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are represented in sequence by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18;

[0017] (2) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are represented in sequence by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 18;

[0018] (3) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are represented in sequence by SEQ ID NO: 13, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24;

[0019] (4) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are represented in sequence by SEQ ID NO: 19, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 17, SEQ ID NO: 18;

[0020] (5) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are represented in sequence by SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 18;

[0021] (6) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are represented in sequence by SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24.

[0022] It is noted that HCDR1, HCDR2 and HCDR3 are amino acid sequences identical to HCDR1, HCDR2, HCDR3 of the same heavy chain variable region defined in the antibody or antigen-binding fragment thereof of the first aspect, and LCDR1, LCDR2 and LCDR3 are amino acid sequences identical to LCDR1, LCDR2, LCDR3 of the same light chain variable region defined in the antibody or antigen-binding fragment thereof of the first aspect.

[0023] In the present application, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, as long as they exhibit the desired biological activity.

[0024] In the present application, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable regions of the heavy and light chains of immunoglobulins, referring to the regions that contain one or more, or even all, of the amino acid residues that play a major role in the binding of the antibody or antigen-binding fragment to its recognized antigen or epitope. In the detailed description of the application, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0025] In the present application, the heavy chain complementarity determining regions are denoted as HCDR, which includes HCDR1, HCDR2 and HCDR3; and the light chain complementarity determining regions are denoted as LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0026] Methods for defining CDRs are well known in the art, and include the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition and the AbM definition. As described herein, the "Kabat definition" refers to the definition system described in Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" is described in Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods can not strictly follow one of the above schemes, but will still overlap at least a portion of the CDR regions defined by the Kabat definition, although they can be shortened or lengthened according to predictions or experimental results for particular residues or groups of residues.

[0027] According to embodiments of the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 are defined by any one or a combination of Kabat, Chothia, IMGT, AbM or Contact systems, preferably by the Kabat system.

[0028] In the present application, the "framework region" or "FR" region, which includes the heavy chain framework region and the light chain framework region, refers to the region of the antibody heavy chain variable region and the light chain variable region except the CDR; wherein the heavy chain framework region can be further subdivided into regions adjacent to the CDRs, designated HFR1, HFR2, HFR3, and HFR4 framework regions; the light chain framework region can be further subdivided into regions adjacent to the CDRs, designated LFR1, LFR2, LFR3, and LFR4 framework regions.

[0029] In the present application, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination:

[0030] LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0031] In some specific embodiments, the antibody or antigen binding fragment thereof of the present application further has framework regions HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4;

[0032] The HFR1 comprises SEQ ID NO: 3 or an amino acid sequence having at least 80% identity thereto;

[0033] The HFR2 comprises SEQ ID NO: 4 or an amino acid sequence having at least 80% identity thereto;

[0034] The HFR3 comprises SEQ ID NO: 5 or an amino acid sequence having at least 80% identity thereto;

[0035] The HFR4 comprises SEQ ID NO: 6 or an amino acid sequence having at least 80% identity thereto;

[0036] The LFR1 comprises SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto;

[0037] The LFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto;

[0038] The LFR3 comprises SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto;

[0039] The LFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto.

[0040] It should be noted that in other embodiments, the amino acid sequences of each framework region of the antibody or antigen-binding fragment thereof to abnormal prothrombin provided by the present application can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the corresponding framework region (SEQ ID NO:XX) described above.

[0041] In a second aspect, the embodiments of the present application provide an antibody or antigen-binding fragment thereof to abnormal prothrombin, comprising a heavy chain variable region and / or a light chain variable region, the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 1, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33; the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 2, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34.

[0042] In an optional implementation, the antibody or antigen-binding fragment thereof of the first aspect and the second aspect described above further comprises a constant region.

[0043] In an optional implementation, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0044] In an optional implementation, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments.

[0045] In an optional implementation, the heavy chain constant region comprises CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0046] In an optional implementation, the IgG is selected from IgG1, IgG2, IgG3, or IgG4.

[0047] In an optional implementation, the light chain constant region is selected from kappa type or lambda type light chain constant region.

[0048] In an optional implementation, the species origin of the constant region is bovine, equine, dairy cattle, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, cockerel, or human.

[0049] In an optional implementation, the species source of the constant region is mice.

[0050] In an optional embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:11, and the light chain constant region sequence (CL) is as shown in SEQ ID NO:12.

[0051] It should be noted that, in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the aforementioned constant regions (SEQ ID NO: 11-12).

[0052] In an optional embodiment, the antigen-binding fragment is selected from any one of the antibody's F(ab)2, F(ab')2, Fab', Fab, Fv, and scFv.

[0053] The antigen-binding fragments of the aforementioned antibodies typically possess the same binding specificity as the source antibody. Those skilled in the art will readily understand, based on the description of this invention, that the antigen-binding fragments of the aforementioned antibodies can be obtained, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the complete antibody structure disclosed in this invention, those skilled in the art can readily obtain the aforementioned antigen-binding fragments.

[0054] The antigen-binding fragments of the aforementioned antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesizing, for example, automated peptide synthesizers sold by Applied BioSystems.

[0055] Thirdly, the present invention provides an antibody conjugate comprising the antibody or its antigen-binding fragment described above.

[0056] In an optional embodiment, the antibody conjugate further includes biotin or a biotin derivative conjugated to the antibody or its antigen-binding fragment.

[0057] In an optional embodiment, the antibody conjugate further includes a marker conjugated to the antibody or its antigen-binding fragment.

[0058] In an optional implementation, the aforementioned marker refers to a type of substance that has properties such as luminescence, color development, and radioactivity that can be directly observed by the naked eye or detected or probed by instruments. Through these properties, qualitative or quantitative detection of the corresponding target can be achieved.

[0059] In optional embodiments, the markers include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0060] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0061] In optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0062] In optional embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0063] In optional embodiments, the radioactive isotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0064] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0065] In optional embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0066] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latexes.

[0067] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0068] In an optional embodiment, the colloidal metal is colloidal gold.

[0069] In an optional embodiment, the antibody conjugate further includes a solid-phase carrier conjugated to the antibody or its antigen-binding fragment.

[0070] In an optional embodiment, the solid support is selected from microspheres, plates, and membranes.

[0071] In optional embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon, and nitrocellulose membranes.

[0072] Fourthly, the present invention provides a reagent or kit comprising the above-described antibody or its antigen-binding fragment or the above-described antibody conjugate.

[0073] As previously stated, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present invention can effectively bind to abnormal prothrombin. Therefore, reagents or kits containing the abnormal prothrombin antibody or its antigen-binding fragment can effectively perform qualitative or quantitative detection of abnormal prothrombin. The reagents or kits provided by the present invention can be used, for example, in immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, latex immunoturbidimetry, and other detection methods involving the specific binding properties of abnormal prothrombin and its antibody. As previously stated, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present invention have higher binding activity or affinity to abnormal prothrombin; therefore, reagents or kits containing the antibody or its antigen-binding fragment have higher detection sensitivity or specificity.

[0074] Fifthly, the present invention provides a method for detecting abnormal prothrombin, comprising: a) contacting the antibody or its antigen-binding fragment, antibody conjugate, reagent or kit with abnormal prothrombin in a sample to be tested under conditions sufficient to induce an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample;

[0075] In an optional embodiment, the immune complex further includes a second antibody that binds to the antibody or its antigen-binding fragment.

[0076] In an optional embodiment, the immune complex further includes a second antibody that binds to abnormal prothrombin.

[0077] In a sixth aspect, the present invention provides the use of the above-mentioned abnormal prothrombin antibody or its antigen-binding fragment, antibody conjugate or the above-mentioned reagent or kit in the preparation of products for detecting abnormal prothrombin.

[0078] In a seventh aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or its antigen-binding fragment.

[0079] Eighthly, the present invention provides a carrier containing the above-mentioned nucleic acid molecules.

[0080] In a ninth aspect, the present invention provides cells containing the above-described carrier.

[0081] In a tenth aspect, the present invention provides a method for preparing an abnormal prothrombin antibody or an antigen-binding fragment thereof, comprising: culturing cells as described above.

[0082] Based on the amino acid sequence of the abnormal prothrombin antibody or its antigen-binding fragment disclosed in this invention, those skilled in the art will readily conceive of preparing the abnormal prothrombin antibody or its antigen-binding fragment using genetic engineering or other techniques (chemical synthesis, recombinant expression). For example, the antibody or its antigen-binding fragment can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the antibody or its antigen-binding fragment as described in any of the preceding claims. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the abnormal prothrombin antibody or its antigen-binding fragment of this invention, it falls within the protection scope of this invention.

[0083] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0085] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligo Nucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0086] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0087] Example 1: Preparation of Abnormal Prothrombin Antibody

[0088] (1) Mouse immunization and antibody detection

[0089] Five 6-8 week old SPF-grade female BALB / c mice were selected. Freund's complete adjuvant and 1 mg / ml PIVKA-II synthetic peptide were mixed in equal volumes and emulsified. The emulsified antigen was used to immunize the 6-8 week old SPF-grade female BALB / c mice, with each mouse receiving 50 μg of antigen protein via paw injection. Two weeks after the initial immunization, the antigen protein was mixed with Freund's incomplete adjuvant and emulsified, and each mouse received 40 μg of antigen protein via paw injection or subcutaneous injection in the back. Two weeks later, blood was collected via tail vein, the supernatant was collected by centrifugation, and serum titer was determined by ELISA. Immunization was repeated every two weeks, and serum titer was measured. After two immunizations, the serum titer, after a million-fold dilution, was higher than 2.0. A serum titer of 10 was selected for screening. 6 Lymphocytes were isolated from the mice mentioned above for cell fusion.

[0090] (3) Hybridoma antibody screening

[0091] The antibody screening protocol is crucial for the successful development of the antibodies of this invention. In the antibody screening stage, the ELISA plate is first coated with a synthetic peptide of PIVKA-II. After the potential target antibody binds to the PIVKA-II peptide, it is then incubated with goat anti-mouse HRP in conjunction with the ELISA reagent to screen for highly reactive hybridoma clones. Secondly, the ELISA plate is coated with prothrombin. If the potential target antibody binds to prothrombin, and then incubation is performed with goat anti-mouse HRP in conjunction with the ELISA reagent, it indicates that the antibody will be interfered with by the aforementioned protein and should be excluded.

[0092] The target clones obtained through screening need to meet the following conditions: 1. Good reactivity to PIVKA-II synthetic peptide; 2. No significant reactivity to prothrombin. The PIVKA-II-A cell line screened in this invention was used for antibody A preparation.

[0093] (4) Production and purification of monoclonal antibodies

[0094] Two groups of 6-8 week old BALB / c mice were selected, and 500 μL of paraffin oil was injected intraperitoneally to suppress the immune response. One week after injection, 0.5 ml of PIVKA-II-A hybridoma cells (approximately 1 × 10⁻⁶ cells) were injected intraperitoneally into one group of mice. 6 Quantity. Ascites fluid collection began two weeks later. The collected ascites fluid was purified by ammonium sulfate precipitation and protein G affinity purification to obtain the target antibody.

[0095] (5) Identification of monoclonal antibody subtypes and cloning of gene sequences

[0096] The SBA Clonotyping System-HRP kit from Southern Biothech was used to identify the heavy and light chain isotypes of monoclonal antibodies, following the manufacturer's instructions. The specific procedure was as follows:

[0097] a. Dilute the capture antibody to 1 μg / mL with coating buffer (0.05 M pH 9.5 carbonate and bicarbonate buffer), add 100 μL / well to the microplate, and coat overnight at 4°C. Wash the plate three times with PBS buffer containing 0.05% Tween-20.

[0098] b. Dilute the culture supernatant of the hybridoma cells to be tested 1:1 with diluent (1% BSA, 0.1% PBST), add 100 μL / well to the ELISA plate, and incubate at 37°C for 30 min. Dilute the corresponding enzyme-labeled antibodies (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lambda-HRP) 1:3000 with diluent.

[0099] After washing the plate three times with washing buffer, add 100 μL of diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing three more times, add chromogenic buffer and incubate for approximately 5 minutes (depending on the reaction strength), then add 2M sulfuric acid to terminate the reaction and read the OD450 absorbance. Identification revealed that the heavy chain isotype of the PIVKA-II-A antibody is IgG1, and the light chain is Kappa. Based on the antibody isotype results, the antibody gene sequence was cloned using a RACE-based method. Hybridoma cells in good growth condition were collected, and total RNA was obtained from the hybridoma cells using a total RNA extraction kit. The mRNA was reverse transcribed into cDNA according to the Takara SMARTer RACE instructions, and the full-length sequence of the target antibody was amplified.

[0100] The obtained antibody was named PIVKA-II-A. PIVKA-II-A was mutated, and five mutant antibodies with equivalent performance were screened out. The antibody sequences (the antibody CDR region is defined using the kabat method) are shown in Table 1 below:

[0101] Table 1 Antibody Sequences

[0102]

[0103] Example 2: Antibody Performance Detection

[0104] 1. Reactivity identification

[0105] The PIVKA-II synthetic peptide was diluted to 1 μg / mL with 50 mM carbonate buffer for coating in microplates, 100 μL per well, and incubated overnight at 4°C. The next day, the plates were washed twice with PBST and patted dry. The above antibody was added, serially diluted 3-fold starting from 1000 ng / mL, and loaded at 100 μL / well, incubated at 37°C for 30 min (with some supernatant added for 1 h). The plates were washed 5 times with PBST and patted dry. Horseradish peroxidase-labeled goat anti-mouse IgG was added at 100 μL / well, incubated at 37°C for 30 min, washed 5 times with PBST and patted dry. Urea peroxide (50 μL / well) and tetramethylbenzidine (50 μL / well) were added, and the reaction was incubated for 10 min. The reaction was stopped by adding dilute hydrochloric acid, 50 μL / well. The OD value was read at 450 nm (reference 630 nm) on a microplate reader. The antibody reactivity was identified as shown in Table 2 below.

[0106] Table 2 Reactivity data

[0107]

[0108] The experimental results show that the antibodies against the above six abnormal prothrombin strains all exhibit good reactivity.

[0109] 2. Functional testing

[0110] A. Biotin-labeled antibody PIVKA-IIA-F

[0111] Weigh out biotin (NHS-LC-LC-Biotin, from Thermo Scientific, 21343), dissolve biotin in DMSO to a concentration of 5.677 mg / mL, take 0.3 mg of PIVKA-II synthetic peptide AF, and a total of 6 antibodies. The required volume of biotin solution for each 1 mg antibody is 3.3 μL. Add biotin to each of the 6 antibodies and mix well. Connect the antibodies to biotin and react at 25°C in the dark for 4 hours.

[0112] B. Alkaline phosphatase-labeled antibody

[0113] Displace 0.3 mg of PIVKA-II antibody (Bio-Mapper, MB340103) into TSE (pH 8.5) buffer, and displace 0.66 mg of ALP (from Sigma, ALPI12G) into ALP dialysis buffer (pH 7.6) solution. Mix well and determine the concentration. Dissolve the weighed Traut's Reagent 2-Iminothiolane·HCl (abbreviated as 2-IT, from Thermo Scientific, 26101) in TSE (pH 8.5) solution to a concentration of 13.76 mg / mL. Dissolve the weighed Sulfo-SMCC (from Thermo Scientific, PG82085) in purified water to a concentration of 3.7 mg / mL. Both activator solutions should be prepared fresh and used within 10 minutes of preparation. Calculate the required volume of 2-IT solution for this step based on a requirement of 5 μL per 1 mg of antibody. Add 2-IT solution to the antibody, mix well, and allow to stand at (25±2)℃ for 20 min; this is antibody activation. After activation, displace the activated antibody into TSE (pH 7.3) solution and determine the antibody concentration. Simultaneously, calculate the required volume of Sulfo-SMCC solution based on 10 μL of SMCC solution required per 1 mg ALP. Add the Sulfo-SMCC solution to the desalted ALP, mix well, and allow to stand at (25±2)℃ for 20 min. After activation, displace the activated ALP into TSMZ (pH 7.3) solution and determine the ALP concentration. Dilute the desalted antibody to 0.3 mg / mL with TSE (pH 7.3) solution, and dilute the desalted ALP to 0.4 mg / mL with TSMZ (pH 7.3) solution. Calculate the antibody and ALP volumes for ligation based on an antibody:ALP mass ratio of 1:0.91. Mix the antibody and ALP according to the calculated volume and incubate at 2–8°C for 12–20 hours. Dissolve maleimide in DMSO to a concentration of 9.7 mg / mL, and dilute 10-fold with TSMZ (pH 7.3) to obtain the stop solution. Add 20 μL of the stop solution required for 1 mL of antibody-ALP conjugate to terminate the reaction.

[0114] C. Performance Evaluation

[0115] (1) Linear Experiment

[0116] The assay reagents include R1, R2, and streptavidin magnetic bead solution:

[0117] R1 includes: biotin-labeled PIVKA-II-A / B / C / D / E / F antibody, at a concentration of 1 μg / mL;

[0118] R2 comprises: alkaline phosphatase-labeled PIVKA-II antibody at a concentration of 1 μg / mL.

[0119] The specific experimental procedures are as follows:

[0120] a. Experimental preparation: 5 mL each of R1 and R2 working solutions, 4 mL of magnetic bead solution, and WRSA-WRS F calibrators.

[0121] b. Place each component into the kit separately, and then place the kit into the EXI1800 fully automated chemiluminescence immunoassay analyzer (from Zhongyuan Huiji Biotechnology Co., Ltd.). Select the anti-PIVKA-II project (30uL sample, 145uL R, 245uL R, 30uL magnetic bead solution) for the experiment.

[0122] Table 3. Results of the linear experiment

[0123] WRS A B C D E F WRS-A 5832 5186 4775 4824 4557 4822 WRS-B 278176 300142 298024 322820 285768 307920 WRS-C 1457265 1338806 1436399 1367102 1383355 1377439 WRS-D 8653771 8564077 8962454 8530574 8795014 8711246 WRS-E 39735341 38509591 37199737 39860317 38948502 38406417 WRS-F 76387407 79618529 76121636 82031457 78439119 79524886

[0124] Therefore, as shown in Table 3, the above six anti-prothrombin antibodies, when used as labeling antibodies in PIVKA-II chemiluminescence assay, all exhibited good linearity.

[0125] 3. Clinical trials

[0126] The specific experimental procedures are as follows:

[0127] (1) Experimental preparation: Take out 20 PIVKA-II samples, thaw them at room temperature, and mix them thoroughly on a vortex mixer;

[0128] (2) Testing: Antibodies against PIVKA-II-A and mutant PIVKA-II-C were extracted and tested in 20 clinical samples. The clinical results are shown in Table 4 below:

[0129] Table 4 Clinical Trial Results

[0130] Sample No. A C PRL-01 23322 25870 PRL-02 20416 19215 PRL-03 17010 20014 PRL-04 21770 24276 PRL-05 23369 26612 PRL-06 39475 42866 PRL-07 14246 16536 PRL-08 28026 28031 PRL-09 32864 32295 PRL-10 29552 33652 PRL-11 33901 29659 PRL-12 20058 23605 PRL-13 40931 41387 PRL-14 36664 43423 PRL-15 29745 35532 PRL-16 33469 29811 PRL-17 39688 37046 PRL-18 27062 26530 PRL-19 78211 69145 PRL-20 64562 55405

[0131] The clinical data above show that the clinical measurements of PIVKA-II-A and the mutant strain PIVKA-II-C meet the requirements and can be used in in vitro diagnostic reagents.

[0132] All data, reagents, and procedures described herein should be understood as illustrative rather than restrictive. Although the invention has been described in conjunction with the specific embodiments described above, many modifications and other variations will be apparent to those skilled in the art. All such modifications and other variations also fall within the scope of the invention.

Claims

1. An antibody or antigen-binding fragment thereof to an abnormal prothrombin, characterized in that, The antibody or antigen-binding fragment thereof comprises the complementarity determining regions of any one of the following groups: (1) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, respectively; (2) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 18, respectively; (3) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 13, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, respectively; (4) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 19, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 17, SEQ ID NO: 18, respectively; (5) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 18, respectively; (6) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, The complementarity determining regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems, or a combination of multiple systems, preferably by the Kabat system.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof further comprises the framework regions HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; The HFR1 comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence at least 80% identical thereto; The HFR2 comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence at least 80% identical thereto; the HFR3 comprises SEQ ID NO: 5 or an amino acid sequence having at least 80% identity thereto; the HFR4 comprises SEQ ID NO: 6 or an amino acid sequence having at least 80% identity thereto; the LFR1 comprises SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto; the LFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; the LFR3 comprises SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto; the LFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto.

4. An antibody or antigen-binding fragment thereof to an abnormal prothrombin, characterized in that, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region, and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as set forth in any one of SEQ ID NO: 1, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33; and the amino acid sequence of the light chain variable region is as set forth in any one of SEQ ID NO: 2, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO:

34.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region is selected from the group consisting of a heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD, or a combination of segments of multiple constant regions; preferably, the species origin of the constant region is bovine, equine, bovine, porcine, ovine, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, cock or human; preferably, the heavy chain constant region comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto, and the light chain constant region comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto.

6. An antibody conjugate, characterized in that, the antibody conjugate comprises the antibody or antigen-binding fragment thereof of any one of claims 1 to 5; preferably, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or antigen-binding fragment thereof; preferably, the antibody conjugate further comprises a label conjugated to the antibody or antigen-binding fragment thereof; preferably, the label is selected from the group consisting of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent and a nanoparticle label; preferably, the antibody conjugate further comprises a solid support conjugated to the antibody or antigen-binding fragment thereof; preferably, the solid support is selected from at least one of a microsphere, a plate or a membrane; preferably, the solid support is selected from at least one of a magnetic microsphere, a plastic microsphere, a plastic microparticle, a microwell plate, glass, a capillary, a nylon or a nitrocellulose membrane.

7. A reagent or kit characterized in that, the reagent or kit comprises the antibody or antigen-binding fragment thereof of any one of claims 1 to 5 or the antibody conjugate of claim 6; Preferably, the method is selected from at least one of immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence, latex immunoturbidimetry.

8. A method of detecting abnormal prothrombin, characterized by, Comprising: a) contacting the antibody or antigen-binding fragment thereof of any one of claims 1-5, the antibody conjugate of claim 6, or the reagent or kit of claim 7 with an abnormal prothrombin in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur to form an immunocomplex; and b) detecting the presence of the immunocomplex, the presence of the complex indicating the presence of the antigen in the test sample; Preferably, the immunocomplex further comprises a second antibody that binds to the antibody or antigen-binding fragment thereof; Preferably, the immunocomplex further comprises a second antibody that binds to abnormal prothrombin.

9. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-5, the antibody conjugate of claim 6, or the reagent or kit of claim 7 in the manufacture of a product for detecting abnormal prothrombin.

10. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 5; a vector comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 5; a cell comprising the nucleic acid or vector; or a method of making the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, the method comprising the cell.

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