Abnormal prothrombin antibody or conjugate thereof and application thereof

Abstract

The invention relates to the technical field of antibodies, in particular to an abnormal prothrombin (PIVKA-II) antibody or a conjugate thereof and application thereof. The antibody or the conjugate thereof comprises a heavy chain variable region VH and a light chain variable region VL, the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH are respectively shown as SEQ ID No: 1-3, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region VL are respectively shown as SEQ ID No: 4-5; the antibody conjugate is formed by coupling the antibody with a marker or a solid-phase carrier. The antibody disclosed by the invention has high binding activity and affinity to PIVKA-II, and the specificity, sensitivity, reliability and accuracy detection of trace PIVKA-II in a sample can be realized in a double-antibody sandwich manner.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically, to an antibody against abnormal prothrombin or its conjugate and its applications. Background Technology

[0002] Abnormal prothrombin, also known as vitamin K deficiency or antagonist-induced protein-II (PIVKA-II) or degamma-carboxyprothrombin (DCP), is an abnormal protein with no clotting function produced in cases of vitamin K deficiency, malabsorption, or hepatocellular carcinoma. In 1984, Liebman et al. first reported in the *New England Journal of Medicine* that abnormal prothrombin was detected in the serum of 91% of hepatocellular carcinoma (HCC) patients, with an average concentration as high as 900 ng / mL, while levels were extremely low or undetectable in chronic active hepatitis, metastatic liver cancer, and healthy individuals. This discovery laid the foundation for its use as a biomarker for liver cancer.

[0003] Under normal circumstances, with the participation of vitamin K, the liver modifies the prothrombin precursor by carboxylation through γ-glutamyl carboxylase to generate normal prothrombin with clotting function. When vitamin K is deficient, malabsorption is impaired, or hepatocellular carcinoma occurs, the carboxylation process is blocked, leading to the release of abnormal prothrombin without clotting function into the blood.

[0004] The clinical significance of PIVKA-II is as follows: (1) Early diagnosis and screening of hepatocellular carcinoma (HCC): The sensitivity of single detection for HCC diagnosis is about 60-74%, and the specificity is 89-90%; for small hepatocellular carcinoma with a diameter of <20 mm, the positive rate of PIVKA-II can reach 50%; the positive rate of lesions with a diameter of 20-30 mm is about 60%, showing its value in early screening; the sensitivity of AFP for early hepatocellular carcinoma is only 10-20%, while that of PIVKA-II can reach 90%. The combined detection of the two can increase the diagnostic sensitivity to 78-84% and the specificity to 98.5%, which is significantly better than a single marker; (2) Efficacy evaluation and recurrence monitoring: After surgical resection, intervention or targeted therapy, the PIVKA-II level in the effective patients drops rapidly; it rises again when there is recurrence or metastasis. Continuous monitoring can detect signs of recurrence early; the serum half-life is only 40-72 hours, which is much shorter than the 5-7 days of AFP, and can reflect changes in treatment more quickly; (3) Prognostic judgment: The pre-PIVKA-II level is significantly correlated with postoperative survival rate and disease-free survival. The higher the level, the worse the prognosis; PIVKA-II positive patients often have a higher risk of intrahepatic metastasis and portal vein invasion; (4) Liver function assessment: In benign liver diseases such as hepatitis and cirrhosis, PIVKA-II may be slightly elevated due to impaired hepatocyte synthesis function, which can help assess liver reserve function.

[0005] PIVKA-II, as an important serum biomarker with high specificity and rapid response in the diagnosis and treatment of liver cancer, is valuable in compensating for the insufficient sensitivity of AFP and providing precise complementarity in diagnosis, efficacy evaluation, prognosis, and recurrence monitoring. Its combined use with AFP and AFP-L3% can maximize diagnostic efficacy. However, the performance of current antibodies targeting PIVKA-II does not yet fully meet the needs of clinical testing reagents. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a monoclonal antibody, kit, and applications for the detection of PIVKA-II. The antibody of this invention exhibits high binding activity and affinity for PIVKA-II, enabling specific, sensitive, reliable, and accurate detection of trace amounts of PIVKA-II in samples through a double-antibody sandwich method. This invention is specifically achieved through the following technical solutions: A first aspect of the present invention provides a monoclonal antibody or conjugate of PIVKA-II, wherein the antibody or conjugate comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH comprises: The amino acid sequence is HCDR1 as shown in SEQ ID No:1; The amino acid sequence is HCDR2 as shown in SEQ ID No:2; The amino acid sequence is HCDR3 as shown in SEQ ID No:3; The light chain variable region VL includes: The amino acid sequence is LCDR1 as shown in SEQ ID No:4; The amino acid sequence is LCDR2 as shown in SEQ ID No:5; The amino acid sequence is LCDR3 as shown in SEQ ID No:6; The antibody-conjugate is formed by conjugating the antibody with a label or a solid-phase carrier.

[0007] In a specific embodiment of the present invention, the heavy chain variable region VH of the antibody or its conjugate is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO. 7.

[0008] In a specific embodiment of the present invention, the light chain variable region VL of the antibody or its conjugate is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO. 8.

[0009] In a specific embodiment of the present invention, the heavy chain variable region VH of the antibody has an amino acid sequence as shown in SEQ ID NO.7, and the light chain variable region VL of the antibody has an amino acid sequence as shown in SEQ ID NO.8.

[0010] Furthermore, the antibody is a full-length antibody or its antigen-binding fragment; the antigen-binding fragment is selected from at least one of the following: Fab fragment, F(ab')2 fragment, Fv fragment, dsFv fragment, scFv fragment, sc(Fv)2 fragment, and diabody.

[0011] Furthermore, the antibody is a full-length antibody, the heavy chain amino acid sequence of which is shown in SEQ ID NO.9; and the light chain amino acid sequence of which is shown in SEQ ID NO.10.

[0012] Furthermore, the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, nanoparticle labeling, and radioactive labeling; the solid support is selected from one or more of microspheres, plates, or membranes.

[0013] A second aspect of the present invention provides a detection reagent or kit for PIVKA-II, the reagent or kit comprising the antibody or conjugate described above.

[0014] A third aspect of the present invention provides a detection kit for PIVKA-II, the kit comprising a capture reagent and a detection reagent, each comprising an antibody or conjugate of PIVKA-II with a different sequence, wherein the antibody or conjugate of PIVKA-II is selected from the antibodies or conjugates described above.

[0015] A fourth aspect of the present invention provides the use of the above-mentioned antibody or its conjugate in the preparation of a product for detecting PIVKA-II.

[0016] The fifth aspect of the present invention provides a method for detecting PIVKA-II for non-diagnostic purposes, comprising the following steps: forming an immune complex with the above-described antibody or its conjugate and the antigen in the sample.

[0017] A sixth aspect of the present invention provides a nucleic acid molecule that encodes the above-described PIVKA-II monoclonal antibody or its antigen-binding fragment.

[0018] A seventh aspect of the present invention provides a vector or host cell comprising the aforementioned nucleic acid molecules.

[0019] The beneficial effects of this invention are as follows: the antibody provided by this invention has high binding activity and affinity for PIVKA-II, and can achieve specific, sensitive, reliable and accurate detection of trace amounts of PIVKA-II in samples through a double antibody sandwich method, which has great clinical application value. Attached Figure Description

[0020] Figure 1 The results show the correlation between the PIVKA-II antibody of this invention and the reference standard. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0022] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0023] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0024] 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.

[0025] 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 formulation or unit dose practice or testing of this document, 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. Where specific conditions are not specified in the examples, they are performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are conventional products that are commercially available.

[0026] The first object of the present invention is to provide a high-quality antibody or conjugate of PIVKA-II, wherein the antibody or conjugate thereof: The first antibody includes: HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID No:1-3; and LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID No:4-6; In this invention, "CDR," "CDRs," or "complementarity-determining region" refers to a highly variable region of the heavy and light chains of an immunoglobulin, specifically a region containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In a specific embodiment of this invention, CDRs refer to the highly variable region of the heavy and light chains of the antibody.

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

[0028] The methods for defining CDRs are well known in the art, including the Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, the "Kabat definition" refers to the definition system described in Kabat et al., USD ept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). In this invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 is defined by the Kabat system.

[0029] In this invention, the antibody conjugate is composed of an antibody coupled to a label or a solid-phase support. The label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, nanoparticle labeling, and radioactive labeling; the solid-phase support is selected from one or more of microspheres, plates, or membranes.

[0030] In optional embodiments, the aforementioned markers refer to substances that possess properties such as luminescence, color development, and radioactivity that can be directly observed with the naked eye or detected by instruments. These properties enable qualitative or quantitative detection of the corresponding target analytes. Examples include, but are not limited to, fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

[0031] 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.

[0032] 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 dyes, 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 dyes) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar dyes).

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, dispersed dyes, dye-labeled microspheres, and latexes.

[0038] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0039] In an optional embodiment, the colloidal metal is colloidal gold.

[0040] In an optional embodiment, the antibody conjugate further includes a solid-phase carrier conjugated to the antibody or its antigen-binding fragment.

[0041] In an optional embodiment, the solid support is selected from microspheres, plates, and membranes.

[0042] 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.

[0043] In this invention, the antibody is a full-length antibody or its antigen-binding fragment; the antigen-binding fragment is selected from at least one of the following: Fab fragment, F(ab')2 fragment, Fv fragment, dsFv fragment, scFv fragment, sc(Fv)2 fragment, and diabody.

[0044] The amino acid sequence of the heavy chain variable region of the first antibody of the present invention is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0045] In this invention, the "frame region" or "FR region" includes the heavy chain frame region and the light chain frame region, referring to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR; wherein, the heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including the HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including the LFR1, LFR2, LFR3 and LFR4 frame regions.

[0046] In this invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0047] In an optional embodiment, the antibody further comprises at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4; It should be noted that, in other embodiments, the amino acid sequences of each frame region of the antibody PIVKA-II provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding frame region.

[0048] In an optional embodiment, the antibody or its antigen-binding fragment further includes a constant region.

[0049] In an optional implementation, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0050] In an optional implementation, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0051] In an optional embodiment, the heavy chain constant region includes CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0052] In an optional implementation, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0053] In an optional implementation, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0054] In an optional implementation, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans.

[0055] In an optional implementation, the species source of the constant region is mice.

[0056] It should be noted that, in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the aforementioned constant region.

[0057] In this embodiment of the invention, the heavy chain amino acid sequence of the first antibody is shown in SEQ ID NO.9; the light chain amino acid sequence is shown in SEQ ID NO.10.

[0058] A second objective of the present invention is to provide a detection reagent or kit for PIVKA-II, wherein the reagent or kit includes the aforementioned first antibody.

[0059] Some detection methodologies include a capture reagent and a detection reagent in their kits, wherein the capture reagent and the detection reagent include a first antibody or a conjugate thereof.

[0060] For example, the above-mentioned kit can be used for detections involving the specific binding properties of PIVKA-II and its antibodies, such as immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, and latex immunoturbidimetry. For instance, in fluorescence immunochromatography, the antigen in the sample binds to the fluorescently labeled first antibody on the binding pad. The solution undergoes chromatography due to the siphon effect of the absorbent pad, moving towards the absorbent pad. When the complex moves to the detection line, it binds to the coated antibody on the T line, forming a "sandwich" type complex that is enriched on the T line. In chemiluminescence immunoassay, the antigen in the sample binds to the first antibody coated on magnetic beads, and after washing, it binds to the enzyme-labeled second antibody (usually horseradish peroxidase HRP or alkaline phosphatase AP), forming a "solid-phase antibody-antigen-enzyme-labeled antibody" sandwich complex. It is understood that the first and second antibodies here do not specifically refer to any particular sequence of the present invention. Theoretically, as long as the two antibodies forming the "double-antibody sandwich" immune complex bind to different epitopes of the antigen, the two antibodies in the capture reagent and the detection reagent are theoretically not specific and can be interchanged.

[0061] A third object of the present invention is to provide the use of a first antibody or its conjugate in the preparation of a product for detecting PIVKA-II.

[0062] The technical solution of the present invention will be further described below through some specific embodiments.

[0063] Example 1: Preparation of PIVKA-II Monoclonal Antibody For mouse immunization and antibody detection, 20-25 6-8 week old SPF-grade female BALB / c mice (Hunan Slack Jingda Experimental Animal Co., Ltd.) were selected. Freund's complete adjuvant and PIVKA-II natural protein (Chongqing Aishengs Biotechnology Co., Ltd.) at a concentration of 1 mg / ml were mixed in equal volumes and emulsified. The emulsified antigen was used to immunize 6-8 week old healthy SPF-grade BALB / c mice via multiple subcutaneous injections at the back, with each mouse receiving 500 μg of antigen protein. Two weeks after the initial immunization, the antigen protein was mixed with incomplete Freund's adjuvant and emulsified, and 200 μg of antigen protein was injected subcutaneously at the back of each mouse again. Three weeks later, blood was collected via tail vein, the supernatant was collected by centrifugation, and serum titer was detected by ELISA. Booster immunizations were performed every two weeks, and serum titer was detected again. One week after the final immunization, the serum titer after a million-fold dilution was higher than 1.5. Mice with a serum titer of 106 or higher were selected, and spleen cells were harvested for cell fusion.

[0064] In the hybridoma antibody screening stage, ELISA plates were first coated with PIVKA-II natural protein. After the potential target antibody bound to PIVKA-II, goat anti-mouse HRP was used in conjunction with ELISA chromogenic solution for color development, and highly reactive hybridoma clones were screened. Five highly reactive and specific antibodies were selected, numbered 1-5#.

[0065] For the production and purification of monoclonal antibodies, 15 6-8 week old BALB / c mice were selected and injected intraperitoneally with 500 μL of paraffin oil to suppress the immune response. One week after injection, 0.5 ml of PIVKA-II monoclonal antibody 1-3# hybridoma cells (3 mice / group) were injected intraperitoneally into each mouse, with a cell count of approximately 1×10⁻⁶. 6 Quantity. Ascites fluid collection began two weeks later. The collected ascites fluid was purified by ammonium sulfate precipitation and affinity purification with protein G to obtain the target antibody.

[0066] Example 2: Evaluation of antibody pairing The purified anti-1-4# antibodies were used as coating antibodies (naked antibodies) and labeled antibodies (Chongqing Aishengs Biotechnology Co., Ltd., catalog number BPI02) to recognize the PIVKA-II protein using a double antibody sandwich method. Antibodies that could be used for pairing were screened out, and the evaluation data are shown in Table 1 below.

[0067] Table 1 Antibody pairing data

[0068] The data above shows that antibody #2, with an OD value above 1.0 and higher reactivity, was selected for the next step of testing.

[0069] Example 3: Antibody gene sequence cloning and performance testing Monoclonal antibody isotype identification and gene sequence cloning were performed using the Southern Biothech SBACOnotyping System-HRP kit, following the manufacturer's instructions, to identify the heavy and light chain isotypes of the PIVKA-II #2 monoclonal antibody. The specific procedures were as follows: 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. 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. c. 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 2 M sulfuric acid to terminate the reaction and read the OD450 absorbance. Based on the antibody subtype results, clone the antibody gene sequence using the RACE-based method. Collect hybridoma cells in good growth condition, obtain total RNA from the hybridoma cells using a total RNA extraction kit, and reverse transcribe the mRNA into cDNA according to the Takara SMARTer RACE instruction manual. Amplify the full-length sequence of the target antibody.

[0070] The subtype information of the identified monoclonal antibody #2 is shown in Table 2.

[0071] Table 2. Monoclonal antibody subtypes

[0072] Stability assays were performed by subjecting the above antibodies to accelerated thermal stress at 37°C for 14 days in a predetermined buffer (PBS, 0.05% ProClin™ 300). The accelerated antibodies were evaluated by an indirect ELISA method, and the test results were shown as the relative deviation of the detection values ​​stored at 4°C to assess the thermal stability of the antibodies. In addition, the freeze-thaw stability of the antibodies was tested by repeatedly freezing and thawing the antibodies at -20°C five times, and the test results were shown as the relative deviation of the detection values ​​stored at 4°C for 0 days. The test results are shown in Table 3 below (the detection method in this experiment is consistent with the "Activity Identification").

[0073] Table 3 Stability Study

[0074] The experimental results show that the No. 2 antibody provided by this invention has good thermal acceleration stability and freeze-thaw stability.

[0075] Functional testing A. Biotin-labeled antibody 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 antibody, the required volume of biotin solution for each 1 mg of antibody is 3.3 μL, add antibody #2 to biotin and mix well, ligate the antibody to biotin, and react at 25°C in the dark for 4 h.

[0076] B. Alkaline phosphatase-labeled antibody Displace 0.3 mg of the labeled antibody (Chongqing Aishengs Biotechnology Co., Ltd., catalog number BPI02) into TSE (pH 8.5) buffer. Displace 0.66 mg of alkaline phosphatase (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 based on 5 μL of 2-IT solution required for every 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 (the volume required for 1 mL of antibody-ALP conjugate) to terminate the reaction.

[0077] Performance Evaluation (1) Linear experiment The assay reagents include R1, R2, and streptavidin magnetic bead solution: R1 includes: a biotin-labeled antibody, used at a concentration of 1 μg / mL; R2 comprises: alkaline phosphatase-labeled antibody at a concentration of 1 μg / mL.

[0078] The specific experimental procedures are as follows: a. Experimental preparation: 5 mL each of R1 and R2 working solutions, 4 mL of magnetic bead solution, and calibrators WRS A to WRS H.

[0079] b. Place each component into the kit separately, and then place the kit into the fully automated chemiluminescence immunoassay analyzer (EXI1800, Zhongyuan Huiji Biotechnology Co., Ltd.). Select the PIVKA-II test (sample 15μL, R1 45μL, R2 20μL, magnetic bead solution 20μL) for the experiment.

[0080] Table 4 Calibration Experiment Results

[0081] Based on the data in Table 4 above and Figure 1 It can be seen that when antibody #2 is used as a coating antibody in the PIVKA-II detection reagent, it has a good detection linear range.

[0082] The data above shows that when these two antibodies are used in combination in the PIVKA-II chemiluminescent immunoassay reagent, they have excellent detection precision and good clinical detection value.

[0083] The partial amino acid sequence of antibody PIVKA-II, which is involved in this application, is shown in Table 5 below (defined by the Kabat system).

[0084] Table 5 Amino acid sequence listing

[0085] 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. A monoclonal antibody or conjugate thereof for PIVKA-II, said antibody or conjugate comprising a heavy chain variable region VH and a light chain variable region VL, said heavy chain variable region VH comprising: The amino acid sequence is HCDR1 as shown in SEQ ID No:1; The amino acid sequence is HCDR2 as shown in SEQ ID No:2; The amino acid sequence is HCDR3 as shown in SEQ ID No:3; The light chain variable region VL includes: The amino acid sequence is LCDR1 as shown in SEQ ID No:4; The amino acid sequence is LCDR2 as shown in SEQ ID No:5; The amino acid sequence is LCDR3 as shown in SEQ ID No:6; The antibody-conjugate is formed by conjugating the antibody with a label or a solid-phase carrier.

2. The monoclonal antibody or its conjugate as claimed in claim 1, wherein the heavy chain variable region VH of the antibody or its conjugate is an amino acid sequence having at least 90% identity with SEQ ID NO. 7; and the light chain variable region VL of the antibody or its conjugate is an amino acid sequence having at least 90% identity with SEQ ID NO.

8.

3. The monoclonal antibody or its conjugate as described in claim 1 or 2, wherein the antibody is a full-length antibody or its antigen-binding fragment; the antigen-binding fragment is selected from at least one of the following: Fab fragment, F(ab')2 fragment, Fv fragment, dsFv fragment, scFv fragment, sc(Fv)2 fragment, and diabody.

4. The monoclonal antibody or its conjugate as described in claim 1 or 2, wherein the antibody is a full-length antibody, the heavy chain amino acid sequence of which is shown in SEQ ID NO. 9; and the light chain amino acid sequence of which is shown in SEQ ID NO.

10.

5. The monoclonal antibody or its conjugate as described in claim 1 or 2, wherein the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, nanoparticle labeling, and radioactive labeling; and the solid support is selected from one or more of microspheres, plates, or membranes.

6. A detection reagent or kit for PIVKA-II, said reagent or kit comprising the antibody or conjugate thereof as described in any one of claims 1 to 5.

7. The detection kit of claim 6, wherein the reagent or kit comprises a capture reagent and a detection reagent, each of the capture reagent and the detection reagent comprising an antibody or conjugate of a different sequence of PIVKA-II, wherein the antibody or conjugate of PIVKA-II is selected from the antibodies or conjugates of any one of claims 1 to 5.

8. Use of the antibody or its conjugate as described in any one of claims 1 to 5 in the preparation of a product for detecting PIVKA-II.

9. A method for detecting PIVKA-II for non-diagnostic purposes, comprising the following steps: forming an immune complex with an antibody or conjugate as described in any one of claims 1 to 5 and an antigen in a sample, and detecting the presence or content of the immune complex.

10. A nucleic acid molecule expressing the antibody of any one of claims 1-5, or a vector or host cell containing said nucleic acid molecule.

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