Thrombin-antithrombin III complex antibody, detection kit and application thereof

Abstract

The present application relates to the technical field of biological medicine, and provides a thrombin-antithrombin III complex antibody, a detection kit and application thereof.The present application specifically discloses a thrombin monoclonal antibody and an antithrombin III monoclonal antibody pairing combination, an intermediate product and a detection kit product containing the same.The antibody combination of the present application can be combined with the thrombin-antithrombin III complex in a double-antibody sandwich method with high sensitivity and strong specificity;the thrombin-antithrombin III complex detection kit suitable for the immunochemiluminescence method is prepared by using the above antibody, and the thrombin-antithrombin III complex in the human body can be detected by a two-step method.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a thrombin-antithrombin III complex antibody, a detection kit and application thereof. BACKGROUND

[0002] Thrombin (T) is a serine protease produced from prothrombin, which plays an important role in blood coagulation, wound repair and hemostasis (J.J.N. Posma, J.J. Posthuma, H.M.H. Spronk.“Coagulation and non-coagulation effects of thrombin.” J Thromb Haemost , 2016; 14(10): 1908-1916.), which can form a fibrin network by activating fibrinogen, promote platelet aggregation and form a blood clot. Antithrombin III (AT-III) is a multifunctional serine protease inhibitor synthesized by the liver and endothelial cells (Zhong Xiaohong, Wei Ping, Huang Min.“Expression levels of plasma thrombin-antithrombin III complex, D-dimer and antithrombin III in patients with atrial fibrillation and their significance in predicting thromboembolism.” Pract Prev Med , 2018; 25(10): 1212-1213, 1276.), which accounts for 50%-60% of the total anticoagulant effect in the body, and its main function is to inhibit the activity of thrombin, which can directly interact with excess thrombin to form denatured thrombin and make it inactive. In addition, AT-III also has a certain inhibitory effect on platelet aggregation. The level of AT-III mainly reflects the strength of thrombin activity, and its level plays an important role in maintaining the dynamic balance of coagulation and anticoagulation, and a decrease in AT-III may lead to the occurrence of thrombosis or DIC (Li Zhanjun, Jiao Tingting, Zhang Chenglei, et al.“Application of thrombomodulin and thrombin-antithrombin complex in the diagnosis of disseminated intravascular coagulation.” Ningxia Med J J Clin Lab Anal. , 2019; 41(2): 183-185.).

[0003] Thrombin-antithrombin complex (TAT) is a molecular complex composed of thrombin and antithrombin III in a 1:1 ratio. An elevated TAT level indicates excessive thrombin generation and activation of the blood coagulation cascade, reflecting a prothrombotic state (Qian Chen, Weiling Shou, Wei Wu, et al. “Performance evaluation of thrombomodulin, thrombin-antithrombin complex, plasmin-α2-antiplasmin complex, and t-PA: PAI-1 complex.” Haematologica. Thrombin has an extremely short half-life in the blood, making its levels difficult to measure directly. In contrast, TAT has a half-life of 3–15 minutes and can be directly measured. Therefore, TAT can be used as a marker of thrombin generation, reflecting the coagulation state in vivo.

[0004] Elevated TAT levels in the body have been shown to be associated with a number of diseases. Studies have shown that TAT levels are elevated in patients with myocardial infarction accompanied by mild inflammation (Réganon E, Vila V, Martínez-Sales V, et al. “Inflammation, fibrinogen and thrombin generation in patients with previous myocardial infarction.” Crit Care. 2002;87(7):740-5; discussion 745.). TAT testing can help identify patients with persistent severe coagulopathy in the early stages of sepsis (Koyama K, Madoiwa S, NunomiyaS, et al. “Combination of thrombin-antithrombin complex, plasminogen activator inhibitor-1, and protein C activity for early identification of severe coagulopathy in initial phase of sepsis: a prospective observational study.” Eur J Neurol.2014; 18(1):R13.), and also helps to evaluate the prognosis severity of brain injury after cerebral hemorrhage (Wu CH, Yang RL, Huang SY, et al. “Analysis of thrombin-antithrombin complex contents in plasma and hematoma fluid of hypertensive intracerebral hemorrhage patients after clot removal.” Chin J Emerg Med 2011; 18(8):1060–1066.)。

[0005] TAT combined with Plasmin-α2-plasmin inhibitor complex (PIC), Tissue plasminogen activator-plasminogen activator inhibitor-1 complex (t-PAIC), Thrombomodulin (TM), D-Dimer and Fibrin Degradation Products (FDP) can detect coagulation, fibrinolysis and endothelial system diseases earlier, more sensitively and more comprehensively, making it possible to identify thrombosis early, monitor the risk of thrombosis development comprehensively and guide the use of coagulation drugs effectively. The Expert Consensus on Diagnosis and Treatment of Acute Hemorrhagic Coagulopathy (2020 Edition) mentioned that for patients with complex clinical conditions, using TAT, PIC, t-PAIC and TM for early coagulation dysfunction assessment has become a consensus in the field (Expert Consensus Group on Diagnosis and Treatment of Acute Hemorrhagic Coagulopathy, Shao M, Xue M, Wang S. “Expert Consensus on Diagnosis and Treatment of Acute Hemorrhagic Coagulopathy. Figure 1 ,2020,29(6):780-787.DOI:10.3760 / cma.j.issn.1671-0282.2020.06.007.)。

[0006] Currently, the four main detection methods of thrombus are enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Enzyme-linked immunosorbent assay requires manual detection by personnel, which is tedious and has problems such as long test period and insufficient sensitivity; Radioimmunoassay requires the use of radioactive substances, which has pollution hazards and unstable test results.

[0007] Chemiluminescence method has the advantages of high sensitivity, strong precision, wide linear range and not easy to be disturbed, and is widely used in clinical disease index detection. The main determinant of the performance of the current TAT detection reagent is the functional characteristics of the TAT binding antibody, and a good antibody pair can effectively improve the diagnosis efficiency of the disease. The number of diagnostic test kits for TAT detection using chemiluminescence method in the current market is still limited, and there are certain deficiencies in sensitivity, linear range and precision. Therefore, it is of great significance to screen suitable antibody pairs for application in TAT diagnosis. SUMMARY

[0008] The present application provides thrombin-antithrombin III complex antibody pairs, intermediate products, test kits containing the antibody pairs and related application schemes to solve the problems of low sensitivity, narrow linear range and poor precision of existing TAT detection antibodies, and improve the performance of TAT test kit products.

[0009] In a first aspect of the present application, a thrombin-antithrombin III complex antibody or an antigen binding fragment thereof is provided, comprising the light and heavy chain variable regions of a thrombin antibody and / or the light and heavy chain variable regions of an antithrombin III antibody; the light and heavy chain variable regions of the thrombin antibody comprise VHCDR1 as shown in SEQ ID NO: 1, VHCDR2 as shown in SEQ ID NO: 2, VHCDR3 as shown in SEQ ID NO: 3, VLCDR1 as shown in SEQ ID NO: 4, VLCDR2 with a sequence of STD, and VLCDR3 as shown in SEQ ID NO: 5; the light and heavy chain variable regions of the antithrombin III antibody comprise VHCDR1 as shown in SEQ ID NO: 6, VHCDR2 as shown in SEQ ID NO: 7, VHCDR3 as shown in SEQ ID NO: 8, VLCDR1 as shown in SEQ ID NO: 9, VLCDR2 with a sequence of SAS, and VLCDR3 as shown in SEQ ID NO: 10.

[0010] As an alternative, in the light and heavy chain variable regions of the thrombin antibody, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 11, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 12; in the light and heavy chain variable regions of the antithrombin III antibody, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 13, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 14.

[0011] As an alternative, the antibody or antigen-binding fragment thereof can specifically recognize and bind to an epitope of the thrombin-antithrombin III complex antigen, such as SEQ ID NO: 15 and / or as shown in SEQ ID NO: 16.

[0012] As an alternative, the thrombin-antithrombin III complex antibody comprises a monoclonal antibody; and an antigen-binding fragment thereof selected from F(ab’)2, F(ab)2, Fab’, Fab, Fv or scFv.

[0013] In a second aspect of the present application, a nucleic acid encoding the above-mentioned thrombin-antithrombin III complex antibody or antigen-binding fragment thereof is provided.

[0014] In a third aspect of the present application, an expression vector comprising the above-mentioned nucleic acid is provided.

[0015] In a fourth aspect of the present application, a transgenic cell line or recombinant bacteria comprising the above-mentioned nucleic acid or the above-mentioned expression vector is provided.

[0016] In a fifth aspect of the present application, an immunoconjugate comprising an antibody moiety and a conjugating moiety conjugated to the antibody moiety is provided, wherein the antibody moiety comprises the above-mentioned thrombin-antithrombin III complex antibody or antigen-binding fragment thereof, and the conjugating moiety is selected from a fluorescent substance, a chemiluminescent substance, a colored substance, an enzyme or a combination thereof.

[0017] In a sixth aspect of the present application, a thrombin-antithrombin III complex detection kit is provided, wherein the kit comprises the above-mentioned thrombin-antithrombin III complex antibody or antigen-binding fragment thereof.

[0018] As an alternative, the detection kit comprises a thrombin antibody and an antithrombin III antibody used in pairs; the thrombin antibody as a capture antibody and the antithrombin III antibody as a detection antibody; or, the antithrombin III antibody as a capture antibody and the thrombin antibody as a detection antibody.

[0019] As an alternative, the detection kit comprises capture antibody-coated magnetic microparticles, detection antibodies labeled with a luminescent label, a substrate suitable for the luminescent label and an excitation solution, and a series of quality control and calibration samples of the thrombin-antithrombin III complex antigen.

[0020] As an alternative, the capture antibody-coated magnetic microparticles are formed by coupling streptavidin-coated magnetic beads with biotinylated thrombin antibodies; and the luminescent label comprises acridinium ester.

[0021] In a seventh aspect, the present application provides use of the thrombin-antithrombin III complex antibody or antigen-binding fragment thereof, the nucleic acid, the expression vector, the transgenic cell line or the recombinant bacteria, or the immunoconjugate as described above in the preparation of a detection reagent or kit for evaluating early coagulation dysfunction.

[0022] The present application provides a thrombin monoclonal antibody and an antithrombin III monoclonal antibody pairing combination, intermediates, and a detection reagent product comprising the same. The antibody combination of the present application can bind to the thrombin-antithrombin III complex with high sensitivity and strong specificity in a double antibody sandwich method; a thrombin-antithrombin III complex detection kit (immunochemiluminescence method) is prepared using the above-mentioned antibodies, and the thrombin-antithrombin III complex in the human body can be detected by a two-step method. Through appropriate reagent preparation, the TAT detection kit has good detection performance, including high accuracy and sensitivity, a wide linear range, and considerable precision, etc. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The detection result analysis curve of the TAT kit linear range in Example 6 of the present application. DETAILED DESCRIPTION

[0024] The present application provides antibodies for the detection of thrombin-antithrombin III complex (TAT). Specifically, it includes an antibody that can bind to thrombin (T) and an antibody that can bind to antithrombin III (AT-III). The two antibodies can form an antibody pairing for the detection of thrombin-antithrombin III complex (TAT) by a double antibody sandwich method.

[0025] For the antibody, in some embodiments, the antibody comprises all or a portion of the constant region of the antibody. In some embodiments, the constant region is selected from various types, including IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 or IgG4), IgM, etc. The present application also discloses an antigen-binding fragment that can specifically bind to anti-TAT. Examples of the antigen-binding fragment include F(ab’)2, F(ab)2, Fab’, Fab, Fv, scFv, etc.

[0026] For the monoclonal antibody described in the present application, it can be obtained from a single clone by any means available or known in the art, including expression in any eukaryotic, prokaryotic or phage system. The monoclonal antibody for use in the present application can be prepared using a wide variety of techniques known in the art, including hybridoma technology, recombinant technology or phage display technology, etc.

[0027] Other products of the present invention also include intermediates used in the preparation of antibodies, including expression vectors, transgenic cell lines, or recombinant bacteria, or compositions comprising the foregoing. Specifically, expression vectors may be recombinant expression vectors, such as eukaryotic expression vectors, while transgenic cell lines or recombinant bacteria may also include eukaryotic or prokaryotic cells, such as Escherichia coli, yeast, or animal cells (e.g., mammalian cells, such as mouse cells or human cells), as well as cell lines.

[0028] The present invention also provides a TAT detection kit or detection reagent, which comprises an antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the kit further comprises a solid substrate for attachment of the antibody or antigen-binding fragment thereof, the solid substrate including but not limited to microplates, magnetic particles, filter paper for immunochromatography, polymers such as polystyrene, glass filters and other insoluble carriers. In some embodiments, the kit further comprises other components, including but not limited to enzymes for labeling, corresponding substrates, radioactive isotopes, fluorescent substances, colored substances, buffers, etc.

[0029] TAT is detected using the above-mentioned kit or detection reagent. In some embodiments, the detection method comprises: binding the antibody or antigen-binding fragment thereof of the present invention to the TAT antigen in the sample to be tested to form an antibody-antigen or antibody fragment-antigen complex; then, detecting the complex in the sample to determine whether the target antigen is present in the sample.

[0030] Specifically, in some embodiments, the detection reagent or kit comprises an antibody or antigen-binding fragment thereof of the present invention; this includes a pair of antibodies: an antibody against thrombin (T) and an anti-thrombin III antibody. One of these antibodies serves as a capture antibody, and the other serves as a detectable, labeled detection antibody. The specific detection method includes adsorbing the capture antibody onto a solid support; then adding a sample potentially containing TAT to the support; adding a labeled detection antibody to the support; and detecting the presence of the labeled antibody to determine the presence of TAT. Alternatively, the specific detection method may include known methods such as enzyme immunoassay, immunochemiluminescence, radioimmunoassay, fluorescence immunoassay, immunochromatography, competitive assay, or sandwich assay.

[0031] In a preferred embodiment, the present invention provides an immunochemiluminescent detection kit prepared using a preferred two-antibody pair (T-5# and AT-III-3#). Through independent screening of the most sensitive and specific monoclonal antibody pair, along with appropriate reagent configuration, this kit achieves excellent performance for TAT detection, with high specificity, accuracy, and sensitivity, a wide linear range, and considerable precision.

[0032] More specifically, the detection kit of the present application has the following advantages:

[0033] 1. The reagent has high accuracy, and the relative deviation of the detection results of each concentration sample is not more than 5%; the specificity is strong, and other interfering components can be excluded.

[0034] 2. The reagent has high sensitivity, wide linear range, and high correlation degree. The lower limit of the linear range (0.4 ng / mL) is lower than the reference value (4 ng / mL), avoiding false positive misjudgment; the actual linear range covers most of the sample concentrations. At the same time, the linear correlation coefficient r of the reagent in this interval is not less than 0.9995, ensuring the accuracy of the detection results in the whole range, and accurately monitoring the index change fluctuation of the patient.

[0035] 3. The reagent has high precision and good repeatability, and the intra- and inter- laboratory precision CVs are both less than 5%, which can provide reliable traceability basis for the detection data.

[0036] 4. The reagent has wide applicability, which is suitable for both high-speed full-automatic chemiluminescence workstations and small-sized chemiluminescence analyzers, and can meet the application scenarios of various hospitals including outpatient, emergency and community hospitals, while maintaining stable detection performance.

[0037] The technical solutions of the present application will be described in detail below in combination with examples.

[0038] The experimental materials used in the embodiments of the present application can be obtained from the market channels if no special description is made, and the experimental operation methods are carried out by the means known in the art if no special description is made.

[0039] Example 1: Recombinant expression of T and AT-III antigens

[0040] According to the thrombin (T) (1TMU_H) and antithrombin III (AT-III) (CAA48690.1) sequence information published by the National Center for Biotechnology Information (NCBI), the full-length sequences thereof were selected and cloned into a pCDNA3.4 eukaryotic expression vector, and the obtained plasmid was transfected into HEK293 cells (human embryonic kidney 293) by PEI transfection reagent (polyethylenimine, Polyethylenimine). After being cultured in a constant temperature shaker at 37℃, 8% CO2 and appropriate rotation speed for 7 days, the cell supernatant was collected by centrifugation, and the recombinant expressed T antigen and AT-III antigen were obtained by nickel medium affinity chromatography purification, with sizes of about 36.0 kDa and 58.2 kDa, respectively.

[0041] Example 2: Immunization of animals with recombinant T and AT-III

[0042] The recombinant T antigen prepared in Example 1 was diluted to 1 mg / mL with 20 mM PBS (phosphate buffered saline) at pH 7.4 and emulsified with Freund's adjuvant at a 1:1 ratio. Six- to eight-week-old Balb / c mice were immunized intraperitoneally with 100 μg of the emulsified recombinant T antigen per mouse, using Freund's complete adjuvant for the primary immunization and Freund's incomplete adjuvant for the booster. After three immunizations, tail vein blood was collected and titered. Those with titers exceeding 1 million were selected for cell fusion. 72 hours prior to fusion, direct immunization of the spleen with 30 μg of T antigen was performed.

[0043] The recombinant AT-III antigen prepared in Example 1 was diluted to 1 mg / mL with 20 mM PBS (phosphate buffered saline) at pH 7.4 and emulsified with Freund's adjuvant at a 1:1 ratio. Six- to eight-week-old Balb / c mice were immunized intraperitoneally with 100 μg of the emulsified recombinant AT-III antigen per mouse. Freund's complete adjuvant was used for the primary immunization, and incomplete adjuvant was used for the booster. After three immunizations, tail vein blood was collected and titered. Cells with titers exceeding 1 million were selected for cell fusion. 72 hours prior to fusion, direct immunization of the spleen with 30 μg of AT-III antigen was performed.

[0044] Example 3: Screening of T and AT-III monoclonal antibodies

[0045] (1) Fusion cells: The recombinant T and AT-III immunized mice to be fused were killed by cervical dislocation, and their spleens were removed, ground and filtered to prepare a spleen cell suspension, which was then transferred to a 50 mL centrifuge tube. The cells were resuspended in serum-free RPMI1640 medium and washed three times, each time by centrifugation at 15,000 rpm for 5 minutes. The myeloma cells were resuspended in serum-free RPMI1640 medium and washed three times, each time by centrifugation at 15,000 rpm for 5 minutes. The spleen cell suspension and myeloma cell suspension were mixed, centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and the bottom of the tube was flicked to disperse the cells. At the same time, 1 mL of PEG1450 pre-warmed to 37°C was slowly added within 60 seconds. After the addition was completed, the mixture was pipetted and mixed for 30 seconds. Then, 40 mL of RPMI 1640 medium pre-warmed to 37°C was immediately added to terminate the fusion reaction. The cells were transferred to RPMI 1640 HAT cell screening medium, mixed, and plated on a 96-well cell culture plate. After 7 days, the RPMI 1640 cell culture medium was replaced, and after another 3 days, part of the supernatant was aspirated for subsequent operations;

[0046] (2) Screening of T monoclonal antibodies: The recombinantly expressed T protein was coated in a polystyrene 96-well ELISA plate at a total amount of 50 ng / well. The supernatant of the recombinant T immune mouse cells obtained in Example 3 (1) was added to the coated 96-well plate. After incubation and washing, the plate was incubated with HRP-labeled goat anti-mouse secondary antibody, reaction substrate and stop solution. The cell wells corresponding to the high-value positive wells were selected for subcloning. After five subclones, seven hybridoma monoclonal cell lines with good reactivity to the recombinant T protein (OD value ≥ 3.0) were finally selected.

[0047] (3) Screening of AT-III monoclonal antibodies: The recombinantly expressed AT-III protein was coated in a polystyrene 96-well ELISA plate at a total amount of 50 ng / well. The supernatant of the recombinant AT-III immune mouse cells obtained in Example 3 (1) was added to the coated 96-well plate. After incubation and washing, the plate was incubated with HRP-labeled goat anti-mouse secondary antibody, reaction substrate, and stop solution. The cell wells corresponding to the high-value positive wells were selected for subcloning. After five subclones, five hybridoma monoclonal cell lines with good reactivity to the recombinant AT-III protein (OD value ≥ 3.0) were finally selected.

[0048] Example 4: Paired screening of T and AT-III monoclonal antibodies

[0049] (1) HRP labeled antibody: Dissolve 30 mg of HRP dry powder in 1.5 mL of pure water, add 1.2 mL of 25 mg / mL NaIO4 aqueous solution, mix well, and react at 4°C for 30 min. Remove and add 0.2 mL of ethylene glycol solution, let it sit at room temperature for 30 min to complete HRP activation, and store at 4°C for use. Dilute AT-III antibody to 2 mg / mL with 20 mM carbonate buffer pH 9.6, mix 2 mL of the AT-III antibody to be labeled with the activated HRP labeling solution, and dialyze overnight in 20 mM carbonate buffer pH 9.6. Remove and add 0.2 mL of 2 mg / mL NaBH4 solution, let it react at 4°C for 2 hours. Add an equal volume of saturated ammonium sulfate solution, let it react at 4°C for 30 min, then centrifuge. Resuspend the precipitate with 1 mL of 20 mM PBS pH 7.4 solution, add an equal volume of glycerol, mix well, and store.

[0050] (2) Paired detection: Use 20mM PBS pH7.4 solution to dilute T antibody to 1μg / mL, 100μL / well antithrombin antibody is coated on a polystyrene 96-well enzyme-labeled plate and incubated at 37℃ for 2 hours. Wash the 96-well plate 5 times with PBST, add blocking solution for blocking, and discard the blocking solution after blocking. Add 100μL / well of TAT diluted to 10ng / mL to the 96-well plate, incubate at 37℃ for 1 hour, wash the 96-well plate 5 times with PBST, add HRP-labeled AT-III antibody, incubate at 37℃ for 30min, wash the 96-well plate 5 times with PBST, and then incubate the reaction substrate and stop solution in sequence, and place it in an enzyme-labeled instrument for reading. Use T antibody as the coating antibody and AT-III antibody as the labeled antibody. After pairwise detection, the monoclonal antibody pair with the highest sensitivity and best specificity is selected, that is, monoclonal antibody T-5# and AT-III-3#.

[0051] Example 5: Sequence determination and binding epitope analysis of monoclonal antibodies

[0052] (1) Antibody sequence determination: Nanjing GenScript Biotech Co., Ltd. was commissioned to perform de-novo protein sequencing on two monoclonal antibodies, T-5# and AT-III-3#. After spectral analysis, the amino acid sequences of the heavy chain and light chain variable regions of the two monoclonal antibodies, T-5# and AT-III-3#, were obtained. The specific amino acid sequences are shown in Table 1. The subtype of the two antibodies was mouse IgG1.

[0053] Table 1 Amino acid sequences of the variable regions of monoclonal antibodies

[0054]

[0055] (2) Antibody binding epitope analysis: Nanjing GenScript Biotech Co., Ltd. was commissioned to conduct binding epitope analysis on two monoclonal antibodies, T-5# and AT-III-3#. The binding epitopes of both antibodies were linear epitopes, and the relevant information is shown in Table 2. Among them, the binding epitope of monoclonal antibody T-5# was the amino acid sequence from position 104 to position 117 of the full-length thrombin protein: KLKKPVAFSDYIHP (Lys-Leu-Lys-Lys-Pro-Val-Ala-Phe-Ser-Asp-Tyr-Ile-His-Pro), and the binding epitope of monoclonal antibody AT-III-3# was the amino acid sequence from position 78 to position 88 of the full-length antithrombin III protein: RRVWELSKANS (Arg-Arg-Val-Trp-Glu-Leu-Ser-Lys-Ala-Asn-Ser).

[0056] Table 2 Binding epitope information of monoclonal antibodies

[0057]

[0058] Example 6: Configuration of TAT detection reagents

[0059] (1) Preparation of antibody buffer: Add 50 mM pH 7.3 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.5% lauryl polyoxyethylene ether, 50 mM tris(hydroxymethylaminomethane) (Tris), 2% sucrose, 4% arginine, 2.5% casein, and 0.1% sodium azide to prepare antibody buffer. Adjust the pH to 7.30 ± 0.05 (25°C). Filter through a 0.8 μm filter to remove large particles.

[0060] (2) Preparation of magnetic bead storage buffer: Add 50 mM pH 7.3 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.5% lauryl polyoxyethylene ether, 50 mM potassium dihydrogen phosphate, 20 mM sodium chloride, 2% sucrose, 4% arginine, 1% bovine serum albumin, and 0.1% sodium azide to prepare magnetic bead storage buffer. The pH is adjusted to 7.40 ± 0.05 (25°C). Filter through a 0.8 μm filter membrane to remove large particles.

[0061] (3) Biotin labeling of antibodies: Dissolve biotin to 1 mg / ml using dimethyl sulfoxide solution, add TAT capture antibody T-5#, mix at a mass ratio of 0.5:50, then dilute with an equal volume of carbonate buffer (pH 9.6), incubate at 37°C for 1 hour to complete labeling. The final concentration of biotin-labeled TAT capture antibody is 50 μg / mL.

[0062] (4) Coupling of magnetic bead reagent: 20 mg of concentrated streptavidin-labeled magnetic beads were magnetically separated and washed three times with antibody buffer. The biotin-labeled TAT capture antibody was added and mixed at a mass ratio of 1250:3. The mixture was reacted at 30°C for 1 h in an antibody buffer environment to complete the coupling of the antibody and magnetic beads. The supernatant was discarded after magnetic separation and resuspended with magnetic bead storage buffer. The volume was diluted to 16 mL to obtain the magnetic bead reagent with antibody coupling.

[0063] (5) Coupling of acridinium ester labeling reagent: Dissolve acridinium ester in dimethyl sulfoxide solution to 5 mg / mL, add TAT detection antibody AT-III-3#, mix at a mass ratio of 2.5:50, and then add an equal volume of carbonate buffer pH 9.6 to dilute. Incubate at 37°C for 1 hour to complete the labeling. The final concentration of acridinium ester-labeled TAT detection antibody is 50 μg / mL.

[0064] (6) Preparation of calibrators and quality control products: TAT antigen lyophilized powder (from Nanjing Bomi No. Biotechnology Co., Ltd.) was prepared into calibrators with calibrator diluent at concentrations of 0.51 ng / mL, 0.99 ng / mL, 4.01 ng / mL, 10.02 ng / mL, 19.92 ng / mL, 40.10 ng / mL, 60.04 ng / mL, 80.31 ng / mL, 99.77 ng / mL, and 119.81 ng / mL. The composition of the calibrator diluent was: 50 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) at pH 7.3, 0.5% polyoxyethylene lauryl alcohol ether, 50 mM potassium chloride, 2% sucrose, 4% arginine, and 0.1% sodium azide. TAT antigen lyophilized powder was prepared into quality control products with calibrator diluent at concentrations of 4 ng / mL and 40 ng / mL, which were used as low-value quality control products and high-value quality control products, respectively.

[0065] Example 7: Accuracy of the kit

[0066] The accuracy of a reagent refers to the degree of agreement between the average value of the quantity obtained by repeated measurements an infinite number of times and a reference value.

[0067] The TAT detection kit described in Example 6 was used to test one sample of a high-concentration (40.10 ± 1.42 ng / mL) and one sample of a low-concentration (4.01 ± 0.16 ng / mL) TAT reference product, respectively, three times. The test results are shown in Table 3. Based on the relative deviation of the test results, the relative deviation of the three test results for the two reference products was within 5%, indicating that the kit has good accuracy.

[0068] Table 3 Test results of the TAT kit accuracy in Example 6

[0069]

[0070] In addition, the two antibodies of the present invention are formulated to form the TAT detection kit in Example 6, which can specifically identify the TAT complex during the detection process, has high specificity, and effectively eliminates interference from other impurities in the sample to be tested, ensuring the specificity and accuracy of the test results.

[0071] Example 8: Linear range of the kit

[0072] The expected linear range of the TAT detection kit in Example 6 is 0.40~120.00 ng / mL. A high value sample (123.21 ng / mL) and a low value sample (0.40 ng / mL) were selected near the upper and lower limits of the range. The high and low value samples were accurately diluted in different proportions with the zero value TAT calibrator to prepare the following five different concentration levels (0.40 ng / mL, 6.54 ng / mL, 31.10 ng / mL, 61.81 ng / mL, 123.21 ng / mL) of the test samples, which were detected using the TAT kit in Example 1.

[0073] The test results are shown in Table 4. After fitting curve and linear analysis, the linear regression equation of the test kit is: y=1.007x-0.2186, r=0.9999>0.995( ​ ), it is preliminarily judged that it meets the expected linear range requirements; the relative deviation between each measured value and the theoretical value of each concentration sample is no more than 10%, and the expected linear range is acceptable.

[0074] Table 4 Detection results of the linear range of the TAT kit in Example 6

[0075]

[0076] Example 9: Precision of the kit

[0077] Precision refers to the degree of agreement between labeled or measured values ​​obtained from repeated measurements of the same or similar analytes under specified conditions. The precision of the kit described in Example 6 was assessed using repeatability, intermediate precision (within-laboratory precision), and reproducibility (between-laboratory precision).

[0078] This performance was evaluated using a high-concentration TAT quality control product 1 (target value 40.00 ng / mL), a low-concentration quality control product 2 (target value 4.00 ng / mL), and a mixed quality control product (target value 22.00 ng / mL) of known concentrations.

[0079] In the same laboratory, using the same detection instrument, three batches of samples were tested separately, with two analytical batches tested every day. Two samples of each concentration in each batch were processed in parallel for testing to evaluate the laboratory precision of the kit.

[0080] In the same laboratory, samples were tested separately using three different commercially available instruments (NRM411, NORMAN-CL 5, and NRM411-S7) and a single batch of the test kit. Each batch of samples was tested continuously for 5 consecutive days, with each batch of samples repeated 5 times daily to evaluate the inter-laboratory precision of the test kit.

[0081] The results of the intra-laboratory precision and inter-laboratory precision tests were analyzed using a two-factor variance analysis model. The results of the variance analysis are shown in Table 5, the results of the standard deviation and CV analysis are shown in Table 6, and the results of the confidence interval analysis are shown in Table 7. All of the above results were as expected and no abnormal values were found.

[0082] Table 5 Precision variance analysis results for the TAT kit in Example 6

[0083]

[0084] Table 5: Related Notes: SS site: Sum of Squares - inter-laboratory (inter-instrument); MS site: Mean Square - inter-laboratory (inter-instrument); SS day: Sum of Squares - inter-day; MS day: Mean Square - inter-day; SS error: Sum of Squares - intra-batch; MS error: Mean Square - intra-batch; SS total: Sum of Squares - total.

[0085] Table 6 Precision analysis results for the TAT kit in Example 6

[0086]

[0087] Table 6: Related Notes: s: standard deviation; CV: coefficient of variation.

[0088] Table 7 Precision confidence interval (95%, a = 0.05) for the TAT kit in Example 6

[0089]

[0090] The present application has been described in detail with reference to specific embodiments and illustrative examples, but these are not to be construed as limiting the application. Those skilled in the art understand that various equivalent substitutions, modifications and improvements can be made to the technical solutions and embodiments of the present application without departing from the spirit and scope of the present application, and these all fall within the scope of the present application.

Claims

1. An antibody or antigen-binding fragment thereof targeting the thrombin portion of the thrombin-antithrombin III complex, characterized in that: Comprising the light and heavy chain variable regions of a thrombin antibody; the light and heavy chain variable regions of the thrombin antibody comprise VHCDR1 as shown in SEQ ID NO: 1, VHCDR2 as shown in SEQ ID NO: 2, VHCDR3 as shown in SEQ ID NO: 3, VLCDR1 as shown in SEQ ID NO: 4, VLCDR2 with a sequence of STD, and VLCDR3 as shown in SEQ ID NO:

5.

2. An antibody or antigen-binding fragment thereof targeting the antithrombin III portion of the thrombin-antithrombin III complex, characterized in that: Comprising the light and heavy chain variable regions of an anti-thrombin III antibody; the light and heavy chain variable regions of the anti-thrombin III antibody comprise VHCDR1 as shown in SEQ ID NO: 6, VHCDR2 as shown in SEQ ID NO: 7, VHCDR3 as shown in SEQ ID NO: 8, VLCDR1 as shown in SEQ ID NO: 9, VLCDR2 with a sequence of SAS, and VLCDR3 as shown in SEQ ID NO:

10.

3. The antibody or antigen-binding fragment thereof targeting the thrombin portion of the thrombin-antithrombin III complex according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the thrombin antibody is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

12.

4. The antibody or antigen-binding fragment thereof targeting the antithrombin III portion of the thrombin-antithrombin III complex according to claim 2, characterized in that The amino acid sequence of the heavy chain variable region of the anti-thrombin III antibody is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

14.

5. The antibody or antigen-binding fragment thereof targeting the thrombin portion of the thrombin-antithrombin III complex according to claim 1, characterized in that The antibody or antigen-binding fragment thereof can specifically recognize and bind to the epitope of the thrombin-antithrombin III complex antigen such as SEQ ID NO:

15.

6. The antibody or antigen-binding fragment thereof targeting the antithrombin III portion of the thrombin-antithrombin III complex according to claim 2, characterized in that The antibody or antigen-binding fragment thereof can specifically recognize and bind to the epitope of the thrombin-antithrombin III complex antigen as shown in SEQ ID NO:

16.

7. The antibody or antigen-binding fragment thereof targeting the thrombin portion of the thrombin-antithrombin III complex according to claim 1, characterized in that The thrombin antibody includes a monoclonal antibody; an antigen-binding fragment thereof is selected from F(ab')2, F(ab)2, Fab', Fab, Fv or scFv.

8. The antibody or antigen-binding fragment thereof targeting the antithrombin III portion of the thrombin-antithrombin III complex according to claim 2, characterized in that The anti-thrombin III antibody includes a monoclonal antibody; an antigen-binding fragment thereof is selected from F(ab')2, F(ab)2, Fab', Fab, Fv or scFv.

9. A nucleic acid encoding an antibody or antigen-binding fragment thereof targeting the thrombin portion of the thrombin-antithrombin III complex according to claim 1, 3, 5 or 7, or an antibody or antigen-binding fragment thereof targeting the antithrombin III portion of the thrombin-antithrombin III complex according to claim 2, 4, 6 or 8.

10. An expression vector comprising the nucleic acid of claim 9.

11. A transgenic cell line or recombinant bacterium comprising the nucleic acid of claim 9 or the expression vector of claim 10.

12. An immunoconjugate, characterized in that The invention comprises an antibody portion and a coupling portion coupled to the antibody portion, wherein the antibody portion comprises an antibody or an antigen-binding fragment thereof targeting the thrombin portion in the thrombin-antithrombin III complex according to claim 1, 3, 5 or 7, or an antibody or an antigen-binding fragment thereof targeting the antithrombin III portion in the thrombin-antithrombin III complex according to claim 2, 4, 6 or 8, and the coupling portion is selected from a fluorescent substance, a chemiluminescent substance, a colored substance, an enzyme or a combination thereof.

13. A thrombin-antithrombin III complex detection kit, characterized in that: An antibody or antigen-binding fragment thereof targeting the thrombin portion of the thrombin-antithrombin III complex according to claim 1, 3, 5 or 7 and an antibody or antigen-binding fragment thereof targeting the antithrombin III portion of the thrombin-antithrombin III complex according to claim 2, 4, 6 or 8.

14. The thrombin-antithrombin III complex detection kit according to claim 13, characterized in that: The thrombin antibody and the anti-thrombin III antibody are used in pairs; the thrombin antibody is used as a capture antibody, and the anti-thrombin III antibody is used as a detection antibody; Alternatively, anti-thrombin III antibody is used as the capture antibody and anti-thrombin antibody is used as the detection antibody.

15. The thrombin-antithrombin III complex detection kit according to claim 13, characterized in that: It includes magnetic particles coated with capture antibodies, detection antibodies labeled with luminescent markers, substrates and excitation fluids suitable for the luminescent markers, and a series of quality control products and calibrators for thrombin-antithrombin III complex antigens.

16. The thrombin-antithrombin III complex detection kit according to claim 15, characterized in that: The capture antibody-coated magnetic particles are formed by coupling streptavidin-coated magnetic beads with biotinylated thrombin antibodies; and the luminescent marker comprises acridinium ester.

17. Use of the antibody or antigen-binding fragment thereof targeting the thrombin portion of the thrombin-antithrombin III complex according to claim 1, 3, 5 or 7, the antibody or antigen-binding fragment thereof targeting the antithrombin III portion of the thrombin-antithrombin III complex according to claim 2, 4, 6 or 8, the nucleic acid according to claim 9, the expression vector according to claim 10, the transgenic cell line or recombinant bacterium according to claim 11, or the immunoconjugate according to claim 12 in the preparation of a detection reagent or kit for evaluating early coagulation dysfunction.

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