WO2022237888A1 - 一种检测血栓或凝血相关疾病的生物标志物及其应用 - Google Patents

一种检测血栓或凝血相关疾病的生物标志物及其应用 Download PDF

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WO2022237888A1
WO2022237888A1 PCT/CN2022/092581 CN2022092581W WO2022237888A1 WO 2022237888 A1 WO2022237888 A1 WO 2022237888A1 CN 2022092581 W CN2022092581 W CN 2022092581W WO 2022237888 A1 WO2022237888 A1 WO 2022237888A1
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antibody
detection
fxiiiap
thrombus
related diseases
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PCT/CN2022/092581
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English (en)
French (fr)
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林哲
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医工瑞思(福建)工程研究中心有限公司
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Priority to CN202280034900.0A priority Critical patent/CN117396759A/zh
Publication of WO2022237888A1 publication Critical patent/WO2022237888A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G01N2800/224—Haemostasis or coagulation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G01N2800/226—Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

Definitions

  • the invention relates to the technical field of detection of thrombus or coagulation-related diseases, in particular to a biomarker for detecting thrombus or coagulation-related diseases and its application.
  • Thrombosis and related coagulation diseases have always been important diagnostic items in clinical diagnosis, involving a wide range of diseases, including cerebral venous thrombosis (CVT), renal vein thrombosis (RVT), venous thrombosis (VTE), myocardial infarction (MI), etc. .
  • CVT cerebral venous thrombosis
  • RVVT renal vein thrombosis
  • VTE venous thrombosis
  • MI myocardial infarction
  • the existing diagnostic methods mainly include computed tomography (CT) and magnetic resonance imaging (MRI), which are very effective in judging the occurrence, type and severity of stroke. efficient.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • CAT scan computerized axial tomography
  • cerebral ischemia usually 24 to 36 hours after stroke onset, and approximately 50% of ischemic brain Strokes are difficult to see on a CAT scan.
  • Biomarkers refer to biochemical indicators that can mark the changes in the structure or function of organs, tissues or cells of organisms. Therefore, the detection of biomarkers can be used for disease diagnosis and provide scientific basis for subsequent treatment plans.
  • Stroke-related molecular markers are disclosed in the invention patent with publication number CN103299191A.
  • the invention patent with publication number CN1339108A discloses a method for diagnosing and distinguishing apoplexy.
  • Thrombosis can be used as an important indicator in the diagnosis of cardiovascular diseases such as ischemic stroke.
  • the currently clinically used biomarker screening test for thrombus is D-dimer detection, which is mainly used in the diagnosis of venous thromboembolism (VTE), deep vein thrombosis (DVT) and pulmonary embolism (PE).
  • VTE venous thromboembolism
  • DVT deep vein thrombosis
  • PE pulmonary embolism
  • D-dimer is derived from cross-linked fibrin clots dissolved by plasminase, which mainly reflects the state of fibrinolysis (thrombosis), rather than directly reflecting thrombus formation. All factors affecting thrombolysis can affect the results of D-dimer detection. For example, patients with thrombolytic disorders, patients taking oral anticoagulants, and patients with VTE symptoms lasting more than 14 days may all be false negatives. In addition, D-dimer reflects the process of thrombolysis and has a long half-life (13-23 hours). False-positive results can also be expected when patients have other triggers that can cause blood clotting, such as inflammation or pregnancy.
  • D-dimer also has a high misdiagnosis rate in clinical tests.
  • D-dimers are missed in 20% of cases, with higher rates (approximately 50%) in early stages (less than 24 hours after symptom onset).
  • false-negative results are prone to occur for early newly formed thrombus D-dimer.
  • D-dimer Due to the long half-life of D-dimer, it cannot accurately and timely reflect whether the body is undergoing bleeding and coagulation when there is a coagulation disease. Additionally, due to poorly defined D-dimer analytes, different assays are based on different monoclonal antibodies that recognize different fibrin fragments or surface structures. There are no reference preparations or calibrators that can be used as international standards. Coupled with its low sensitivity and specificity, the application of D-dimer is still controversial.
  • test results lack the meaning of treatment guidance, and cannot timely and accurately reflect the coagulation status in the body, and cannot indicate the treatment strategy, such as the effect of anticoagulation, and it is also difficult to confirm whether new thrombus has occurred, because old thrombus can still be dissolved to produce D-dimerization body.
  • the present invention proposes a biomarker that can directly detect thrombus formation itself, which can Alone or in combination with D-dimer detection, it can detect and screen with high sensitivity and high accuracy for thrombosis or coagulation-related diseases.
  • the invention provides a biomarker, which can be used for detection of thrombus or blood coagulation-related diseases.
  • the present invention also provides the application of the above-mentioned biomarkers in detection products of thrombosis or coagulation-related diseases and therapeutic effect evaluation products;
  • the present invention also provides a kit for detecting thrombus or coagulation-related diseases.
  • the main technical solutions adopted in the present invention include:
  • the embodiments of the present invention provide a biomarker that can be used for the detection of thrombus or coagulation-related diseases; the marker includes FXIIIAP or FXIIIA, wherein FXIIIAP is the activating peptide of coagulation factor 13, and FXIIIA is A of coagulation factor 13 Subunit.
  • said biomarkers include a combination of FXIIIAP and D dimer.
  • the coagulation-related diseases include stroke, cerebral venous thrombosis, renal vein thrombosis, venous thrombosis or myocardial infarction.
  • the present invention also provides a method for diagnosing thrombosis or cardiovascular-related diseases, which includes the following steps:
  • the sample to be tested is blood or plasma.
  • a specific antibody is prepared using a partial sequence of FXIIIAP as an antigen; the partial sequence of FXIIIAP includes the following sequence SETSRTAFGGRRAVPNNNSNAAEDDLPTVELQGVV or RTAFGGRRAVPPNN.
  • the embodiment of the present invention provides an application of the above-mentioned biomarker in detection products and treatment effect evaluation products of thrombus or blood coagulation-related diseases.
  • the detection products and treatment effect evaluation products of thrombosis or coagulation-related diseases include kits or reagents.
  • an embodiment of the present invention provides a kit for detecting thrombus or coagulation-related diseases, and the capture antibody or/and detection antibody in the kit is a specific antibody prepared from the above-mentioned biomarker as an antigen .
  • the kit for detecting thrombus or coagulation-related diseases is a double-antibody sandwich ELISA detection kit or a dry immunofluorescence detection kit, including a capture antibody and a detection antibody.
  • the capture antibody and detection antibody are prepared using the whole or partial sequence of FXIIIAP as an antigen.
  • the partial sequence includes the following sequence or a fragment containing the following sequence: SETSRTAFGGRRAVPNNNSNAAEDDLPTVELQGVV or RTAFGGRRAVPPNN.
  • the capture antibody and detection antibody are extracted from experimental animals immunized with FXIIIAP.
  • the antibody of the present invention in addition to being applied to enzyme-linked immunosorbent assay (ELISA), can also be used in colloidal gold method, radioimmunoassay (radioimmunoassay, RIA), chemiluminescence immunoassay (chemiluminescent immunoassay, CLIA), electro-chemiluminescence immunoassay (electro-chemiluminescence immunoassay, ECLI), fluorescence immunoassay such as dry-type immunofluorescence method (dry-type immunofluorescence method), time-resolved fluoroisnmunoassay (time-resolved fluoroisnmunoassay) , TRFIA) to detect the above biomarkers.
  • radioimmunoassay radioimmunoassay
  • CLIA chemiluminescent immunoassay
  • electro-chemiluminescence immunoassay electro-chemiluminescence immunoassay
  • fluorescence immunoassay such
  • the present invention mainly provides a biomarker that can be used to detect thrombus or blood coagulation-related diseases. Because it can be combined with the traditional marker D-dimer, compared with the prior art, it can significantly improve the sensitivity (detection rate) .
  • the marker FXIIIAP pointed out in the present invention has a better detection effect on thrombus in the acute phase (less than 48 hours).
  • the rapid degradation of FXIIIAP due to the rapid degradation of FXIIIAP, it cannot be detected 48 hours after the onset of symptoms, and the missed diagnosis rate is very high.
  • the appearance of the biomarkers proposed by the present invention is not related to the process of fibrinolysis. Compared with the prior art, it can be applied to the detection of patients with insufficient fibrinolysis;
  • the biomarker proposed by the present invention can better reflect whether there is new thrombus formation in the body. Therefore, it can also be used to evaluate the effect of treatment and the severity of the disease.
  • the biomarkers proposed by the present invention can rapidly diagnose coagulation diseases including stroke, improve the sensitivity and specificity of existing diagnosis, predict the time and degree of thrombus formation, and predict the severity and prognosis of the disease.
  • the application of the present invention can provide information for treatment strategies, such as providing guidance for anticoagulant or thrombolytic therapy, and effectively monitor the effect of anticoagulant drugs.
  • the detection kit proposed by the present invention can detect finger blood and corresponding long-term detection, and has extremely high commercial value.
  • the detection kit provided by the present invention makes up for the gap of FXIIIAP detection kits on the market.
  • Fig. 1 is a diagram of the detection results of ischemic stroke patient samples with the traditional marker D dimer as the control of the present invention
  • Fig. 2 is a diagram showing the detection results of the marker FXIIIAP of the present invention on samples from patients with ischemic stroke;
  • Fig. 3 is a comparison chart of the sensitivity of the marker FXIIIAP according to the present invention and the traditional marker D-dimer in the detection results of samples from patients with early ischemic stroke;
  • Fig. 4 is a sensitivity diagram of the detection results of the FXIIIAP and D-dimer marker combination proposed by the present invention on samples from patients with early ischemic stroke;
  • Figure 5 shows the antibody described in embodiment 3-1-1 (original antibody, denoted as 1-37), the antibody described in embodiment 3-1-2 (improved antibody 1, denoted as 1-35) and embodiment 3 -The antibody described in 1-4 (improved antibody 2, denoted as 5-18) is used for the contrast chart of the sensitivity of FXIIIAP detection by ELISA;
  • Figure 6 shows the antibody described in embodiment 3-1-1 (original antibody, denoted as 1-37), the antibody described in embodiment 3-1-2 (improved antibody 1, denoted as 1-35) and embodiment 3 -1-4 described antibody (improved antibody 2, denoted as 5-18) is used in ELISA to FXIIIAP detection specificity contrast figure;
  • Figure 7 shows the basic information of patient A
  • Fig. 8 shows the thrombus monitoring data 1 of patient A
  • Fig. 9 shows the thrombus monitoring data 2 of patient A
  • Figure 10 is a monitoring change diagram of the patient's A white blood cell and promyelocyte ratio
  • Figure 11 is a monitoring change chart of FXIIIAP and fibrinogen of patient A
  • Fig. 12 is a monitoring change diagram of D dimer and fibrin monomer of patient A;
  • Figure 13 shows the basic information of patient B
  • Figure 14 shows the thrombus monitoring data 1 of patient B
  • Figure 15 shows the thrombus monitoring data two of patient B
  • Fig. 16 is a monitoring change diagram of the ratio of white blood cells and promyelocytes in patient B;
  • Figure 17 is a monitoring change diagram of FXIIIAP and fibrinogen of patient B;
  • Fig. 18 is a monitoring change diagram of D dimer and fibrin monomer of patient B;
  • Figure 19 shows the basic information of patient C
  • Figure 20 shows patient C's thrombus monitoring data
  • Figure 21 shows the basic information of patient D
  • Figure 22 shows Patient D's thrombus monitoring data.
  • a biomarker for detection of thrombosis or coagulation-related diseases FXIIIAP coagulation factor 13 activating peptide.
  • a biomarker for detection of thrombosis or coagulation-related diseases FXIIIA (subunit A of coagulation factor 13).
  • a biomarker for detecting thrombosis or coagulation-related diseases is a combination of FXIIIAP and FXIIIA.
  • a biomarker for detection of thrombosis or coagulation-related diseases is a combination of FXIIIAP and D-dimer.
  • a biomarker for detection of thrombosis or coagulation-related diseases it is a combination of FXIIIA and D-dimer.
  • a biomarker for the detection of thrombosis or coagulation-related diseases a combination of FXIIIAP, FXIIIA and D-dimer.
  • the combination of FXIIIAP or FXIIIA described in Embodiments 1-4, Embodiments 1-5 and Embodiments 1-6 and the biomarker formed by D dimer can be more accurately applied to the detection of thrombus or Coagulation-related disorders.
  • the appearance of FXIIIAP is not associated with the fibrinolytic process. Therefore, patients with insufficient fibrinolysis can also be detected by this method.
  • FXIIIAP appeared earlier than D-dimer. In clinical tests, it was found that FXIIIAP has a better detection effect on acute thrombus (less than 2 days). On the other hand, D-dimer starts to increase on the second day after the onset of symptoms, and increases significantly on the third day.
  • FXIIIAP will be degraded quickly in the body, so its increase reflects the current situation of thrombus formation in the body. Compared with D-dimer, it can better reflect whether there is new thrombus formation in the body. Therefore, it can also be used for the evaluation of the treatment effect and the severity of the disease. However, because it is often rapidly degraded, it may not be detected in patients with advanced symptoms, which has caused a certain rate of missed diagnosis. Therefore, it must be used in combination with D-dimer to reduce the risk of missed diagnosis.
  • FXIIIAP is not only effective for stroke caused by thrombus, but also theoretically has a considerable effect on hemorrhagic stroke.
  • the permeability of the blood-brain barrier increases, and coagulation factors can pass through the blood-brain barrier.
  • the molecular weight of FXIIIAP is very small (only 3.91KD), it can pass through the blood-brain barrier smoothly.
  • the brain is rich in tissue factor, when the blood-brain barrier is damaged, tissue factor will be released into the body, causing blood coagulation in the body. Therefore, some studies have used D-dimer to detect intracranial hemorrhage, but the results are not satisfactory.
  • coagulation-related diseases referred to in the present invention include but not limited to stroke, cerebral venous thrombosis, renal vein thrombosis, venous thrombosis, myocardial infarction, and may be all diseases caused by thrombosis.
  • Embodiment 1-1 The application of the biomarker as described in Embodiment 1-1 in detection products and therapeutic effect evaluation products of thrombus or coagulation-related diseases.
  • the product includes kits or reagents.
  • the product includes kits or reagents.
  • the product includes kits or reagents.
  • the product includes kits or reagents.
  • the product includes kits or reagents.
  • the product includes kits or reagents.
  • kits for detecting thrombus or coagulation-related diseases which includes a capture antibody or/and a detection antibody, and the capture antibody and detection antibody are prepared using the biomarker described in Embodiment 1-1 as an antigen specific antibodies. Further, in other embodiments, the kit is more specifically prepared as a double-antibody sandwich ELISA detection kit including a capture antibody and a detection antibody.
  • the capture antibody and detection antibody are prepared using the entire sequence of FXIIIAP as an antigen.
  • the capture antibody and detection antibody are prepared using a partial sequence of FXIIIAP as an antigen, and the partial sequence is: SETSRTAFGGRRAVPNNNSNAAEDDLPTVELQGVV.
  • the capture antibody and detection antibody are prepared using a partial sequence of FXIIIAP as an antigen, and the partial sequence is: a partial fragment of SETSRTAFGGRRAVPNNNSNAAEDDLPTVELQGVV.
  • the capture antibody and detection antibody are prepared using a partial sequence of FXIIIAP as an antigen, and the partial sequence is: RTAFGGRRAVPPNN.
  • the capture antibody and detection antibody are prepared using a partial sequence of FXIIIAP as an antigen, and the partial sequence is: a partial fragment of RTAFGGRRAVPPNN.
  • a kit for detecting thrombus or coagulation-related diseases which includes a capture antibody and a detection antibody, and the capture antibody and detection antibody are specific antibodies made using the biomarkers described in Embodiment 1-2 as antigens. Sexual antibodies. Further, in other embodiments, the kit is more specifically prepared as a double-antibody sandwich ELISA detection kit including a capture antibody and a detection antibody.
  • a kit for detecting thrombus or coagulation-related diseases which includes a capture antibody and a detection antibody, and the capture antibody and detection antibody are specific antibodies made using the biomarkers described in Embodiments 1-3 as antigens. Sexual antibodies. Further, in other embodiments, the kit is more specifically prepared as a double-antibody sandwich ELISA detection kit including a capture antibody and a detection antibody.
  • a kit for detecting thrombus or coagulation-related diseases which includes a capture antibody and a detection antibody, and the capture antibody and detection antibody are specific antibodies made using the biomarkers described in Embodiments 1-4 as antigens. Sexual antibodies.
  • the kit is more specifically prepared as a double-antibody sandwich ELISA detection kit including a capture antibody and a detection antibody.
  • the capture antibody and detection antibody are extracted from the experimental animals immunized with FXIIIAP, to be precise, extracted from the IgG of these experimental animals immunized with FXIIIAP.
  • a kit for detecting thrombus or coagulation-related diseases which includes a capture antibody and a detection antibody, and the capture antibody and detection antibody are specific antibodies made using the biomarkers described in Embodiments 1-5 as antigens. Sexual antibodies.
  • the kit is more specifically prepared as a double-antibody sandwich ELISA detection kit including a capture antibody and a detection antibody.
  • the capture antibody and detection antibody are extracted from the experimental animals immunized with FXIIIAP, to be precise, extracted from the IgG of these experimental animals immunized with FXIIIAP.
  • a kit for detecting thrombus or coagulation-related diseases which includes a capture antibody and a detection antibody, and the capture antibody and detection antibody are specific antibodies made using the biomarkers described in Embodiments 1-6 as antigens. Sexual antibodies.
  • the kit is more specifically prepared as a double-antibody sandwich ELISA detection kit including a capture antibody and a detection antibody.
  • the capture antibody and detection antibody are extracted from the experimental animals immunized with FXIIIAP, to be precise, extracted from the IgG of these experimental animals immunized with FXIIIAP.
  • a kit for detecting thrombus or coagulation-related diseases which includes a capture antibody and a detection antibody, and the capture antibody and detection antibody are specific antibodies made using the biomarkers described in Embodiments 1-6 as antigens. Sexual antibodies. Further, in other embodiments, the kit is more specifically prepared as a double-antibody sandwich ELISA detection kit including a capture antibody and a detection antibody.
  • the capture antibody and detection antibody are extracted from the experimental animals immunized with FXIIIAP, to be precise, extracted from the IgG of these experimental animals immunized with FXIIIAP.
  • coagulation factor 13 is the last coagulation factor activated during the coagulation process, and its main function is to crosslink the fibrin clot to stabilize the thrombus. Its activation reflects the completion of the coagulation process and the formation of a stable thrombus. During its activation, each FXIII releases two coagulation factor 13 activating peptides (FXIIIAP).
  • FXIIIAP is a polypeptide of only 37 amino acids (approximately 3.91 KD). Its function is to stabilize the dimer structure of FXIIIA 2 . After being activated by thrombin, FXIIIAP leaves FXIII and enters the blood, becoming free FXIIIAP.
  • the structural conformation of FXIIIAP in the free state is obviously different from that of FXIIIAP bound to FXIII.
  • the specific antibody prepared using the synthesized FXIIIAP as the antigen cannot combine with the FXIIIAP in the unactivated FXIII (FXIIIAP in the bound state).
  • the specific antibody of the present invention can effectively detect the FXIIIAP in the free state, without combining with unactivated FXIII. This feature guarantees diagnostic specificity, enabling accurate detection of activated FXIII. It has been proved by clinical experiments that the released FXIIIAP can be detected by ELISA or fluorescent immunoassay.
  • the core component of the kit proposed by the invention is the FXIIIAP-specific antibody produced by using the improved FXIIIAP amino acid sequence.
  • FXIIIAP is cleaved by Thrombin and released into the blood, forming a double ⁇ -strands structure with an amino acid sequence of 1-35, while the amino acids at 36 and 37 do not contribute to the formation of this structure.
  • the instability of these two amino acids directly leads to the variable structure of the carboxy-terminal of the synthesized FXIIIAP, the produced antibody cannot efficiently detect FXIIIAP in vivo.
  • the present invention uses synthetic FXIIIAP with a more stable structure as an antigen (1-35 amino acid sequence), and the specific sequence is SETSRTAFGGRRAVPNNNSNAAEDDLPTVELQGVV to generate specific antibodies against stable double ⁇ -strands structure. Can effectively improve the sensitivity of antibodies.
  • the specific antibody produced by this method can effectively improve the sensitivity to natural FXIIIAP.
  • the sensitivity is significantly improved; more preferably, cysteine is added to the carboxy-terminal or amino-terminal of the above partial sequence to bind to the carrier protein.
  • the result of the sensitivity experiment of the improved antibody shows that the specific antibody produced by this method can detect more than 80% of the natural FXIIIAP.
  • this diagnostic reagent is easier to standardize and popularize.
  • the FXIIIAP detection kit provided by the present invention for this purpose uses FXIIIAP as the specific antibody prepared by the antigen, which can effectively bind the free state FXIIIAP in the plasma, and cannot be combined with unactivated FXIIIAP.
  • FXIIIAP in the plasma (FXIIIAP in the bound state) is bound to effectively detect the free state of FXIIIAP in the plasma.
  • Activated FXIII can be accurately detected and used to judge thrombus or cardiovascular and cerebrovascular diseases.
  • the kit proposed by the present invention has been proved through clinical experiments that the released FXIIIAP can be detected by ELISA or fluorescent immunoassay.
  • a cysteine sequence is added to the N-terminus of the standard full sequence of FXIIIAP to bind to the carrier protein and increase the immunogenicity of the synthesized FXIIIAP.
  • the standard full sequence of FXIIIAP (SETSRTAFGGRRAVPNNNSNAAEDDLPTVELQGVVPR, PDB Entry1F13) can be used to facilitate standardization, which is easier to standardize than D-dimer and easy to promote.
  • the synthetic FXIIIAP partial sequence with a more stable structure is used as the antigen (1-35 amino acid sequence), and the specific sequence is SETSRTAFGGRRAVPNNNSNAAEDDLPTVELQGVV to generate specific antibodies against the stable double ⁇ -strands structure.
  • the specific sequence is SETSRTAFGGRRAVPNNNSNAAEDDLPTVELQGVV to generate specific antibodies against the stable double ⁇ -strands structure.
  • cysteine is added to the carboxyl terminus of the above-mentioned partial sequence.
  • the capture antibody and the detection antibody are extracted from the IgG of the experimental animal immunized with FXIIIAP.
  • the detection antibody needs to be combined with biotin, and then combined with alkaline phosphatase-labeled streptavidin (Streptavidine alkaline phosphatase) to further increase the sensitivity of detection.
  • FXIIIA is also reduced in the blood due to thrombus formation, which consumes the A subunit of coagulation factor 13 (FXIIIA). Although it cannot have the same clear diagnostic significance as FXIIIAP and D-dimer, the decrease of its content can show the amount and severity of thrombus formation, and its decrease indicates more thrombosis and serious condition.
  • the invention provides a detection method for thrombosis and coagulation-related diseases, which comprises the following steps:
  • the sample to be tested is blood or plasma.
  • a specific antibody is prepared using a partial sequence of FXIIIAP as an antigen; the partial sequence of FXIIIAP includes the following sequence SETSRTAFGGRRAVPNNNSNAAEDDLPTVELQGVV or RTAFGGRRAVPPNN.
  • the sample to be tested is blood or plasma.
  • the antibody preparation in the detection kit uses the FXIIIAP partial sequence (SETSRTAFGGRRAVPNNNSNAAEDDLPTVELQGVV) as the antigen, and adds cysteine to its amino terminal to bind to the carrier protein to prepare specific antibodies.
  • the method for producing specific antibody can adopt the method of generating antibody induced by common antigen.
  • the antibody preparation in the detection kit is based on the complete sequence of FXIIIAP (SETSRTAFGGRRAVPNNNSNAAEDDLPTVELQGVVPR) as the antigen, and cysteine is added to its amino terminal to bind to the carrier protein to prepare specific antibodies.
  • the method for producing specific antibody can adopt the method of generating antibody induced by common antigen.
  • FXIIIAP detection kit includes: washing solution, sample diluent, chromogenic substrate, microtiter plate coated with capture antibody and detection antibody.
  • Example 1 The specific antibody in Example 1 was used as the capture antibody, and the full sequence of FXIIIAP in Example 2 was used as the detection antibody.
  • the preparation method of the detection antibody is as follows: use the standard full sequence FXIIIAP (SETSRTAFGGRRAVPNNNSNAAEDDLPTVELQGVVPR) to bind to the carrier protein, immunize mice, and the obtained antibody is purified with G protein.
  • the detection antibody must bind to biotin (Biotin).
  • biotin biotin
  • the detection antibody also needs to be combined with alkaline phosphatase-labeled streptavidin (Streptavidine alkaline phosphatase) to increase the sensitivity.
  • the use process of the above kit at least includes the following steps:
  • a biotin-bound detection antibody (Detection Antibody) is added, and after adding alkaline phosphatase-labeled streptavidin, a chromogenic substrate is added;
  • Results were analyzed with standard ELISA analysis equipment.
  • a combined detection kit comprising the FXIIIAP detection kit and the D-dimer quantitative detection kit of Example 3; wherein the D-dimer quantitative detection kit can be a conventional detection kit.
  • the combination of detection kits in this embodiment can simultaneously detect FXIIIAP and D dimer in blood or plasma samples of patients, which can greatly improve the detection rate of thrombus and coagulation-related diseases.
  • Test samples 122 samples (early ischemic stroke patients) were tested;
  • Plasma samples were collected on the day when the patient developed symptoms as day 1, and samples were divided into day 2, day 3, and day 4 according to the time of collection.
  • the combined detection kit of Example 4 was used for detection; in this detection, it was set that D dimer in the patient's plasma sample exceeded 500 ng/ml as positive, and in this experiment, FXIIIAP exceeding 7 ng/ml was tentatively determined as positive.
  • Sensitivity also known as sensitivity or true positive rate refers to the proportion of positive samples that are judged positive (in this experiment, the patients who really suffered from ischemic stroke, according to FXIIIAP and/or D Dimer test results, the proportion judged as ischemic stroke), calculated by dividing the ratio of true positives by true positives + false negatives (actually positive, but judged as negative).
  • Fig. 1 is with traditional marker D dimer as the contrast of the present invention, to the detection result figure of patient's sample of ischemic stroke; Can obtain in 122 patient's samples from Fig. 1, D2
  • the concentration of the polymer changed as follows: D-dimer increased significantly on the third day, exceeding 500 ng/ml, while the average concentration on the first day was lower than 500 ng/ml.
  • Figure 2 is a diagram of the detection results of the marker FXIIIAP of the present invention on ischemic stroke patient samples; from Figure 2, it can be obtained that the concentration change of FXIIIAP is opposite to that of D dimer, and the first day shows The higher concentration was 15.8 ng/ml, which dropped to 7.2 ng/ml on day 2 and 4 ng/ml on days 3 and 4.
  • Figure 3 is a sensitivity comparison chart of the detection results of the marker FXIIIAP of the present invention and the traditional marker D-dimer in early ischemic stroke patient samples; the sensitivity comparison is shown in Figure 3: On day 1, the sensitivity of FXIIIAP was higher than that of D-dimer, and it was similar on day 2. The sensitivity of D-dimer was higher than that of FXIIIAP on days 3 and 4. If the two are combined, that is, D dimer exceeds 500ng/ml or FXIIIAP exceeds 7ng/ml, it is positive. Then the sensitivities on day 1 and day 2 can be increased to 72% and 68%. Sensitivity was significantly improved compared to D-dimer alone (39% on day 1 and 50% on day 2).
  • Fig. 4 is the sensitivity graph of the marker combination of FXIIIAP and D dimer proposed by the present invention to the detection results of early ischemic stroke patient samples; The diagnosis of stroke has a good effect.
  • Test samples The combination of FXIIIAP detection kit and D-dimer quantitative detection kit was used to detect 27 plasma samples (for early hemorrhagic stroke patients, samples were taken on the day when symptoms appeared), and the following results were obtained:
  • the combined sensitivity of detecting FXIIIAP and D-dimer reached 100%, with no missed diagnosis.
  • the missed diagnosis rate of D-dimer detected only by D-dimer detection kit was 22%. Therefore, the combined detection kit proposed by the present invention helps to reduce the missed diagnosis rate of D-dimer for hemorrhagic stroke.
  • Figure 5 is the antibody described in Embodiment 3-1-1 (original antibody, denoted as 1-37), the antibody described in Embodiment 3-1-2 (improved antibody 1, denoted as 1-35 ) and the antibody described in Embodiment 3-1-4 (improved antibody 2, denoted as 5-18) are used for the sensitivity comparison chart of ELISA detection of FXIIIAP.
  • Embodiment 3-1-1 original antibody, denoted as 1-37
  • Embodiment 3-1-2 improved antibody 1, denoted as 1-35
  • Embodiment 3-1-4 improved antibody 2, denoted as 5-18
  • FXIIIAP in 85% and 87% of the plasma samples could be detected in the in vitro test using the "improved antibody 1" or improved antibody 2 detection kit, respectively.
  • Laboratory results show that using improved antibody 1 or improved antibody 2 as the capture antibody can better recognize FXIIIAP in the body, and does not cross-react with FXIII, which can reflect the true level of FXIIIAP in the sample to be tested.
  • the sensitivity for the initial detection of FXIIIAP in patients was increased by about 30% due to the improvement of the capture antibody.
  • the above-mentioned detection kits achieve a better detection rate for early and early coagulation-related diseases or thrombus. On the contrary, there is a missed diagnosis rate for late coagulation-related diseases or thrombus.
  • Test samples 40 cases of normal human plasma were used as the control group, and 40 cases of plasma samples from hospitalized leukemia patients with thrombus were used as the experimental group for detection. to test.
  • the data of each experimental group or control group are the mean or mean ⁇ standard deviation of 40 parallel experiments.
  • the FXIIIAP detection kit includes: washing solution, sample diluent, chromogenic substrate, microtiter plate coated with capture antibody and detection antibody.
  • the capture antibodies are the antibodies described in embodiment 3-1-1 (original antibody, SETSRTAFGGRRAVPNNNSNAAEDDLPTVELQGVVPR), the antibody described in embodiment 3-1-2 (improved antibody 1, SETSRTAFGGRRAVPNNNSNAAEDDLPTVELQGVV) and the antibody described in embodiment 3-1-4
  • the above antibody improved antibody 2, RTAFGGRRAVPPNN
  • the detection antibody is the full sequence of FXIIIAP (SETSRTAFGGRRAVPNNNSNAAEDDLPTVELQGVVPR)
  • the preparation method is: use the standard full sequence FXIIIAP (SETSRTAFGGRRAVPNNNSNAAEDDLPTVELQGVVPR) to combine with the carrier protein, immunize mice, and the obtained antibody is purified with G protein.
  • the detection antibody must bind to biotin (Biotin).
  • the use process of the FXIIIAP detection kit includes at least the following steps:
  • a biotin-conjugated detection antibody is added, and after the addition of alkaline phosphatase-labeled streptavidin, a chromogenic substrate is added;
  • Results were analyzed with standard ELISA analysis equipment.
  • FXIIIAP in the free state can be effectively detected without binding to FXIIIAP in the unactivated FXIII (FXIIIAP in the bound state); non-specificity means that FXIIIAP in the bound state on the unactivated FXIII is compatible with the antibody function, thus affecting the experimental value.
  • Table 1 is the antibody described in embodiment 3-1-1 (original antibody, denoted as 1-37), the antibody described in embodiment 3-1-2 (improved antibody 1, denoted as 1-35) and The antibody described in embodiment 3-1-4 (improved antibody 2, denoted as 5-18) is used for the specificity comparison data of ELISA detection of FXIIIAP.
  • Figure 6 is the antibody described in Embodiment 3-1-1 (original antibody, denoted as 1-37), the antibody described in Embodiment 3-1-2 (improved antibody 1, denoted as 1- 35) and the specificity comparison chart of the antibody described in Embodiment 3-1-4 (improved antibody 2, denoted as 5-18) used in ELISA for the detection of FXIIIAP.
  • the antibody preparation in the detection kit uses the partial sequence of FXIIIAP (RTAFGGRRAVPPNN) as the antigen, and adds cysteine to its amino terminal to bind to the carrier protein to prepare specific antibodies.
  • the method for producing specific antibody can adopt the method of generating antibody induced by common antigen.
  • FXIIIAP detection kit dry immunofluorescence quantitative method
  • PVC bottom plate fluorescent binding pad, nitrocellulose membrane, sample pad and absorbent pad, sample pad, fluorescent binding pad, nitrocellulose membrane, and absorbent pad are fixed in the horizontal direction On the PVC floor.
  • the detection antibody labeled with fluorescent microspheres is coated on the fluorescent binding pad, and the detection line composed of capture antibody and the quality control line composed of rabbit anti-mouse IgG antibody are sequentially arranged on the nitrocellulose membrane.
  • Example 5 The specific antibodies of Example 5 were used as capture antibodies and detection antibodies.
  • the preparation method of the detection antibody is as follows: After binding the partial sequence of FXIIIAP (improved antibody 2, RTAFGGRRAVPPNN) to the carrier protein, immunize mice, and the obtained antibody is purified with G protein.
  • FXIIIAP improved antibody 2
  • RTAFGGRRAVPPNN partial sequence of FXIIIAP
  • the basic principle of the fluorescent immunochromatography kit above is the double-antibody sandwich method, that is, the analyte in the sample combines with the fluorescent microsphere-labeled detection antibody (improved antibody 2-fluorescent microsphere) dispersed on the binding pad to form a complex. Under the action of chromatography, the complex diffuses forward along the reaction membrane, and the complex is captured by the corresponding capture antibody (improved antibody 2) immobilized on the detection line of the reaction membrane.
  • the fluorescent microsphere-labeled detection antibody that reacts with the T line is chromatographed to the quality control line, and combined with the rabbit anti-mouse IgG antibody to develop color. According to the standard curve, the concentration of FXIIIAP in the sample to be tested can be calculated.
  • the use process of the above kit at least includes the following steps:
  • detection kits and related detection methods of the present invention the initial clinical data of the detection kits are listed below.
  • the following are test results for early diagnosis of abnormal coagulation thrombus, thrombotic platelet purpura and femoral vein thrombosis and ischemic stroke caused by M3 leukemia.
  • the FXIIIAP detection kit and the D-dimer quantitative detection kit of Example 6 were used to monitor the abnormal coagulation thrombus caused by M3 leukemia in real time, wherein the FXIIIAP detection kit was used to monitor the change of FXIIIAP, and the D-dimer quantitative detection The kit is used to monitor changes in D-dimer.
  • the patients were patients A and B with abnormal coagulation thrombus caused by M3 leukemia, and the changes of various physical and chemical indicators (including FXIIIAP and D dimer) were monitored within 18 to 20 days of admission and treatment.
  • Figure 7 is the basic information of patient A
  • Figure 8 and Figure 9 are the thrombus monitoring data of patient A
  • Figure 10 is a monitoring change chart of the ratio of white blood cells and promyelocytes of patient A
  • FIG. 11 is a monitoring change chart of FXIIIAP and fibrinogen in patient A
  • FIG. 12 is a monitoring change chart of patient A's D dimer and fibrin monomer.
  • Figure 13 is the basic information of patient B
  • Figure 14 and Figure 15 are the thrombus monitoring data of patient B
  • Figure 16 is a monitoring change chart of the proportion of white blood cells and promyelocytes of patient B
  • FIG. 17 is a monitoring change chart of FXIIIAP and fibrinogen in patient B
  • FIG. 18 is a monitoring change chart of patient B's D dimer and fibrin monomer.
  • Thrombotic platelet purpura and femoral vein thrombosis were monitored in real time by using the FXIIIAP detection kit and the D-dimer quantitative detection kit in Example 6 respectively, wherein the FXIIIAP detection kit was used to monitor changes in FXIIIAP, D-dimer Quantitative detection kits are used to monitor changes in D-dimer.
  • the patient was patient C with thrombotic thrombocytopenic purpura and femoral vein thrombosis.
  • the change levels of various physical and chemical indicators were monitored after the patient was admitted to the hospital and treated for a week after his condition was relieved, and when he relapsed.
  • FIG. 19 shows the basic information of patient C
  • FIG. 20 shows the thrombus monitoring data of patient C.
  • the combination of the FXIIIAP detection kit and the D-dimer quantitative detection kit in Example 6 was used for real-time monitoring of ischemic stroke, wherein the FXIIIAP detection kit was used to monitor changes in FXIIIAP, and the D-dimer quantitative detection kit Used to monitor changes in D-dimer.
  • the patient is patient D with ischemic stroke, and the change levels of various physical and chemical indicators (including FXIIIAP and D dimer) of the patient after admission to the emergency department and treatment are monitored.
  • FIG. 21 shows the basic information of patient D
  • FIG. 22 shows the thrombus monitoring data of patient D.
  • the combination of FXIIIAP and D dimer markers proposed by the present invention can effectively reduce the missed diagnosis rate and provide feedback on the formation and stability of thrombus in the body;
  • the antibody of the present invention can be applied to a variety of existing methods Science, such as enzyme-linked immunosorbent assay, dry immunofluorescence, etc., to effectively detect thrombus or coagulation-related diseases.

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Abstract

本发明涉及血栓或凝血相关疾病检测技术领域，尤其涉及一种生物标志物及其在血栓或凝血相关疾病检测工作方面的应用。标志物包括凝血因子13激活肽(FXIIIAP)或/和凝血因子13的A亚基(FXIIIA)。本发明提供的检测试剂盒包括捕获抗体和检测抗体，为以FXIIIAP为抗原制得的特异性抗体。本发明的生物标志物在对包含脑卒中在内的血栓或凝血相关疾病进行快速筛查诊断方面有很好的应用前景，可大大提高现有诊断的灵敏度和特异性，推测血栓形成时间及程度，并可对疾病的严重程度及预后进行预测。

Description

一种检测血栓或凝血相关疾病的生物标志物及其应用 技术领域
本发明涉及血栓或凝血相关疾病检测技术领域，尤其涉及一种检测血栓或凝血相关疾病的生物标志物及其应用。
背景技术
血栓形成及其相关凝血性疾病一直是临床诊断中重要诊断项目，涉及广泛的疾病，包括脑静脉血栓(CVT)，肾静脉血栓(RVT)，静脉血栓(VTE)，心肌梗塞(MI)等等。其中脑静脉血栓、心肌梗塞的早期诊断与早期治疗尤为重要。以脑静脉血栓(缺血性中风的一个重要原因)为例，现有的诊断方法主要包括计算机断层扫描(CT)和磁共振成像(MRI)，在判断中风的发生、类型和严重性方面非常有效。然而，这些技术首先较为昂贵并且耗时，其次对早期发现的筛查作用不明显，容易错过最佳治疗时机。此外对于出血性脑卒中的诊断，CAT扫描(计算机化轴位断层扫描)有较好的效果。尽管CAT扫描对出血性中风的鉴别相对灵敏，但它在中风评估中对脑缺血不十分灵敏，通常在中风发作后24到36小时的时候才呈阳性，并且大约50％的缺血性脑卒中难以在CAT扫描看到。其结果往往是，当现有的诊断技术确诊中风时，早期治疗的机会已经丧失。
对于急性缺血性及出血性脑卒中的诊断，目前这些临床常用的方法在灵敏度，准确度和时效性上存在不足，容易产生误诊或漏诊，从而延误治疗时机。
近年来研究人员发现，利用分子标志物快速筛选诊断并区分脑卒中的类型，是可供解决这些问题的新思路。
生物标志物(biomarker)是指可以标记生物体器官，组织或细胞结构或功能改变的生化指标，因此生物标志物的检测可用于疾病诊断、并为后续治疗方案提供科学依据。
中风的多种标志物是已知的。在公开号为CN103299191A的发明专利中公开了中风相关的分子标志物。在公开号为CN1339108A的发明专利中公开了诊断和区分中风的方法。
血栓形成可以作为诊断例如缺血性脑卒中等心血管疾病的一个重要指标。目前临床使用的对血栓的生物标志物筛选检查是D二聚体检测，主要应用在静脉血栓栓塞(VTE)、深静脉血栓形成(DVT)和肺栓塞(PE)的诊断。在很多情况下，D二聚体的检查已显示出良好的发展前景。但由于多种因素都可以影响D二聚体的检测，以及D二聚体本身的缺陷，因此在临床上使用D二聚体检测仍存在很多争议。首先D二聚体来源于纤溶酶溶解的交联纤维蛋白凝块，主要反映纤维蛋白溶解(血栓溶解)的状况，而并非直接反映血栓形成。所有影响血栓溶解的因素均可影响D二聚体检测结果。例如具有溶栓障碍的患者，口服抗凝药的患者，VTE症状持续14天以上的患者均可能为假阴性。另外，D二聚体反映血栓溶解过程且其半衰期较长(13-23小时)当患者有其他可引起凝血的诱因时，例如炎症或怀孕时，也可以预期出现假阳性结果。同时D二聚体在临床测试中也有很高的漏诊率。例如，对于CVT，D二聚体存在20％的漏诊，早期(症状出现小于24小时)漏诊率更高(大约50％)。对于静脉血栓一般的D二聚体诊断也存在50％的漏诊。特别是对早期新生成的血栓D二聚体容易出现假阴性结果。
由于D二聚体半衰期长，不能在已有凝血疾病时，准确及时反映机体是否正在发生出血及凝血。另外，由于D二聚体分析物的定义不明确，不同的测定方法基于识别不同纤维蛋白片段或表面结构的不同单克隆抗体。没有可以用作国际标准的参考制剂或校准剂。加上其敏感度和特异性不高的缺点，D二聚体的应用仍存在争议。此外检测结果缺乏治疗指导意义，并不能及时准确的反映体内凝血状况，无法指示治疗策略，例如抗凝的效果如何，也难以确认是否出现新血栓，因为陈旧血栓依旧可以被溶解而产生D二聚体。
发明内容
(一)要解决的技术问题
鉴于现有技术的上述缺点、不足，并且与上述生物标志物(间接检测组织对中风的应激反应)不同 地，本发明提出了一种可以对血栓形成本身进行直接检测的生物标志物，可以单独或与D二聚体检测结合，对血栓或凝血相关疾病进行高敏感度和高准确率的检测和筛查。
具体而言：
本发明提供了一种生物标志物，可用于血栓或凝血相关疾病的检测。
相应地，本发明还提供上述生物标志物血栓或凝血相关疾病的检测产品和治疗效果评估产品中的应用；
进一步地，本发明还提供一种用于检测血栓或凝血相关疾病的试剂盒。
(二)技术方案
为了达到上述目的，本发明采用的主要技术方案包括：
第一方面，本发明实施例提供一种生物标志物，可用于血栓或凝血相关疾病的检测；所述标志物包括FXIIIAP或FXIIIA，其中FXIIIAP为凝血因子13激活肽，FXIIIA为凝血因子13的A亚基。
可选地，所述的生物标志物包括FXIIIAP和D二聚体组合。
可选地，所述凝血相关疾病包括中风、脑静脉血栓、肾静脉血栓、静脉血栓或心肌梗塞。
第二方面，本发明还提供一种血栓或心血管相关疾病的诊断方法，其包括以下步骤：
S1以FXIIIAP的全序列或部分序列为抗原制得特异性抗体；
S2将所得到的特异性抗体在待测样品中结合游离状态的FXIIIAP得到结合物；
S3通过检测到的所述结合物，可确定FXIII被激活并且诊断为血栓或心血管相关疾病。
可选地，所述待测样品为血液或血浆。
可选地，以FXIIIAP的部分序列为抗原制得特异性抗体；所述FXIIIAP的部分序列包括以下序列SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV或RTAFGGRRAVPPNN。
第三方面，本发明实施例提供一种如上所述的生物标志物在血栓或凝血相关疾病的检测产品和治疗效果评估产品中的应用。
可选地，所述血栓或凝血相关疾病的检测产品和治疗效果评估产品包括试剂盒或试剂。
第四方面，本发明实施例提供一种用于检测血栓或凝血相关疾病的试剂盒，所述试剂盒中的捕获抗体或/和检测抗体为上述的生物标志物为抗原制得的特异性抗体。
可选地，所述的用于检测血栓或凝血相关疾病的试剂盒为双抗体夹心ELISA检测试剂盒或干式免疫荧光检测试剂盒，包括捕获抗体和检测抗体。
可选地，所述的用于检测血栓或凝血相关疾病的试剂盒中，所述捕获抗体和检测抗体为以FXIIIAP的全序列或部分序列为抗原制备而得。
可选地，所述的用于检测血栓或凝血相关疾病的试剂盒中，所述部分序列中包括以下序列或含有以下序列的片段：SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV或RTAFGGRRAVPPNN。
可选地，所述的用于检测血栓或凝血相关疾病的试剂盒中，所述捕获抗体和检测抗体提取自FXIIIAP免疫的试验动物。
可选地，本发明的抗体，除了应用于酶联免疫吸附测定法(enzyme-linked immunosorbent assay，ELISA)，还可用于胶体金法，放射免疫分析法(radioimmunoassay，RIA)、化学发光免疫分析法(chemiluminescent immunoassay，CLIA)、电化学发光免疫分析法(electro-chemiluminescence immunoassay，ECLI)，荧光免疫法例如干式免疫荧光法(dry-type immunofluorescence method)、时间分辨荧光免疫分析法(time-resolved fluoroisnmunoassay，TRFIA)以检测上述生物标志物。
(三)有益效果
本发明的有益效果主要包括：
本发明主要提供了可用于检测血栓或凝血相关疾病的生物标志物，由于其可以与传统标志物D二聚体结合，相对于现有技术，可以实现对敏感度(检出率)的显著提高。
本发明指出的标志物FXIIIAP相对于现有技术而言(或更具体地，与D二聚体相对比)，对于急性期的血栓(小于48小时)有更好的检测效果。另一方面，由于FXIIIAP快速降解，在病人出现症状48小时后，无法检测到，漏诊率非常高，因此，本发明还提出将FXIIIAP与D二聚体联合应用，可有效 地、大幅地降低在整个发病周期包括早期和晚期内，对血栓或凝血相关疾病的检测漏诊率。
其中，
1.本发明提出的生物标志物的出现与纤溶过程不相关，相对于现有技术而言，其可适用于纤溶不足的患者的检测；
2.本发明提出的生物标志物与D二聚体相比能更好的反映体内是否有新血栓形成的状况。因此也可用于评估治疗效果及疾病的严重情况的评估。
3.本发明提出的生物标志物对包含脑卒中在内的凝血疾病进行快速诊断，提高现有诊断的灵敏度和特异性，能推测血栓形成时间及程度，对疾病的严重程度及预后进行预测。
4.本发明的应用能够对治疗策略提供信息，例如为抗凝或溶栓治疗提供指导，并有效监测抗凝药物的效果。
5.本发明提出的检测试剂盒可以对指血进行检测，及相应的长期检测，具有极高的商业价值。
6.本发明提供的检测试剂盒，弥补了市场上FXIIIAP的检测试剂盒的空白。
附图说明
图1为以传统标志物D二聚体作为本发明对照，对缺血性脑卒中病人样本的检测结果图；
图2为本发明所述的标志物FXIIIAP对缺血性脑卒中病人样本的检测结果图；
图3为本发明所述的标志物FXIIIAP与传统标志物D二聚体在早期缺血性脑卒中病人样本检测结果的敏感性对比图；
图4为本发明提出的FXIIIAP与D二聚体的标志物组合对早期缺血性脑卒中病人样本检测结果的敏感性图；
图5为实施方式3-1-1所述抗体(原有抗体，记为1-37)、实施方式3-1-2所述抗体(改进抗体1，记为1-35)以及实施方式3-1-4所述抗体(改进抗体2，记为5-18)用于ELISA对FXIIIAP检测的敏感性对比图；
图6为实施方式3-1-1所述抗体(原有抗体，记为1-37)、实施方式3-1-2所述抗体(改进抗体1，记为1-35)以及实施方式3-1-4所述抗体(改进抗体2，记为5-18)用于ELISA对FXIIIAP检测的特异性对比图；
图7示出了患者A的基本信息；
图8示出了患者A的血栓监测数据一；
图9示出了患者A的血栓监测数据二；
图10为患者的A白细胞以及早幼粒细胞比例的监测变化图；
图11为患者A的FXIIIAP以及纤维蛋白原的监测变化图；
图12为患者A的D二聚体以及纤维蛋白单体的监测变化图；
图13示出了患者B的基本信息；
图14示出了患者B的血栓监测数据一；
图15示出了患者B的血栓监测数据二；
图16为患者B的白细胞、早幼粒细胞比例的监测变化图；
图17为患者B的FXIIIAP以及纤维蛋白原的监测变化图；
图18为患者B的D二聚体以及纤维蛋白单体的监测变化图；
图19示出了患者C的基本信息；
图20示出了患者C的血栓监测数据；
图21示出了患者D的基本信息；
图22示出了患者D的血栓监测数据。
具体实施方式
为了更好的解释本发明，以便于理解，下面结合附图，通过具体实施方式，对本发明作详细描述。
【实施方式系列1】
[实施方式1-1]
一种用于检测血栓或凝血相关疾病的生物标志物：FXIIIAP凝血因子13激活肽。
[实施方式1-2]
一种用于检测血栓或凝血相关疾病的生物标志物：FXIIIA(凝血因子13的A亚基)。
[实施方式1-3]
一种用于检测血栓或凝血相关疾病的生物标志物：其为FXIIIAP与FXIIIA的组合。
[实施方式1-4]
一种用于检测血栓或凝血相关疾病的生物标志物：其为FXIIIAP与D二聚体的组合。
[实施方式1-5]
一种用于检测血栓或凝血相关疾病的生物标志物：其为FXIIIA与D二聚体的组合。
[实施方式1-6]
一种用于检测血栓或凝血相关疾病的生物标志物：FXIIIAP、FXIIIA与D二聚体的组合。
其中，实施方式1-4、实施方式1-5和实施方式1-6所述的FXIIIAP或FXIIIA与D二聚体形成的生物标志物组合，其可以更准确的应用于检测不同时期的血栓或凝血相关疾病。与D二聚体不同，FXIIIAP的出现与纤溶过程不相关。因此纤溶不足的患者也可以用此方法检测。此外，FXIIIAP的出现要早于D二聚体。在临床测试中发现，FXIIIAP对于急性期的血栓(小于2天)有更好的检测效果。而D二聚体则在症状出现的第二天开始升高，第三天明显升高的。与D二聚体相反，在症状出现的第一天，D二聚体尚且较低的情况下(<500ng/mL)，FXIIIAP已显示出很高的水平(15.8ng/mL)，而正常人血中FXIIIAP仅4ng/mL。第二天，FXIIIAP明显下降至7.2ng/mL，而第三天降至正常水平。这一结果显示出D二聚体与FXIIIAP有较好的互补性。
研究显示FXIIIAP在体内会被很快降解，因此其升高反映的是体内血栓形成的现状。与D二聚体相比能更好的反映体内是否有新血栓形成。因此也可用于对治疗效果及疾病的严重情况的评估。但由于其往往被快速降解，因此在出现晚期症状的病人体内很可能无法检测到，这就造成了一定漏诊率。因此必须与D二聚体联合应用以减少漏诊风险。
FXIIIAP不仅对血栓引起的中风有效，对于出血性脑卒中理论上也将有相当效果。在发生颅内出血时，血脑屏障的渗透性增加，凝血因子可以通过血脑屏障。由于FXIIIAP的分子量非常小(仅为3.91KD)，可以顺利通过血脑屏障。同时由于颅内富含组织因子，当血脑屏障受损时，会释放组织因子进入体内，引起体内凝血。因此有研究用D二聚体检测颅内出血，但结果并不理想。目前尚无利用FXIIIAP诊断出血性脑卒中的实验检测。理论上可能出现FXIIIAP持续较高浓度，并且不随着时间延长而下降。同时FXIIIA浓度减低，D二聚体浓度升高。(高AP，低FXIIIA和高D二聚体)。
其中，本发明所指的凝血相关疾病包括但不仅限于中风、脑静脉血栓、肾静脉血栓、静脉血栓、心肌梗塞，可以是所有由血栓引起的疾病。
【实施方式系列2】
[实施方式2-1]
如实施方式1-1所述的生物标志物在血栓或凝血相关疾病的检测产品和治疗效果评估产品中的应用。
进一步地，在另一些实施方式中，所述产品包括试剂盒或试剂。
[实施方式2-2]
如实施方式1-2所述的生物标志物在血栓或凝血相关疾病的检测产品和治疗效果评估产品中的应用。进一步地，在另一些实施方式中，所述产品包括试剂盒或试剂。
[实施方式2-3]
如实施方式1-3所述的生物标志物在血栓或凝血相关疾病的检测产品和治疗效果评估产品中的应用。进一步地，在另一些实施方式中，所述产品包括试剂盒或试剂。
[实施方式2-4]
如实施方式1-4所述的生物标志物在血栓或凝血相关疾病的检测产品和治疗效果评估产品中的应用。进一步地，在另一些实施方式中，所述产品包括试剂盒或试剂。
[实施方式2-5]
如实施方式1-5所述的生物标志物在血栓或凝血相关疾病的检测产品和治疗效果评估产品中的应用。进一步地，在另一些实施方式中，所述产品包括试剂盒或试剂。
[实施方式2-6]
如实施方式1-6所述的生物标志物在血栓或凝血相关疾病的检测产品和治疗效果评估产品中的应用。进一步地，在另一些实施方式中，所述产品包括试剂盒或试剂。
【实施方式系列3】
[实施方式3-1]
一种用于检测血栓或凝血相关疾病的试剂盒，其中包括了捕获抗体或/和检测抗体，而所述捕获抗体和检测抗体为以实施方式1-1所述的生物标志物为抗原制得的特异性抗体。进一步地，在另一些实施方式中，所述试剂盒更具体地被制备为包括有捕获抗体和检测抗体的双抗体夹心ELISA检测试剂盒。
[实施方式3-1-1]
在实施方式3-1的基础上，更具体地，所述捕获抗体和检测抗体为以FXIIIAP的全部序列为抗原制备而得。
[实施方式3-1-2]
在实施方式3-1的基础上，更具体地，所述捕获抗体和检测抗体为以FXIIIAP的部分序列为抗原制备而得，所述部分序列为：SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV。
[实施方式3-1-3]
在实施方式3-1的基础上，更具体地，所述捕获抗体和检测抗体为以FXIIIAP的部分序列为抗原制备而得，所述部分序列为：SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV的其中部分片段。
[实施方式3-1-4]
在实施方式3-1的基础上，更具体地，所述捕获抗体和检测抗体为以FXIIIAP的部分序列为抗原制备而得，所述部分序列为：RTAFGGRRAVPPNN。
[实施方式3-1-5]
在实施方式3-1的基础上，更具体地，所述捕获抗体和检测抗体为以FXIIIAP的部分序列为抗原制备而得，所述部分序列为：RTAFGGRRAVPPNN的其中部分片段。
[实施方式3-2]
一种用于检测血栓或凝血相关疾病的试剂盒，其中包括了捕获抗体和检测抗体，而所述捕获抗体和检测抗体为以实施方式1-2所述的生物标志物为抗原制得的特异性抗体。进一步地，在另一些实施方式中，所述试剂盒更具体地被制备为包括有捕获抗体和检测抗体的双抗体夹心ELISA检测试剂盒。
[实施方式3-3]
一种用于检测血栓或凝血相关疾病的试剂盒，其中包括了捕获抗体和检测抗体，而所述捕获抗体和检测抗体为以实施方式1-3所述的生物标志物为抗原制得的特异性抗体。进一步地，在另一些实施方式中，所述试剂盒更具体地被制备为包括有捕获抗体和检测抗体的双抗体夹心ELISA检测试剂盒。
[实施方式3-4]
一种用于检测血栓或凝血相关疾病的试剂盒，其中包括了捕获抗体和检测抗体，而所述捕获抗体和检测抗体为以实施方式1-4所述的生物标志物为抗原制得的特异性抗体。进一步地，在另一些实施方式中，所述试剂盒更具体地被制备为包括有捕获抗体和检测抗体的双抗体夹心ELISA检测试剂盒。进一步地，在另一些实施方式中，所述捕获抗体和检测抗体提取自FXIIIAP免疫的试验动物，准确地说，提取自这些经FXIIIAP免疫的试验动物的IgG。
[实施方式3-5]
一种用于检测血栓或凝血相关疾病的试剂盒，其中包括了捕获抗体和检测抗体，而所述捕获抗体和检测抗体为以实施方式1-5所述的生物标志物为抗原制得的特异性抗体。进一步地，在另一些实施方式 中，所述试剂盒更具体地被制备为包括有捕获抗体和检测抗体的双抗体夹心ELISA检测试剂盒。进一步地，在另一些实施方式中，所述捕获抗体和检测抗体提取自FXIIIAP免疫的试验动物，准确地说，提取自这些经FXIIIAP免疫的试验动物的IgG。
[实施方式3-6]
一种用于检测血栓或凝血相关疾病的试剂盒，其中包括了捕获抗体和检测抗体，而所述捕获抗体和检测抗体为以实施方式1-6所述的生物标志物为抗原制得的特异性抗体。进一步地，在另一些实施方式中，所述试剂盒更具体地被制备为包括有捕获抗体和检测抗体的双抗体夹心ELISA检测试剂盒。进一步地，在另一些实施方式中，所述捕获抗体和检测抗体提取自FXIIIAP免疫的试验动物，准确地说，提取自这些经FXIIIAP免疫的试验动物的IgG。
[实施方式3-7]
一种用于检测血栓或凝血相关疾病的试剂盒，其中包括了捕获抗体和检测抗体，而所述捕获抗体和检测抗体为以实施方式1-6所述的生物标志物为抗原制得的特异性抗体。进一步地，在另一些实施方式中，所述试剂盒更具体地被制备为包括有捕获抗体和检测抗体的双抗体夹心ELISA检测试剂盒。
进一步地，在另一些实施方式中，所述捕获抗体和检测抗体提取自FXIIIAP免疫的试验动物，准确地说，提取自这些经FXIIIAP免疫的试验动物的IgG。
与D二聚体不同，凝血因子13(FXIII)是在凝血过程中被最后激活的凝血因子，主要功能是交联纤维蛋白凝块以稳定血栓。其被激活反映凝血过程已完成，稳定的血栓已形成。在其激活的过程中，每个FXIII释放两个凝血因子13激活肽(FXIIIAP)。FXIIIAP是仅有37个氨基酸的多肽(大约3.91KD)。其功能为稳定FXIIIA 2的二聚体结构。在被凝血酵素(Thrombin)激活后，FXIIIAP离开FXIII进入血液，成为游离状态的FXIIIAP。而游离状态的FXIIIAP的结构构象与结合在FXIII上的FXIIIAP明显不同。本发明中以合成的FXIIIAP为抗原制得的特异性抗体，不能与未被激活的FXIII中的FXIIIAP(结合状态的FXIIIAP)结合。本发明中特异性抗体能有效的检测游离状态的FXIIIAP，而不与未被激活FXIII结合。此特点可保证诊断特异性，可以准确检测出被激活的FXIII。经过临床实验已证明，被释放出的FXIIIAP可以被ELISA或荧光免疫法检测出。
本发明提出的试剂盒，其核心成分为采用改进的FXIIIAP氨基酸序列生产的FXIIIAP特异性抗体。根据计算机模拟实验：FXIIIAP被Thrombin切割，并释放到血液中，以1-35的氨基酸序列形成双β-strands结构，而位于36和37的氨基酸并未对此结构的形成有帮助。相反的，由于这两个氨基酸的不稳定直接导致合成FXIIIAP的羧基端的结构多变，生产的抗体，不能高效的检测到体内FXIIIAP。本发明以具有更加稳定的结构的合成FXIIIAP为抗原(1-35的氨基酸序列)，具体序列为SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV，产生针对稳定的双β-strands结构的特异性抗体。可以有效地提高抗体的敏感性。
特别要说明的是，发明人在进一步分析FXIIIAP的结构时发现，位于5-18位的氨基酸序列对于FXIIIAP与FXIIIA亚基的结合非常关键，且抗原性较好，便于生产抗体。在与FXIIIA结合状态下，与抗体的结合位点会被遮蔽。成为游离状态的FXIIIAP后暴露出与抗体的结合位点。因此含有5-18氨基酸序列(RTAFGGRRAVPPNN)的多肽可以被用于制作抗体。
以此方法制作的特异性抗体相对于以FXIIIAP全序列(1-37的氨基酸序列)制得的抗体(单克隆抗体)可有效地提升对天然FXIIIAP的敏感性。在利用正常血浆的体外测试中显著提高敏感性；更优选地，在上述的部分序列的羧基端或氨基端加上半胱氨酸以结合载体蛋白。改进的抗体的灵敏度实验结果显示，以此方法制造的特异性抗体作可以检测到80％以上的天然FXIIIAP。此外，由于在抗体的制备过程中，应用了FXIIIAP的标准序列。此诊断试剂与D二聚体相比更容易标准化，也便于推广。
目前，市场上无FXIIIAP检测试剂盒，本发明为此提供的FXIIIAP检测试剂盒，其利用FXIIIAP为抗原制得的特异性抗体能够有效的结合血浆中游离状态的FXIIIAP，不能与未被激活的FXIII中的FXIIIAP(结合状态的FXIIIAP)结合，从而有效的检测血浆中的游离状态的FXIIIAP。可以准确检测出被激活的FXIII，用于判断血栓或者心脑血管疾病等。本发明提出的试剂盒经过临床实验已证明，被释放出的FXIIIAP可以被ELISA或荧光免疫法检测出。
其中，为便于抗体的生产，FXIIIAP的标准全序列的氨基端加入半胱氨酸序列以结合载体蛋白，增加合成的FXIIIAP的免疫原性。由于在特异性抗体的制备过程中，应用FXIIIAP的标准全序列(SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVVPR，PDB Entry1F13)，可利于标准化，相比D二聚体更容易标准化，也便于推广。
进一步地，特异性抗体的制备过程中应用具有更加稳定的结构的合成FXIIIAP部分序列为抗原(1-35氨基酸序列)，具体序列为SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV，产生针对稳定的双β-strands结构的特异性抗体。可以有效地提高抗体的敏感性。更优选地，在上述的部分序列的羧基端加上半胱氨酸，改进的所制得特异性抗体的灵敏度实验结果显示，以此方法制造的特异性抗体用于检测可以检测到80％以上的天然的游离FXIIIAP。
其中，捕获抗体和检测抗体提取自FXIIIAP免疫的试验动物的IgG。检测抗体需与生物素结合后，与碱性磷酸酶标记的链霉亲和素(Streptavidine alkaline phosphatase)结合应用，以进一步增加检测的敏感性。
应用本发明的检测方法和检测试剂盒可进行定量分析。由于血栓形成，消耗凝血因子13的A亚基(FXIIIA)，FXIIIA在血液中也会减少。虽然其不能像FXIIIAP和D二聚体一样具有明确的诊断意义，但其含量减低可以显示血栓形成的多少，及严重程度，其降低预示血栓形成多，病情严重。
【实施方式4】
本发明提供一种血栓及凝血相关疾病的检测方法，其包括以下步骤：
S1以FXIIIAP为抗原制得特异性抗体；
S2将所得到的特异性抗体在待测样品中结合游离状态的FXIIIAP得到结合物；
S3检测到所得到的结合物，可确定FXIII被激活并且诊断为血栓或心血管相关疾病。
可选地，所述待测样品为血液或血浆。
可选地，以FXIIIAP的部分序列为抗原制得特异性抗体；所述FXIIIAP的部分序列包括以下序列SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV或RTAFGGRRAVPPNN。
其中，待测样品为血液或血浆。
为了更好的理解上述技术方案，下面将参照附图更详细地描述本发明的示例性实施例。虽然附图中显示了本发明的示例性实施例，然而应当理解，可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更清楚、透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
实施例1
检测试剂盒中的抗体制备，是以FXIIIAP部分序列(SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV)为抗原，并且在其氨基端加入半胱氨酸以结合载体蛋白，制备得到特异性抗体。其中制得特异性抗体的方法可以采用普通的抗原诱导生成抗体的方法。
实施例2
检测试剂盒中的抗体制备，是以FXIIIAP全序列(SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVVPR)为抗原，并且在其氨基端加入半胱氨酸以结合载体蛋白，制备而得到特异性抗体。其中制得特异性抗体的方法可以采用普通的抗原诱导生成抗体的方法。
实施例3
FXIIIAP检测试剂盒包括：洗涤液、样品稀释液、显色底物、包被捕获抗体的酶标板和检测抗体。
采用实施例1的特异性抗体为捕获抗体，采用实施例2的FXIIIAP全序列为检测抗体。
检测抗体(detection antibody)的制备方法如下：用标准全序列FXIIIAP(SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVVPR)，结合载体蛋白后，免疫小鼠，得到的抗体以G蛋白纯化。检测抗体须结合生物素(Biotin)。在检测中，检测抗体还需结合碱性磷酸酶标记的链霉亲和素(Streptavidine alkaline phosphatase)以增加敏感性。
上述试剂盒的使用过程至少包括如下步骤：
以100μl的捕获抗体包被ELISA酶标板的每孔；
以牛血清白蛋白对每孔作非特异性结合即封闭；
捕获抗体与病人血浆结合后，添加结合生物素的检测抗体(Detection Antibody)，并且在添加碱性磷酸酶标记的链霉亲和素后，加入显色底物；
以标准ELISA分析设备分析结果。
实施例4
一种组合检测试剂盒，包括实施例3的FXIIIAP检测试剂盒和D二聚体定量检测试剂盒；其中D二聚体定量检测试剂盒可以是常规的检测试剂盒。
通过本实施例的检测试剂盒组合可以同时检测患者血液或血浆样品中的FXIIIAP和D二聚体，可以极大地提高对血栓及凝血相关疾病的检出率。
为验证本发明试剂盒以及相关检测方法的可行性，我们列出检测试剂盒进行检测的初期临床数据。以下为在对于缺血型脑卒中的早期诊断的测试结果。
试验一：检测样本：对122个样本(为缺血型脑卒中早期病人)进行检测；
1.统计方法：
病人出现症状当天采集血浆样品为第1天，根据采集时间样品分为第2天，第3天和第4天。并采用实施例4的组合检测试剂盒进行检测；在此次检测中，设定患者血浆样品中D二聚体超过500ng/ml为阳性，本实验将FXIIIAP超过7ng/ml暂定为阳性。
2.敏感性的比较
敏感性(Sensitivity，也称为灵敏度或真阳性率)是指实际为阳性的样本中，判断为阳性的比例(此次实验中真正患有缺血型脑卒中的患者，根据FXIIIAP和/或D二聚体检测结果，被判断为缺血型脑卒中的比例)，计算方式是真阳性除以真阳性+假阴性(实际为阳性，但判断为阴性)的比值。
试验结果：
请参照图1，图1为以传统标志物D二聚体作为本发明对照，对缺血性脑卒中病人样本的检测结果图；从如图1中可以得到在122个病人样品中，D二聚体浓度变化如下：D二聚体第3天明显升高，超过500ng/ml，而在第1天平均浓度低于500ng/ml。
请参照图2，图2为本发明所述的标志物FXIIIAP对缺血性脑卒中病人样本的检测结果图；从如图2中可以得到FXIIIAP浓度变化与D二聚体相反，第1天显示较高的浓度为15.8ng/ml，第2天则降到7.2ng/ml，而第3天和第4天则降至4ng/ml。
请参照图3，图3为本发明所述的标志物FXIIIAP与传统标志物D二聚体在早期缺血性脑卒中病人样本检测结果的敏感性对比图；敏感性比较如图3所示：第1天，FXIIIAP的敏感性高于D二聚体，第2天两者相似，第3天及第4天的D二聚体敏感性高于FXIIIAP。如果将两者结合，即D二聚体超过500ng/ml或者FXIIIAP超过7ng/ml为阳性。则可以将第1天和第2天的敏感性提高至72％和68％。相较于仅以D二聚体为诊断标准(第1天，39％，第2天，50％)，敏感性明显提高。
图4为本发明提出的FXIIIAP与D二聚体的标志物组合对早期缺血性脑卒中病人样本检测结果的敏感性图；如图4所示，FXIIIAP结合D二聚体对早期缺血型脑卒中的诊断有很好的效果。
试验二：
检测样本：采用FXIIIAP检测试剂盒和D二聚体定量检测试剂盒的组合对27个血浆样本(为出血性脑卒中早期病人，症状出现当天取样)进行检测，得到以下的结果：
检测FXIIIAP和D二聚体的组合敏感性达到100％，无漏诊。而仅仅用D二聚体检测试剂盒检测D二聚体的漏诊率为22％。因此，本发明提出的组合检测试剂盒有助于降低D二聚体对出血性脑卒中的漏诊率。
试验三：
请参照图5，图5为实施方式3-1-1所述抗体(原有抗体，记为1-37)、实施方式3-1-2所述抗体(改进抗体1，记为1-35)以及实施方式3-1-4所述抗体(改进抗体2，记为5-18)用于ELISA对FXIIIAP检测的敏感性对比图。从图5数据可见，“原有抗体”的检测试剂盒在体外测试中(以正常血浆为样本)， 约40％血浆样品中的FXIIIAP不能被测到。而敏感性不佳也导致“原有抗体”在临床测试中效果未达理想状态。这可能由于所得到的特异性捕获抗体不能高效的捕获血浆中的FXIIIAP。
而基于相同的血浆样品，采用了“改进抗体1”或改进抗体2的检测试剂盒在体外测试中，分别有85％和87％血浆样品中的FXIIIAP可以被检测到。实验室结果显示，采用改进抗体1或改进抗体2作为捕获抗体能更好的识别体内的FXIIIAP，且不与FXIII产生交叉反应，能够反映待测样品中FXIIIAP的真实水平。
因此，由于捕获抗体的改进，对于患者初期检出FXIIIAP的敏感性提高了约30％。此外，由于FXIIIAP的快速降解的特性，使上述检测试剂盒对于初期和早期的凝血相关疾病或血栓检出率达到较佳的状态，相反，对于后期的凝血相关疾病或血栓存在漏诊率。
试验四：
1、检测样本：以40例正常人血浆为对照组，以40例患有血栓的住院白血病患者的血浆样本为实验组进行检测，在患者出现症状当天(确诊的第一天)采集血浆样本并进行检测。各实验组或对照组的数据均为40个平行试验的平均值或平均值±标准差。
2、FXIIIAP检测试剂盒包括：洗涤液、样品稀释液、显色底物、包被捕获抗体的酶标板和检测抗体。
其中，捕获抗体分别为实施方式3-1-1所述抗体(原有抗体，SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVVPR)、实施方式3-1-2所述抗体(改进抗体1，SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV)以及实施方式3-1-4所述抗体(改进抗体2，RTAFGGRRAVPPNN)；检测抗体为FXIIIAP全序列(SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVVPR)，制备方法为：用标准全序列FXIIIAP(SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVVPR)，结合载体蛋白后，免疫小鼠，得到的抗体以G蛋白纯化。检测抗体须结合生物素(Biotin)。
FXIIIAP检测试剂盒的使用过程至少包括如下步骤：
以100μl的捕获抗体包被ELISA酶标板的每孔；
以牛血清白蛋白对每孔作非特异性结合即封闭；
捕获抗体与病人血浆结合后，添加结合生物素的检测抗体，并且在添加碱性磷酸酶标记的链霉亲和素后，加入显色底物；
以标准ELISA分析设备分析结果。
3.特异性的比较
特异性系指能有效的检测游离状态的FXIIIAP，而不与未被激活的FXIII中的FXIIIAP(结合状态的FXIIIAP)结合；非特异性系指未被激活的FXIII上的结合状态的FXIIIAP与抗体相作用，从而对实验数值的影响。
需要说明的是，由于是特异性测试，故采用了FXIIIAP偏低的血栓白血病患者的血液样本，因此并未对中风患者的血样进行测试(脑中风患者FXIIIAP很高(>7ng/ml)，不适合作特异性比较)。与脑中风患者不同(由于血脑屏障打开，大量组织因子进入血液，从而引起广泛的血栓，造成FXIIIAP急速大量升高)，白血病患者局部血栓的FXIIIAP浓度较低且变化要小(FXIIIAP个体差异小)，因此对抗体的特异性要求更高，可以更直观地反映检测数据的真实、有效性。
4.试验结果：
请参照表1，为实施方式3-1-1所述抗体(原有抗体，记为1-37)、实施方式3-1-2所述抗体(改进抗体1，记为1-35)以及实施方式3-1-4所述抗体(改进抗体2，记为5-18)用于ELISA对FXIIIAP检测的特异性对比数据。
表1：
Figure PCTCN2022092581-appb-000001
Figure PCTCN2022092581-appb-000002
请参照表图6，图6为实施方式3-1-1所述抗体(原有抗体，记为1-37)、实施方式3-1-2所述抗体(改进抗体1，记为1-35)以及实施方式3-1-4所述抗体(改进抗体2，记为5-18)用于ELISA对FXIIIAP检测的特异性对比图。
从表1和图6数据可知，所有抗体均可检测出样本中的FXIIIAP，但是抗体1在正常对照组测得的FXIIIAP浓度比原有抗体低，其中改进抗体1测得的FXIIIAP浓度为2.62ng/ml，改进抗体2测得的FXIIIAP浓度为0.43ng/ml。理论上FXIIIAP只有被激活才可以被检测到，因此在正常人体内FXIIIAP是处于非常低的数值(除了本发明所述抗体及其试剂盒，目前国内外无精准的FXIIIAP检测方法，因此无标准浓度)。结合对照组和实验组数据可知，改进抗体2对照组与实验组的差异有统计学的显著差异(P<0.01)，改进抗体1对照组与实验组的差异有统计学的差异(P<0.05)，而原有抗体(全序列1-37)对照组与实验组的差异无统计学差异(P>0.05)。由此可见，在检测体内血栓时，本发明改进抗体(1和2)测得的FXIIIAP浓度更接近于真实数值，能够广泛应用于住院病人血栓的实时、有效的检测。
实施例5
检测试剂盒中的抗体制备，是以FXIIIAP部分序列(RTAFGGRRAVPPNN)为抗原，并且在其氨基端加入半胱氨酸以结合载体蛋白，制备而得到特异性抗体。其中制得特异性抗体的方法可以采用普通的抗原诱导生成抗体的方法。
实施例6
FXIIIAP检测试剂盒(干式免疫荧光定量法)包括：PVC底板、荧光结合垫、硝酸纤维素膜、样品垫和吸水垫，样品垫、荧光结合垫、硝酸纤维素膜、吸水垫按水平方向固定于PVC底板上。其中，荧光结合垫上有包被的由荧光微球标记的检测抗体，硝酸纤维素膜上依次设置有由捕获抗体构成的检测线和由兔抗鼠IgG抗体构成的质控线。
采用实施例5的特异性抗体为捕获抗体和检测抗体。
检测抗体(detection antibody)的制备方法如下：用FXIIIAP部分序列(改进抗体2，RTAFGGRRAVPPNN)结合载体蛋白后，免疫小鼠，得到的抗体以G蛋白纯化。
上述荧光免疫层析试剂盒的基本原理为双抗体夹心法，即样本中的待测物与分散在结合垫的荧光微球标记的检测抗体(改进抗体2-荧光微球)结合形成复合物。在层析作用下，复合物沿着反应膜向前扩散，复合物被固定在反应膜检测线上对应的捕获抗体(改进抗体2)所捕获。样本中待测物越多，反应形成的复合物也越多，检测线上积聚的复合物则越多，显色就更加明显，荧光信号的强度反应了被捕获的待测物的数量；未与T线反应的荧光微球标记的检测抗体则层析至质控线，与兔抗鼠IgG抗体结合显色。根据标准品曲线，可以计算出待测样本中的FXIIIAP浓度。
上述试剂盒的使用过程至少包括如下步骤：
将待测样品加入试纸条的样本孔中，反应15分钟后，使用干式免疫荧光分析仪读取醋酸纤维素膜上T线和C线荧光信号，计算出待测样本中的FXIIIAP浓度。
为验证本发明所述生物标志物、检测试剂盒以及相关检测方法的可行性，下面列出检测试剂盒进行检测的初期临床数据。以下为在对于M3白血病引起的凝血异常血栓、血栓性血小板紫癜及股静脉血栓、缺血型脑卒病例中的早期诊断的测试结果。
试验五：
分别采用实施例6的FXIIIAP检测试剂盒和D二聚体定量检测试剂盒的对M3白血病引起的凝血异常血栓进行实时监测，其中FXIIIAP检测试剂盒用于监测FXIIIAP的变化，D二聚体定量检测试剂盒用于监测D二聚体的变化。患者为由M3白血病引起的凝血异常血栓患者A和B，对患者在入院及治疗18至20天内的各理化指标(包括FXIIIAP和D二聚体)的变化水平进行监测。
请参照图7至图12，其中，图7为患者A的基本信息，图8和图9为患者A的血栓监测数据，图10为患者的A白细胞以及早幼粒细胞比例的监测变化图，图11为患者A的FXIIIAP以及纤维蛋白原的监测变化图，图12为患者A的D二聚体以及纤维蛋白单体的监测变化图。
由图8和图10的监测数据可知，在治疗过程中早幼粒细胞比例下降、白细胞比例上升、细胞释放促凝物质，导致微血栓的形成及诱导分化综合症[8-14天(对应于图10至图12中的背景灰色高亮区域)出现诱导分化综合症症状，提示血栓形成]。
由图9和图11的监测数据可知，在诱导分化综合症期间，FXIIIAP水平上升，和白细胞水平变化趋势一致；纤维蛋白原的变化趋势和FXIIIAP的变化趋势吻合，因为血栓形成导致纤维蛋白原消耗，佐证体内血栓形成，进一步证明FXIIIAP监测的准确性；经初期治疗后，可以看到FXIIIAP水平降低，提示血栓凝血异常改善。
由图9和图12的监测数据可知，D二聚体(血栓降解产物，提示血栓溶解)及纤维蛋白单体(FM，提示高凝状态)变化趋势和FXIIIAP的变化趋势吻合，但因为这两个标志物(D二聚体及纤维蛋白单体)都不是直接检测血栓形成，且在发病初期及诱导分化综合症期间均超过最大检测值，因此难以用于监测体内血栓形成。
请参照图13至图18，其中，图13为患者B的基本信息，图14和图15为患者B的血栓监测数据，图16为患者B的白细胞、早幼粒细胞比例的监测变化图，图17为患者B的FXIIIAP以及纤维蛋白原的监测变化图，图18为患者B的D二聚体以及纤维蛋白单体的监测变化图。
对凝血异常及血栓风险的监控是M3型白血病治疗过程中的关键，由图14至图18的数据可知，经过维A酸治疗后，可以看到生物标志物FXIIIAP水平迅速降低，显示在患者对维A酸药物反应良好，血栓及凝血异常得到控制，因此无需增加化疗药物。由图18的数据可知，D二聚体及纤维蛋白单体下降后迅速进入平台期，失去血栓监测效果。
由此可见，在监测体内血栓时，采用本发明改进抗体监测FXIIIAP浓度，能精准把控用药时机，减少甚至避免不必要的化疗，降低感染风险，降低住院费用，提升治愈率。
试验六：
分别采用实施例6的FXIIIAP检测试剂盒和D二聚体定量检测试剂盒的组合对血栓性血小板紫癜及股静脉血栓进行实时监测，其中FXIIIAP检测试剂盒用于监测FXIIIAP的变化，D二聚体定量检测试剂盒用于监测D二聚体的变化。患者为血栓性血小板紫癜及股静脉血栓患者C，对该患者在入院并经治疗一周病情缓解后，及复发时的各理化指标(包括FXIIIAP和D二聚体)的变化水平进行监测。
请参照图19和图20，其中，图19为患者C的基本信息，图20为患者C的血栓监测数据。
由图20的数据可知，患者在入院时的血栓形成高峰期，FXIIIAP值高，经治疗缓解后迅速降低。后复发入院时FXIIIAP水平又回升。由此可知，FXIIIAP水平和病情变化相吻合，证明其可作为生物标志物用于体内血栓监测。
试验七：
分别采用实施例6的FXIIIAP检测试剂盒和D二聚体定量检测试剂盒的组合对缺血性卒中进行实时监测，其中FXIIIAP检测试剂盒用于监测FXIIIAP的变化，D二聚体定量检测试剂盒用于监测D二聚体的变化。患者为缺血性卒中患者D，对该患者在入院急诊并经治疗后的各理化指标(包括FXIIIAP和D二聚体)的变化水平进行监测。
请参照图21和图22，其中，图21为患者D的基本信息，图22为患者D的血栓监测数据。
由图22的数据可知，患者在入院时FXIIIAP水平高，而D二聚体尚在正常范围内(D二聚体检测纤溶产物因此存在滞后性，因此传统利用D二聚体检测的方法容易有漏诊风险)；经治疗稳定后，FXIIIAP水平迅速下降，D二聚体则上升。
综上试验数据可知，本发明提出的FXIIIAP与D二聚体的标志物组合，能有效减低漏诊率，并反馈体内血栓形成及稳定的状态；本发明的抗体可以应用于多种现有的方法学，例如酶联免疫吸附测定法、干式免疫荧光法等，以对血栓或凝血相关疾病进行有效检测。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (10)

  1. 一种检测血栓或凝血相关疾病的生物标志物，其特征在于，所述标志物包括凝血因子13激活肽或凝血因子13的A亚基。
  2. 如权利要求1所述的检测血栓或凝血相关疾病的生物标志物，其特征在于，所述标志物包括凝血因子13激活肽和D二聚体的组合。
  3. 如权利要求1或2所述的检测血栓或凝血相关疾病的生物标志物，其特征在于：所述凝血相关疾病包括中风、脑静脉血栓、肾静脉血栓、静脉血栓或心肌梗塞。
  4. 如权利要求1所述的生物标志物在血栓或凝血相关疾病的检测产品和治疗效果评估产品中的应用。
  5. 如权利要求4所述的生物标志物在血栓或凝血相关疾病的检测产品和治疗效果评估产品中的应用，其特征在于：所述产品包括试剂盒或试剂。
  6. 一种用于检测血栓或凝血相关疾病的试剂盒，其特征在于：所述试剂盒中包括的捕获抗体或/和检测抗体为以权利要求1或2所述的生物标志物为抗原制得的特异性抗体。
  7. 如权利要求6所述的用于检测血栓或凝血相关疾病的试剂盒，其特征在于：所述试剂盒为双抗体夹心ELISA检测试剂盒或干式免疫荧光检测试剂盒，包括捕获抗体和检测抗体。
  8. 如权利要求6所述的用于检测血栓或凝血相关疾病的试剂盒，其特征在于：所述捕获抗体和检测抗体为以FXIIIAP的全序列或部分序列为抗原制备而得。
  9. 如权利要求8所述的用于检测血栓或凝血相关疾病的试剂盒，其特征在于，所述部分序列中包括以下序列或含有以下序列的片段：SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV或RTAFGGRRAVPPNN。
  10. 如权利要求7所述的用于检测血栓或凝血相关疾病的试剂盒，其特征在于：所述捕获抗体和检测抗体均提取自FXIIIAP免疫的试验动物。
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