Patent Application
20240255532
The present invention relates to the technological field of thrombosis or coagulation-related disorder detection, and in particular to a biomarker for detecting thrombosis or coagulation-related disorders and an application thereof.
Thrombus formation and associated coagulation disorders are pivotal diagnostic elements in clinical settings, encompassing a wide spectrum of diseases such as Cerebral Venous Thrombosis (CVT), Renal Vein Thrombosis (RVT), Venous Thromboembolism (VTE), and Myocardial Infarction (MI). Particularly, the early diagnosis and treatment of conditions like cerebral venous thrombosis and myocardial infarction are of significant importance. For instance, in the context of cerebral venous thrombosis, a major cause of ischemic stroke, established diagnostic methods like Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) are notably effective in determining the incidence, type, and severity of strokes. However, these modalities are not only costly and time-consuming but also lack efficiency in early detection, often resulting in missed opportunities for optimal treatment. In contrast, CAT scans (Computerized Axial Tomography) are better suited for diagnosing hemorrhagic strokes. Despite the relative sensitivity in differentiating hemorrhagic stroke thereof, CAT scans are less sensitive to cerebral ischemia during stroke assessments, usually becoming positive 24-36 hours after stroke, with approximately 50% of ischemic strokes being undetectable via CAT scans. Consequently, by the time existing diagnostic technologies confirm a stroke, the window for early intervention is often lost.
For diagnosing acute ischemic and hemorrhagic strokes, the prevalent clinical methods lack sufficient sensitivity, accuracy, and timeliness, leading to potential misdiagnoses or missed diagnoses, thereby delaying the treatment.
Recent researches indicate that employing molecular biomarkers for rapid screening and differential diagnosis of stroke types offers a novel approach to address these challenges.
Biomarkers refer to biochemical indicators that can mark changes in the structure or function of an organism's organs, tissues or cells. Therefore, the detection of biomarkers can be used for disease diagnosis and provide a scientific basis for subsequent treatment options.
Various stroke biomarkers are already known. Molecular biomarkers related to stroke have been disclosed in the invention patent with publication number CN103299191A. Methods for diagnosing and differentiating strokes are disclosed in the invention patent with publication number CN1339108A. Thrombus formation serves as a crucial diagnostic indicator for cardiovascular diseases like ischemic strokes. The current clinical screening for thrombi predominantly involves D-dimer test, primarily utilized in diagnosing Venous Thromboembolism (VTE), Deep Vein Thrombosis (DVT), and Pulmonary Embolism (PE). D-dimer testing has shown promise in many instances. However, due to various influencing factors and intrinsic limitations of D-dimers themselves, there are still many controversies in the clinical use of D-dimer test. Primarily, D-dimers originate from plasmin-dissolved crosslinked fibrin clots, reflecting the status of fibrinolysis (thrombolysis) rather than direct thrombus formation. All factors affecting thrombolysis can influence D-dimer test outcomes. For example, for the patients with fibrinolytic disorders, those on oral anticoagulants, or those with VTE symptoms persisting for over 14 days, it may yield false-negative results. Moreover, D-dimers, indicative of the thrombus dissolution process and having a prolonged half-life (13-23 hours), can produce false-positive results when other coagulation triggers are present, like inflammation or pregnancy. D-dimers also exhibit a high rate of missed diagnosis in clinical tests. For instance, there's a 20% missed diagnosis rate for CVT, higher in early stages (symptoms appearing in less than 24 hours) at about 50%. For general venous thrombosis, D-dimer diagnosis also shows a 50% missed diagnosis rate. Particularly, for early-stage newly formed thrombi, it can give false-negative results.
Due to the extended half-life of D-dimers, they fail to accurately and timely reflect the occurrence of bleeding and coagulation when there is existing coagulation disorders. Moreover, the ambiguous definition of the D-dimer analyte, along with varying detection methodologies based on monoclonal antibodies that recognize different fibrin fragments or surface structures, result in the absence of international standard reference preparations or calibrators. Coupled with the low sensitivity and specificity thereof, the application of D-dimers is still controversial. Additionally, the detection results lack therapeutic guidance, failing to accurately and promptly reflect the body's coagulation state, thereby not providing indications for treatment strategies, such as the effectiveness of anticoagulant therapies, or confirming the presence of new thrombi, since old thrombi can still be dissolved to produce D-dimers.
Recognizing the deficiencies of existing technologies, the present invention introduces a biomarker for directly detecting thrombus formation, distinct from existing biomarkers that indirectly detect tissue stress responses to stroke. The biomarker, either used alone or in combination with D-dimer test, offers high sensitivity and accuracy in detecting and screening thrombosis or coagulation-related diseases.
More particularly, the invention provides a biomarker applicable for detecting thrombosis or coagulation-related diseases.
Additionally, the invention includes an application of said biomarker in products designed for detecting thrombosis or coagulation-related diseases and for evaluating treatment efficacy.
Further, the invention exhibits a reagent kit tailored for detecting thrombosis or coagulation-related diseases.
To achieve the aforementioned objectives, the invention the following principal technical solutions:
In a first aspect, the invention provides a biomarker for detecting thrombosis or coagulation-related diseases, comprising FXIIIAP or FXIIIA, wherein FXIIIAP is an activation peptide of Coagulation Factor XIII, and FXIIIA is an A subunit of Coagulation Factor XIII.
Optionally, the biomarker may include a combination of FXIIIAP and D-dimer.
Optionally, the relevant coagulation disorders include conditions like stroke, cerebral venous thrombosis, renal vein thrombosis, venous thrombosis, or myocardial infarction.
In a second aspect, the invention also introduces a diagnostic method for thrombosis or cardiovascular diseases, involving:
Optionally, the test sample can be blood or plasma.
Optionally, specific antibodies are generated using a partial sequence of FXIIIAP, including sequences SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV or RTAFGGRRAVPPNN.
In a third aspect, the invention provides an application of the said biomarker in products for detection and treatment effect assessment of thrombosis or coagulation-related diseases.
Optionally, the products for detection and treatment effect assessment of thrombosis or coagulation-related diseases may include a reagent kit or a reagent.
In a fourth aspect, the invention offers a reagent kit for detecting thrombosis or coagulation-related diseases, wherein the kit's capture antibodies and/or detection antibodies are specific antibodies produced using the biomarker as an antigen.
Optionally, the reagent kit can be a double-antibody sandwich ELISA detection kit or a dry-type immunofluorescence detection kit, comprising a capture antibody and a detection antibody.
Optionally, the antibodies in the kit are produced using complete or partial sequence of FXIIIAP an antigen.
Optionally, in the reagent kit for detecting thrombosis or coagulation-related disorders, the partial sequence includes the following sequence or a fragment comprising the following sequence: SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV or RTAFGGRRAVPPNN.
Optionally, the capture antibodies and the detection antibodies are derived from animals immunized with FXIIIAP.
Optionally, the antibodies of the invention are not limited to ELISA (Enzyme-Linked Immunosorbent Assay) but are also applicable in Colloidal Gold Method, Radioimmunoassay (RIA), Chemiluminescent Immunoassay (CLIA), Electro-Chemiluminescence Immunoassay (ECLI), Fluorescent Immunoassay like Dry-Type Immunofluorescence Method, Time-Resolved Fluoroimmunoassay (TRFIA) for detecting the aforementioned biomarkers.
The invention's beneficial effects include:
The invention provides a biomarker for detecting thrombosis or coagulation-related diseases, which, in combination with the traditional D-dimer biomarker, can significantly enhance sensitivity and detection rates over current technologies.
The biomarker FXIIIAP, as indicated in the invention, demonstrates superior detection performance for acute thrombosis (less than 48 hours) compared to existing technologies, specifically in contrast to D-dimer. However, due to rapid degradation of FXIIIAP 48 hours after symptom onset, it can't be detected, resulting in a very high missed diagnosis rate. Therefore, the present invention proposes combining FXIIIAP with D-dimer to effectively and substantially lower the missed diagnosis rate throughout the disease cycle, including early and late stages.
Wherein,
1. The biomarker proposed in the invention does not correlate with the fibrinolysis process, allowing detection in patients with insufficient fibrinolysis.
2. The biomarker proposed in the invention provides a more accurate reflection of new thrombus formation inside the body compared to D-dimer and is thus applicable in evaluating treatment effects and disease severity.
3. The biomarker proposed in the invention facilitates rapid diagnosis of coagulation diseases including stroke, enhances existing diagnostic sensitivity and specificity, enables inference on thrombus formation timing and extent, and predicts disease severity and prognosis.
4. The application include in the invention provides information on treatment strategies, such as guidance for anticoagulant or thrombolytic therapy, and effectively monitor the effectiveness of anticoagulant drugs.
5. The detection kits proposed in the invention are suitable for finger blood testing and long-term monitoring, possessing a significant commercial value.
6. The detection kits proposed in the invention fills a market gap for FXIIIAP diagnostic kits.
To better explain and understand the present invention, the following detailed description is provided in conjunction with accompanying drawings:
A biomarker for detecting thrombosis or coagulation-related diseases: FXIIIAP (coagulation factor XIII activation peptide).
A biomarker for detecting thrombosis or coagulation-related diseases: FXIIIA (A subunit of coagulation factor XIII).
A biomarker for detecting thrombosis or coagulation-related diseases: a combination of FXIIIAP and FXIIIA.
A biomarker for detecting thrombosis or coagulation-related diseases: a combination of FXIIIAP and D-dimer.
A biomarker for detecting thrombosis or coagulation-related diseases: a combination of FXIIIA and D-dimer.
A biomarker for detecting thrombosis or coagulation-related diseases: a combination of FXIIIAP, FXIIIA, and D-dimer.
In the disclosed embodiments 1-4, 1-5, and 1-6, the biomarker combinations comprising FXIIIAP or FXIIIA and D-dimer exhibited enhanced accuracy for detecting thrombotic or coagulation-related disorders across various stages. Distinct from D-dimer, the presence of FXIIIAP was independent of the fibrinolytic process, thereby enabling the application thereof in patients exhibiting suboptimal fibrinolysis. Moreover, FXIIIAP appeared earlier than D-dimer. Clinical evaluations had demonstrated superior efficacy of FXIIIAP in identifying acute-phase thrombosis (less than 2 days after onset). While D-dimer levels began to increase from the second day after symptom onset, and increased significantly on the third day. FXIIIAP levels were already elevated (15.8 ng/ml) on the first day when D-dimer levels remained comparatively lower (<500 ng/mL). This elevation in healthy individuals was typically around 4 ng/mL. On the second day, FXIIIAP concentrations notably decreased to 7.2 ng/ml and returned to the normal on the third day, illustrating a complementary relationship between D-dimer and FXIIIAP.
Studies indicated rapid in vivo degradation of FXIIIAP, suggesting that the elevated levels thereof reflected the immediate thrombotic state in the body. Compared to D-dimer, FXIIIAP more effectively reflected the formation of new thrombi, thus aiding in evaluating treatment efficacy and disease severity. However, due to the expedited degradation thereof, FXIIIAP might not be detectable in patients with advanced symptoms, resulting in a certain misdiagnosis rate. Therefore, it was necessary to combine with D-dimer to reduce the risk of missed diagnosis.
FXIIIAP is not only effective for stroke caused by thrombosis, but theoretically it will also have considerable effects on hemorrhagic stroke. When intracranial hemorrhage occurs, the permeability of the blood-brain barrier increases, and coagulation factors can pass through the blood-brain barrier. Given the very low molecular weight thereof (merely 3.91 KD), FXIIIAP readily traverses the blood-brain barrier. The brain, abundant in tissue factors, releases these upon blood-brain barrier compromise, which induce systemic coagulation. While D-dimer has been utilized to detect intracranial hemorrhage, outcomes have been suboptimal. Presently, no experimental assays employing FXIIIAP for hemorrhagic stroke diagnosis exist. Theoretically, it is possible that FXIIIAP may maintain elevated levels without reduction over time, alongside diminished FXIIIA and elevated D-dimer concentrations (high AP level, low FXIIIA level, and high D-dimer level).
The present invention comprises coagulation-related conditions, including but not limited to stroke, cerebral venous thrombosis, renal vein thrombosis, venous thrombosis, and myocardial infarction, thereby covering a spectrum of thrombosis-induced disease.
This embodiment relates to an application of the biomarkers as described in Embodiment 1-1 in detection products and therapeutic efficacy evaluation products for thrombosis or coagulation related diseases.
Further, in certain embodiments, the products comprise reagent kits or reagents.
This embodiment relates to an application of the biomarkers as described in Embodiment 1-2 in detection products and therapeutic efficacy evaluation products for thrombosis or coagulation related diseases.
Further, in some embodiments, the products comprise reagent kits or reagents.
This embodiment relates to an application of the biomarkers as described in Embodiment 1-3 in detection products and therapeutic efficacy evaluation products for thrombosis or coagulation related diseases.
Further, in some embodiments, the products comprise reagent kits or reagents.
This embodiment relates to an application of the biomarkers as described in Embodiment 1-4 in detection products and therapeutic efficacy evaluation products for thrombosis or coagulation related diseases.
Further, in some embodiments, the products comprise reagent kits or reagents.
This embodiment relates to an application of the biomarkers as described in Embodiment 1-5 in detection products and therapeutic efficacy evaluation products for thrombosis or coagulation related diseases.
Further, in some embodiments, the products comprise reagent kits or reagents.
This embodiment relates to an application of the biomarkers as described in Embodiment 1-6 in detection products and therapeutic efficacy evaluation products for thrombosis or coagulation related diseases.
Further, in some embodiments, the products comprise reagent kits or reagents.
A reagent kit for detecting thrombotic or coagulation-related disorders, comprising a capture antibody and/or a detection antibody, wherein the antibodies are specific antibodies generated using the biomarkers as described in Embodiment 1-1 as antigens. In some embodiments, the kit is specifically formulated to include a dual-antibody sandwich ELISA kit comprising both capture and detection antibodies.
On the basis of Embodiment 3-1, more specifically, the capture and detection antibodies are produced using the complete sequence of FXIIIAP as the antigen.
On the basis of Embodiment 3-1, more specifically, the capture and detection antibodies are created using a partial sequence of FXIIIAP as the antigen, and the is partial sequence SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV.
On the basis of Embodiment 3-1, more specifically, the capture and detection antibodies are created using a partial sequence of FXIIIAP as the antigen, and the partial sequence is a partial segment of SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV.
On the basis of Embodiment 3-1, more specifically, the capture and detection antibodies are derived using a partial sequence of FXIIIAP as the antigen, and the partial sequence is RTAFGGRRAVPPNN.
On the basis of Embodiment 3-1, more specifically, the capture and detection antibodies are specifically engineered using a fragment of the FXIIIAP sequence as the antigen, and the partial sequence is a partial segment of RTAFGGRRAVPPNN.
A reagent kit for detecting thrombotic or coagulation-related disorders, comprising a capture antibody and/or a detection antibody, wherein the antibodies are specific antibodies generated using the biomarkers as described in Embodiment 1-2 as antigens. In some embodiments, the kit is specifically formulated to include a dual-antibody sandwich ELISA kit comprising both capture and detection antibodies.
A reagent kit for detecting thrombotic or coagulation-related disorders, comprising a capture antibody and/or a detection antibody, wherein the antibodies are specific antibodies generated using the biomarkers as described in Embodiment 1-3 as antigens. In some embodiments, the kit is specifically formulated to include a dual-antibody sandwich ELISA kit comprising both capture and detection antibodies.
A reagent kit for detecting thrombotic or coagulation-related disorders, comprising a capture antibody and/or a detection antibody, wherein the antibodies are specific antibodies generated using the biomarkers as described in Embodiment 1-4 as antigens. In some embodiments, the kit is specifically formulated to include a dual-antibody sandwich ELISA kit comprising both capture and detection antibodies.
Further, in some embodiments, the antibodies are sourced from animals immunized with FXIIIAP, more accurately, extracting IgG from such immunized test animals.
A reagent kit for detecting thrombotic or coagulation-related disorders, comprising a capture antibody and/or a detection antibody, wherein the antibodies are specific antibodies generated using the biomarkers as described in Embodiment 1-5 as antigens. In some embodiments, the kit is specifically formulated to include a dual-antibody sandwich ELISA kit comprising both capture and detection antibodies. In some embodiments, the kit is specifically formulated to include a dual-antibody sandwich ELISA kit comprising both capture and detection antibodies.
Further, in some embodiments, the antibodies are sourced from animals immunized with FXIIIAP, more accurately, extracting IgG from such immunized test animals.
A reagent kit for detecting thrombotic or coagulation-related disorders, comprising a capture antibody and/or a detection antibody, wherein the antibodies are specific antibodies generated using the biomarkers as described in Embodiment 1-6 as antigens. In some embodiments, the kit is specifically formulated to include a dual-antibody sandwich ELISA kit comprising both capture and detection antibodies. In some embodiments, the kit is specifically formulated to include a dual-antibody sandwich ELISA kit comprising both capture and detection antibodies.
Further, in some embodiments, the antibodies are sourced from animals immunized with FXIIIAP, more accurately, extracting IgG from such immunized test animals.
A reagent kit for detecting thrombotic or coagulation-related disorders, comprising a capture antibody and/or a detection antibody, wherein the antibodies are specific antibodies generated using the biomarkers as described in Embodiment 1-7 as antigens. In some embodiments, the kit is specifically formulated to include a dual-antibody sandwich ELISA kit comprising both capture and detection antibodies. In some embodiments, the kit is specifically formulated to include a dual-antibody sandwich ELISA kit comprising both capture and detection antibodies.
Further, in some embodiments, the antibodies are sourced from animals immunized with FXIIIAP, more accurately, extracting IgG from such immunized test animals.
Different from D-dimer, Coagulation Factor XIII (FXIII) is the last coagulation factor activated in coagulation process, primarily responsible for cross-linking fibrin clots to stabilize thrombi. The activation thereof reflects the completion of the coagulation process, indicating formation of a stable thrombus. During the activation thereof, each FXIII molecule releases two Factor XIII Activation Peptides (FXIIIAPs). FXIIIAP, a peptide consisting of only 37 amino acids (approximately 3.91 KD), stabilizes the dimeric structure of FXIIIA2. Upon activation by Thrombin, FXIIIAP disassociates from FXIII and enters the bloodstream as free FXIIIAP. The structural conformation of free FXIIIAP markedly differs from that bound to FXIII. The specific antibodies, produced in the present invention using synthetic FXIIIAP as an antigen, do not bind to FXIIIAP in unactivated FXIII (i.e., bound FXIIIAP). The specific antibodies in the present invention can effectively detect free FXIIIAP without binding to unactivated FXIII. This feature ensures diagnostic specificity and can accurately detect activated FXIII. Clinical experiments have demonstrated that released FXIIIAP can be detected by using ELISA or fluorescent immunological methods.
For the reagent kit proposed in the present invention, core component thereof is FXIIIAP specific antibodies produced using an improved FXIIIAP amino acid sequence. According to computer-simulated experiments: FXIIIAP, when cleaved by Thrombin and released into the bloodstream, forms a double β-strands structure with an amino acid sequence of 1-35, whereas amino acids 36 and 37 do not contribute to this structure formation. On the contrary, the two amino acids' instability directly causes variability in the carboxyl end of synthetic FXIIIAP, and the produced antibodies cannot efficiently detect native FXIIIAP. The present invention utilizes synthetic FXIIIAP with a more stable structure as an antigen (amino acid sequence 1-35), more specifically, Using the sequence SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV to generate specific antibodies against the stable double β-strands structure, effectively enhancing antibody sensitivity.
More particularly, upon further analysis of FXIIIAP's structure, the inventors found amino acid sequence 5-18 to be critical for the binding of FXIIIAP to FXIIIA subunit and antigenically favorable for antibody production. In the bound state with FXIIIA, the antibody binding site is masked. Once FXIIIAP becomes free, the antibody binding site is exposed. Therefore, a peptide containing the amino acid sequence 5-18 (RTAFGGRRAVPPNN) can be used for antibody production.
Specific antibodies produced using this method, relative to antibodies generated using the complete sequence of FXIIIAP (amino acid sequence 1-37, monoclonal antibody), can significantly improve sensitivity to native FXIIIAP. Enhanced sensitivity is observed in vitro tests using normal plasma; more preferably, cysteine is added at the carboxyl or amino end of the above partial sequence for binding carrier proteins. Sensitivity experiments with the improved antibodies show that antibodies produced using this method can detect more than 80% of native FXIIIAP. Additionally, as the standard sequence of FXIIIAP was used in antibody preparation, this diagnostic reagent is easier to standardize and promote compared to D-dimer.
Currently, there are no FXIIIAP detection kits on the market. The FXIIIAP detection kit provided by the present invention, utilizing specific antibodies generated using FXIIIAP as an antigen, can effectively bind free FXIIIAP in plasma without binding to FXIIIAP in unactivated FXIII (bound FXIIIAP), thus effectively detecting free FXIIIAP in plasma. The FXIIIAP detection kit can accurately detect activated FXIII for diagnosing of thrombosis or cardiovascular diseases. The kit proposed in the present invention has been clinically proven to detect released FXIIIAP using ELISA or fluorescent immunological methods.
For ease of antibody production, the standard complete sequence of FXIIIAP has cysteine added at the amino terminus to bind to the carrier protein, increasing the immunogenicity of the synthesized FXIIIAP. As the standard complete sequence of FXIIIAP (SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVVPR, PDB Entry 1F13) is used in the preparation of specific antibodies, standardization is facilitated, making it easier to promote compared to D-dimer.
Further, in the specific antibody preparation process, a partial sequence of the synthetic FXIIIAP with more stable structure (amino acid sequence 1-35) is used the as an antigen, specifically sequence SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV, to produce specific antibodies against the stable double β-strands structure, effectively enhancing antibody sensitivity. More preferably, cysteine is added to the carboxyl end of the above partial sequence, and sensitivity experiments with the improved antibodies demonstrate that antibodies produced using this method can detect more than 80% of native free FXIIIAP.
The capture and detection antibodies are extracted from IgG of animals immunized with FXIIIAP. The detection antibody, after binding with biotin, is used in conjunction with Streptavidine alkaline phosphatase for enhanced sensitivity.
The method and the detection kit provide by the present invention can be used for quantitative analysis. As thrombus formation consumes FXIIIA, the A subunit of Coagulation Factor XIII (FXIIIA), FXIIIA also decreases in the blood. Although FXIIIA does not have a definitive diagnostic significance like FXIIIAP and D-dimer, the decrease thereof can indicate extent and severity of thrombus formation, and suggests more significant thrombus formation and severe condition.
The present invention provides a method for detecting thrombosis and coagulation-related diseases, comprising the following steps:
Optionally, the test sample may be blood or plasma.
Optionally, specific antibodies are produced using a partial sequence of FXIIIAP as an antigen; the partial sequence of FXIIIAP includes sequence SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV or RTAFGGRRAVPPNN.
Wherein, the test sample is blood or plasma.
For better understanding of the above technical solutions, the exemplary embodiments of the present invention are described in more detail below with reference to the accompanying drawings. It should be understood that the present invention can be implemented in various forms and is not limited to the embodiments described herein. Instead, the embodiments are provided for clearer and more thorough understanding of the present invention and to fully convey the scope of the invention to the technical personnel in the field.
For preparation of the antibodies in the detection kit, the FXIIIAP partial sequence (SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV) was used as an antigen, and cysteine was added at the amino terminus to bind to a carrier protein, resulting in specific antibodies. The method for producing the specific antibodies could be common antigen-induced antibody generation techniques.
For preparation of the antibodies in the detection kit, the FXIIIAP complete sequence (SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVVPR) was used as an antigen, and cysteine was added at the amino terminus to bind to a carrier protein, resulting in specific antibodies. The method for producing the specific antibodies could be common antigen-induced antibody generation techniques.
The FXIIIAP detection kit included: washing solution, sample diluent, chromogenic substrate, enzyme-labeled plate coated with capture antibodies, and detection antibodies.
The specific antibodies from Example 1 were used as capture antibodies, and the complete sequence of FXIIIAP from Example 2 was used as detection antibodies.
The method for preparing detection antibodies was as follows: the standard complete sequence FXIIIAP (SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVVPR) was bound to a carrier protein, mice were immunized, and the resulting antibodies were purified with G protein. The detection antibodies must be conjugated with biotin. In the detection process, the detection antibodies were necessary to be combined with Streptavidine alkaline phosphatase to increase sensitivity.
The use of the above detection kit included at least the following steps:
A combined detection kit comprising the FXIIIAP detection kit of Example 3 and a D-dimer quantitative detection kit, wherein the D-dimer quantitative detection kit can be a conventional detection kit.
The combined detection kit in this example could simultaneously detect FXIIIAP and D-dimer in patients' blood or plasma samples, significantly enhancing the detection rate of thrombosis and coagulation-related diseases.
To validate the feasibility of the detection kit and the related detection method in the present invention, initial clinical data from testing the detection kits were given. The following results are for the early diagnosis of ischemic stroke.
122 samples from patients with early ischemic stroke were tested.
Plasma samples were collected on the day symptoms appeared, and were labeled as Day 1, and subsequent samples were set as Day 2, Day 3, and Day 4 according to the collection time. Testing was conducted by using the combined detection kit of Example 4. In this test, the D-dimer level exceeding 500 ng/ml was considered as positive, and the FXIIIAP level more than 7 ng/ml was tentatively considered as positive.
Sensitivity, also known as true positive rate, refers to the proportion of samples that are actually positive and are correctly identified as positive. In this experiment, the patients with actual ischemic stroke were identified based on FXIIIAP and/or D-dimer test results. The sensitivity was calculated as the ratio of true positives to the sum of true positives and false negatives (true positives but identified as negatives).
Refer to
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27 plasma samples (collecting on the day symptoms appear) from patients with early hemorrhagic stroke were tested using a combination of the FXIIIAP detection kit and the D-dimer quantitative detection kit. The results showed that the combined sensitivity of detecting FXIIIAP and D-dimer reached 100%, without missed diagnoses. In contrast, testing with only D-dimer detection kit showed a missed diagnosis rate of 22%. Therefore, the combined detection kit proposed in the present invention is beneficial to reduce the missed diagnosis rate for hemorrhagic stroke using D-dimer.
Refer to
Conversely, for the same plasma samples, FXIIIAP was detected in 85% and 87% of the plasma samples in vitro test using the detection kit containing either improved antibody 1 or improved antibody 2. Laboratory results demonstrated that improved antibodies 1 or 2 as capture antibodies better identified FXIIIAP in vivo, without cross-reacting with FXIII, thereby accurately reflecting the real levels of FXIIIAP in test samples.
Therefore, due to the improvement in capture antibodies, the sensitivity for early detection of FXIIIAP in patients increased by about 30%. Furthermore, due to the rapid degradation characteristics of FXIIIAP, the aforementioned detection kits achieved better detection rates for early-stage coagulation-related diseases or thrombosis. In contrast, there exists a missed diagnosis rate for later-stage coagulation-related diseases or thrombosis.
Plasma samples from 40 normal individuals were selected as the control group, and plasma samples from 40 hospitalized leukemia patients with thrombosis were used the test group. Samples were collected on the day symptoms appeared (the day of diagnosis) and tested. Data were the average values or average values±standard deviation of 40 parallel experiments.
FXIIIAP detection kit Included washing solution, sample diluent, chromogenic substrate, enzyme-labeled plate coated with capture antibody, and detection antibody.
Wherein, the capture antibodies were as described in Embodiment 3-1-1 (original antibody, SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVVPR), Embodiment 3-1-2 (improved antibody 1, SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVV), and Embodiment 3-1-4 (improved antibody 2, RTAFGGRRAVPPNN).
The detection antibody was the complete sequence of FXIIIAP (SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVVPR). The method for preparing the detection antibodies was as follows: the standard complete sequence FXIIIAP (SETSRTAFGGRRAVPPNNSNAAEDDLPTVELQGVVPR) was bound to a carrier protein, mice were immunized, and the resulting antibodies are purified with G protein. The detection antibodies must be conjugated with biotin.
The use of the detection kit included at least the following steps:
Specificity refers to effective detection of free-state FXIIIAP, without binding to unactivated FXIIIAP (bound-state FXIIIAP). Non-specificity refers to interaction of bound-state FXIIIAP on unactivated FXIII with antibodies, thereby affecting experimental values.
It is important to note that due to the specificity test, blood samples from thrombotic leukemia patients with lower FXIIIAP levels were used, and samples from stroke patients were not tested (FXIIIAP levels in stroke patients are very high [>7 ng/ml], unsuitable for specificity comparison). Unlike stroke patients (wherein the opening of the blood-brain barrier allows a large amount of tissue factors into the blood, causing widespread thrombosis and a rapid increase in FXIIIAP), thrombotic leukemia patients have lower and less variable FXIIIAP concentrations in localized thrombosis, thus requiring higher specificity in antibodies for a more direct and valid reflection of test data.
Refer to Table 1 for specificity comparison data of FXIIIAP detection using ELISA with antibodies described in Embodiment 3-1-1 (original antibody, labeled as 1-37), Embodiment 3-1-2 (improved antibody 1, labeled as 1-35), and Embodiment 3-1-4 (improved antibody 2, labeled as 5-18).
Refer to
From the data in Table 1 and
For preparation of antibodies in the detection kit, the partial sequence of FXIIIAP (RTAFGGRRAVPPNN) was used as an antigen, and cysteine was added at amino terminus thereof to bind to a carrier protein, resulting in specific antibodies. The method for producing the specific antibodies can be the conventional antigen-induced antibody production techniques.
The FXIIIAP detection kit (dry-type immunofluorescence quantitative method) included a PVC base plate, a fluorescence conjugate pad, a nitrocellulose membrane, a sample pad, and an absorbent pad, wherein the fluorescence conjugate pad, the nitrocellulose membrane, the sample pad, and the absorbent pad were all fixed on the PVC base plate. The fluorescence conjugate pad contains detection antibodies labeled with fluorescent microspheres, and the nitrocellulose membrane sequentially involves test lines composed of capture antibodies and quality control lines composed of rabbit anti-mouse IgG antibodies.
Specific antibodies from Embodiment 5 were used as both capture antibodies and detection antibodies.
The preparation method for the detection antibody (detection antibody) was as follows: The partial sequence of FXIIIAP (improved antibody 2, RTAFGGRRAVPPNN) was combined with a carrier protein, mice was immunized, and the obtained antibodies were purified using G protein.
The basic principle the aforementioned of fluorescent immunochromatographic detection kit is double-antibody sandwich method, wherein analyte in the sample is able to bind to the detection antibodies labeled with fluorescent microspheres (improved antibody 2-fluorescent microspheres) dispersed in the conjugate pad, thereby forming complexes. The complexes migrate forward along the reaction membrane under chromatography, and are captured by the corresponding capture antibodies (improved antibody 2) at the test line on the reaction membrane. The more analytes in the sample, the more complexes formed, leading to more accumulation of complexes at the test line and a more pronounced color or fluorescence signal, which indicates the quantity of the captured analytes. Unreacted detection antibodies marked with fluorescent microspheres migrate to the quality control line and then bind to the rabbit anti-mouse IgG antibodies, indicating validity. Based on standard curves, the concentration of FXIIIAP in the test samples can be calculated.
The use of the aforementioned kit included the following steps at least:
The test sample was added to the sample well of the test strip. After a 15-minute reaction, a dry immunofluorescence analyzer was used to read the fluorescence signals at the T (Test) and C (Control) lines on the nitrocellulose membrane to determine the concentration of FXIIIAP in the sample.
To verify the feasibility of the biomarkers, detection kits, and detection methods described in the present invention, initial clinical data from applications of the detection kits were presented below. The results pertained to early diagnosis tests in cases of coagulation abnormal thrombosis caused by M3 leukemia, thrombotic thrombocytopeniaurpura, and deep vein thrombosis, and ischemic stroke.
Real-time monitoring of coagulation abnormalities and thrombosis caused by M3 leukemia was conducted using the FXIIIAP detection kit of Embodiment 6 and D-dimer quantitative detection kit, wherein the FXIIIAP detection kit was used to monitor changes in FXIIIAP, and the D-dimer quantitative detection kit was for D-dimer changes. Patients A and B, suffering from coagulation abnormalities and thrombosis due to M3 leukemia, were monitored for various biochemical indicators (including FXIIIAP and D-dimer) from admission to 18-20 days of treatment.
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Monitoring coagulation abnormalities and thrombotic risks was crucial in treatment of M3 leukemia, as indicated in the data from
This indicated that monitoring thrombosis in vivo using the improved antibodies for FXIIIAP detection in the present invention can accurately control medication timing, reducing or even avoiding unnecessary chemotherapy, lowering infection risks, reducing hospitalization costs, and increasing cure rates.
The combination of FXIIIAP detection kit of Embodiment 6 and D-dimer quantitative detection kit combination were used for real-time monitoring of thrombotic thrombocytopenia purpura and deep vein thrombosis, wherein the FXIIIAP detection kit was applied to monitor FXIIIAP changes and the D-dimer quantitative detection kit was for D-dimer changes. Patient C, suffering from thrombotic thrombocytopeniaurpura and deep vein thrombosis, was monitored for various biochemical indicators (including FXIIIAP and D-dimer) at admission, after one week of treatment to condition improvement, and upon disease relapse.
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The combination of FXIIIAP detection kit of Embodiment 6 and D-dimer quantitative detection kit combination were used for real-time monitoring of ischemic stroke, wherein the FXIIIAP detection kit was used to monitor FXIIIAP changes and the D-dimer quantitative detection kit was for D-dimer changes. Patient D, suffering from ischemic stroke, was monitored for various biochemical indicators (including FXIIIAP and D-dimer) from emergency hospital admission and throughout treatment.
Refer to
In conclusion, the data from the above experiments indicated that the combination of FXIIIAP and D-dimer biomarkers proposed in the present invention was able to effectively reduce the missed diagnosis rate and provided feedback on the formation and the stabilization of thrombosis in vivo. The antibodies of the present invention was able to be applied in various existing methodologies, such as enzyme-linked immunosorbent assay (ELISA) and dry immunofluorescence, in order to effectively detect thrombotic or coagulation-related diseases.
Importantly, it should be noted: the embodiments described above are intended to illustrate the technical solutions of the present invention and are not limitations thereof. Despite detailed descriptions of the invention through the aforementioned embodiments, it should be understood by those skilled in the art that modifications to the technical solutions recorded in the aforementioned embodiments, or partial or complete equivalent replacements of some or all of the technical features thereof, do not depart from the essence of the technical solutions of the embodiments of the present invention, which was covered by the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110526415.2 | May 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/092581 | 5/13/2022 | WO |
