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| Product Name | Human Activated Coagulation Factor X (FXa) ELISA Kit |
| Catalog No. | CFTK-HMM-0077 |
| Test Species | Human |
| Application | This kit is for the in vitro quantitative analysis of human activated coagulation factor X (FXa) in serum, plasma, tissue homogenates and related fluid samples. |
| Shelf Life | 6 months |
| Storage | 2-8°C |
| Detection Principle | The kit uses a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). To pre-coated microtiter wells pre-coated with a capture antibody to human activated coagulation factor X (FXa), the specimen, standard, and HRP-labeled detection antibody are sequentially added, incubated and washed thoroughly. The color is developed with the substrate TMB, which is converted to blue by catalysis of peroxidase and to final yellow by acid. The shade of color is positively correlated with human activated coagulation factor X (FXa) in the sample. The absorbance (OD value) is measured at 450 nm wavelength using an enzyme meter and the concentration of the sample is calculated. |
| Sample Processing | 1. Serum: Place whole blood specimens collected in serum separator tubes at room temperature for 2 hours or overnight at 4°C, then centrifuge at 1000×g for 20 minutes and remove the supernatant, or store the supernatant at -20°C or -80°C, but avoid repeated freezing and thawing. 2. Plasma: Collect the specimen with EDTA or heparin as anticoagulant and centrifuge the specimen at 1000×g for 15 minutes at 2-8°C within 30 minutes after collection, and then remove the supernatant for testing, or store the supernatant at -20°C or -80°C, but avoid repeated freezing and thawing. 3. Tissue Homogenization: Rinse the tissue with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood (lysed erythrocytes in the homogenate will affect the measurement), weigh the tissue, and then cut the tissue into pieces. Mix the minced tissue with the corresponding volume of PBS (generally 1:9 weight-to-volume ratio, e.g., 1g of tissue sample corresponds to 9mL of PBS, the specific volume can be adjusted according to the experimental needs, and make a record. It is recommended to add protease inhibitors to PBS) into a glass homogenizer and grind it thoroughly on ice. In order to further lyse the tissue cells, the homogenate can be ultrasonically broken, or repeatedly frozen and thawed. Centrifuge the homogenate at 5000×g for 5-10 minutes and take the supernatant for testing. 4. Cell Culture Supernatant or Other Biological Specimens: Please centrifuge the supernatant at 1000×g for 20 minutes, and then take the supernatant for testing, or store the supernatant at -20°C or -80°C, but should avoid repeated freezing and thawing. Note: Hemolyzed specimens are not suitable for this test. |
| Self-contained Reagents / Instruments / Consumables | Enzyme labeler (450 nm) High-precision spikers and tips: 0.5-10 µL, 2-20 µL, 20-200 µL, 200-1000 µL 37°C thermostat Distilled or deionized water |
| Standard Concentration | 1600, 800, 400, 200, 100, 50 pg/mL |
| Reagent Preparation | The kit should be equilibrated at room temperature before use when removed from the refrigerated environment. Dilution of 20 x Wash Buffer: distilled water is diluted 1:20, i.e. 1 part 20 x Wash Buffer to 19 parts distilled water. |
| Procedures | 1. Remove the required plates from the aluminum foil pouch after equilibrating at room temperature for 20 min, and seal the remaining plates in a self-sealing bag and return them to 4°C. 2. Set up standard wells and sample wells, add 50 µL of standards of different concentrations to each standard well. 3. Add 50 µL of the sample to be tested into the sample wells; do not add to the blank wells. 4. Except for the blank wells, add 100 µL of horseradish peroxidase (HRP)-labeled detection antibody to each of the standard and sample wells, seal the reaction wells with a sealing membrane, and incubate for 60 min at 37°C in a water bath or thermostat. 5. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 µL), let stand for 1 min, shake off the washing solution, pat dry on absorbent paper, and repeat the plate washing for 5 times (plate washer can also be used to wash the plate). 6. Add 50 µL of substrate A and B to each well and incubate at 37°C for 15 min. 7. Add 50 µL of termination solution to each well, and measure the OD value of each well at 450 nm within 15 min. |
| Calculation of Results | The experimental results were calculated by taking the OD value of the measured standard as the horizontal coordinate and the concentration value of the standard as the vertical coordinate, drawing the standard curve on the coordinate paper or using the relevant software and obtaining the linear regression equation, substituting the OD value of the samples into the equation and calculating the concentration of the samples. |
| Detection Range | 50 pg/mL-1600 pg/mL |
| Sensitivity | < 10 pg/mL |
| Specificity | Does not cross-react with other soluble structural analogs. |
| Repeatability | The intraplate coefficient of variation is less than 10% and the interplate coefficient of variation is less than 15%. |
| Note | 1. After a large number of normal specimens, the normal concentration values of the specimens are within the detection range provided by the kit, and 50 µL of the sample can be sampled directly during the experiment. When the value of some samples exceeds the maximum concentration of the standard, the sample diluent can be used to dilute the specimen appropriately and then carry out the experiment. 2. Strictly follow the specified time and temperature for incubation to ensure accurate results. All reagents must be brought to room temperature of 20-25°C before use. keep reagents refrigerated immediately after use. 3. Incorrect plate washing can lead to inaccurate results. Ensure that the wells are as well drained as possible before adding substrate. Do not allow the wells to dry out during incubation. 4. Eliminate liquid residues and fingerprints on the bottom of the plate, otherwise the OD value will be affected. 5. The substrate chromogenic solution should be colorless or very light in color; substrate solution that has turned blue cannot be used. 6. Avoid cross contamination of reagents and specimens to avoid false results. 7. Avoid direct exposure to strong light during storage and incubation. 8. Equilibrate to room temperature before opening the sealed bag to prevent water droplets from condensing on the cold slats. 9. Any reaction reagents should not come into contact with bleaching solvents or strong gases emitted from bleaching solvents. Any bleaching components will destroy the biological activity of the reaction reagents in the kit. 10. Expired products must not be used. 11. If there is a possibility of spreading disease, all samples should be managed and samples and test devices should be handled according to prescribed procedures. |
Coagulation is a sophisticated, tightly regulated physiological process that maintains blood fluidity under normal conditions while preventing excessive blood loss upon vascular injury. At the core of this cascade lies Activated Coagulation Factor X (FXa), a key serine protease that serves as a critical junction in the common pathway of blood coagulation. Derived from its inactive zymogen (Factor X) via proteolytic activation—triggered by either the extrinsic (tissue factor-initiated) or intrinsic (contact activation-driven) pathways—FXa plays an indispensable role in driving the coagulation process forward.
Its primary biological function is to catalyze the conversion of prothrombin (Factor II) to thrombin (Factor IIa), a central enzyme that mediates the final steps of clot formation: converting soluble fibrinogen to insoluble fibrin strands and activating platelets to reinforce the clot structure. Beyond its foundational role in hemostasis, FXa has become a focal point of scientific research due to its involvement in a range of physiological and pathological processes, making it a vital target for investigative studies:
Dysregulated FXa activity is closely associated with thrombotic disorders, where excessive thrombin generation leads to abnormal clot formation—driving research into anticoagulant therapies and disease mechanisms.
Deficiencies or functional impairments of FXa are linked to bleeding phenotypes, spurring investigations into coagulation factor biology and genetic disorders.
Emerging evidence highlights FXa’s involvement in non-hemostatic processes, including inflammation, angiogenesis, and tumor progression, expanding its relevance across disciplines such as cardiovascular biology, immunology, and oncology.
The need for reliable, sensitive tools to quantify FXa in research settings has grown dramatically as the scientific community seeks to:
Unravel the molecular mechanisms governing coagulation cascade regulation and dysregulation.
Evaluate the efficacy of novel FXa-targeting compounds in preclinical drug discovery.
Characterize FXa expression and activity in cell models, animal models, and human biological samples.
Explore the role of FXa in cross-pathway interactions (e.g., coagulation-inflammation crosstalk).
Tailored specifically for research applications, the Human Activated Coagulation Factor X (FXa) ELISA Kit addresses these critical needs. It provides a robust, quantitative platform for measuring FXa levels in diverse biological samples, empowering researchers to generate accurate, reproducible data that advances scientific understanding of coagulation and related fields.
Versatile Sample Compatibility: Optimized for the analysis of FXa in serum, plasma, tissue homogenates, and cell culture supernatants, supporting a wide range of research models and experimental designs.
Exceptional Sensitivity: Offers a detection limit of < 10 pg/mL, enabling quantification of low-abundance FXa in samples with minimal target concentrations—critical for capturing subtle changes in research settings.
Superior Specificity: Rigorously validated to avoid cross-reactivity with other coagulation factors, soluble structural analogs, or non-target biomolecules, ensuring precise measurement of FXa alone.
Broad Detection Range: Covers 50 pg/mL to 1600 pg/mL, accommodating physiological, pathological, and experimentally manipulated FXa levels without requiring excessive sample dilution or concentration.
Consistent Reproducibility: Delivers intraplate coefficient of variation (CV) < 10% and interplate CV < 15%, ensuring reliable results across experiments, batches, and research teams.
Streamlined Protocol: Follows a user-friendly double-antibody one-step sandwich ELISA format with clear, step-by-step instructions, reducing experimental complexity and minimizing human error.
Stable Storage: Maintains optimal performance for 6 months when stored at 2–8°C, facilitating long-term inventory management for research laboratories.
Efficient Workflow: The entire assay, including incubations and detection, can be completed within a manageable timeframe, supporting high-throughput sample processing for large-scale research projects.
Research-Grade Precision: Engineered with high-quality antibodies and optimized reaction conditions to deliver accurate FXa quantification, ensuring data integrity for publications and research conclusions.
Cost-Effective Design: Available in 48T and 96T formats, providing sufficient reagent volumes for research-scale experiments while offering value for high-throughput studies.
Minimal Sample Requirement: Only 50 µL of sample per well is needed, conserving valuable research specimens (e.g., limited animal samples, rare cell culture supernatants, or patient-derived research samples).
Reduced Interference Risk: Includes detailed sample processing guidelines to mitigate issues like hemolysis, ensuring reliable results even with complex biological matrices commonly used in research.
Broad Research Applicability: Suitable for diverse research areas, including coagulation biology, drug discovery (anticoagulant development), disease model characterization, and cross-pathway interaction studies.
Simple Reagent Preparation: The 20× Wash Buffer requires only dilution with distilled or deionized water, eliminating time-consuming, complex reagent preparation steps.
Standard Lab Equipment Compatibility: Works seamlessly with common laboratory tools (450 nm microplate readers, 37°C thermostats), requiring no specialized or expensive equipment investments.
Rigorous Quality Control: Each batch undergoes strict quality testing to ensure consistency in sensitivity, specificity, and reproducibility, meeting research-grade ELISA performance standards.
For research use only, not for clinical use.
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