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| Product Name | Human Anti-Coagulation Factor 3 (FIII) ELISA Kit |
| Catalog No. | CFTK-HMM-0064 |
| Test Species | Human |
| Application | This kit is used for the in vitro quantitative analysis of human anticoagulation factor 3 (FIII) 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 human anti-coagulation factor 3 (FIII) capture antibody, 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 anticoagulant factor 3 (FIII) 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 | 2000, 1000, 500, 250, 125, 62.5 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 | 62.5 pg/mL-2000 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 Factor 3 (FIII), also known as Tissue Factor (TF) or CD142, is a critical cell surface glycoprotein encoded by the F3 gene (with a human gene ID of 2152) that plays an indispensable role in initiating the human blood coagulation cascade. Unlike other coagulation cofactors that circulate as inactive precursors, FIII is a fully functional initiator when expressed on cell surfaces—specifically, it acts as a high-affinity receptor for Coagulation Factor VII (FVII). The formation of the FIII-FVII complex triggers a catalytic cascade, initiating limited proteolysis of downstream coagulation proteases and ultimately leading to thrombin formation, which is essential for normal blood clotting to prevent excessive bleeding.
In research contexts, the quantitative detection of FIII in biological samples is of great significance for studying multiple physiological and pathological processes. Abnormal expression or activity of FIII has been linked to various conditions, including thrombotic disorders (such as deep vein thrombosis and myocardial infarction), inflammatory responses, and even tumor progression—since some cancer cells overexpress FIII to promote angiogenesis and metastasis. As a result, reliable tools for measuring FIII levels are in high demand for academic research, preclinical studies, and drug development related to coagulation biology, cardiovascular diseases, and oncology.
Our Human Anti-Coagulation Factor 3 (FIII) ELISA Kit is designed to meet this critical research need. It enables accurate, in vitro quantitative analysis of FIII in common biological samples, including serum, plasma (with EDTA or heparin as anticoagulants), tissue homogenates, and cell culture supernatants. The kit leverages the well-established double-antibody one-step sandwich ELISA technology, a gold standard in immunoassay techniques, to ensure high specificity, sensitivity, and reproducibility—addressing the core requirement of researchers for reliable data in FIII-related studies. Notably, FIII is the only coagulation factor for which a congenital deficiency has not been described in humans, making its detection primarily focused on measuring variable expression levels rather than absence, further highlighting the need for precise quantitative tools like this ELISA kit.
Broad Sample Compatibility: The kit supports quantitative analysis of FIII in multiple common biological samples, including serum, plasma (EDTA or heparin-anticoagulated), tissue homogenates, and cell culture supernatants. This eliminates the need for researchers to purchase separate kits for different sample types, reducing experimental costs and simplifying workflow.
Optimal Detection Performance: It offers a detection range of 62.5 pg/mL to 2000 pg/mL, which aligns with the typical FIII concentration range in most normal human biological samples—allowing direct sampling of 50 µL of sample for most experiments (with easy dilution using sample diluent for samples exceeding the upper limit). Additionally, the kit has a sensitivity of <10 pg/mL, ensuring accurate detection even for low-abundance FIII in dilute samples (such as cell culture supernatants).
High Specificity & Reproducibility: The kit does not cross-react with other soluble structural analogs of FIII (e.g., other coagulation factors like FVII, FVIII, or unrelated cytokines), ensuring that detected signals are exclusively from FIII. For reproducibility, it maintains an intraplate coefficient of variation (CV) of <10% and an interplate CV of <15%, guaranteeing consistent results across different wells, experiments, and operators.
User-Friendly Operation: The kit provides clear, step-by-step protocols, with a total time-to-result of approximately 4-5 hours (including incubation and washing steps) and a hands-on time of around 1 hour 20 minutes—balancing efficiency and accuracy. All reagents are ready to use after simple preparation (e.g., 20× Wash Buffer only requires 1:20 dilution with distilled water), and no specialized or complex equipment beyond a standard enzyme labeler (450 nm), 37°C thermostat, and common pipettes is needed.
Stable Storage & Long Shelf Life: The kit can be stored at 2-8°C for 6 months, eliminating the need for long-term cryogenic storage and reducing the risk of reagent degradation from repeated freeze-thaw cycles. Before use, simply equilibrate the kit to room temperature, ensuring convenience for routine laboratory use.
Reliable Data Output: Built on the double-antibody one-step sandwich ELISA principle, the kit uses pre-coated capture antibodies and HRP-labeled detection antibodies that specifically bind to FIII. The color development (from TMB substrate, converting from blue to yellow with acid) is positively correlated with FIII concentration, and measuring absorbance at 450 nm ensures objective, quantifiable results—avoiding the subjectivity of qualitative or semi-quantitative methods.
Minimized Experimental Errors: The kit includes detailed sample processing guidelines (e.g., centrifugation speeds and times for serum, plasma, and tissue homogenates; avoidance of hemolyzed specimens) and critical operation notes (e.g., strict adherence to incubation time/temperature, proper plate washing to prevent non-specific binding). These guidelines help reduce common errors caused by sample handling or operational inconsistencies, especially for less experienced researchers.
Cost-Effective for Both Small and Large Experiments: The kit is available in 48T and 96T formats, allowing laboratories to choose the appropriate size based on their experimental scale. For small-scale pilot studies, the 48T format avoids reagent waste; for large-scale experiments (e.g., analyzing multiple samples from a cohort study), the 96T format provides economies of scale. Additionally, the long shelf life means kits can be stored for future use without frequent repurchasing.
Flexible for Diverse Research Needs: Whether studying FIII expression in normal vs. pathological tissues, evaluating the effect of drugs on FIII secretion in cell cultures, or measuring FIII levels in patient-derived serum/plasma (for research purposes only), the kit’s broad sample compatibility and optimal detection range make it adaptable to various research scenarios in coagulation biology, immunology, and oncology.
Comprehensive Support for Experimental Success: The kit provides clear instructions for reagent preparation, standard curve generation (using 6 standard concentrations: 2000, 1000, 500, 250, 125, 62.5 pg/mL), and result calculation (via coordinate paper or software-based linear regression). This ensures that researchers—even those new to ELISA—can efficiently generate valid data without extensive method development.
For research use only, not for clinical use.
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