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| Product Name | Human Plasminogen Precursor Fragment F2 (F2) ELISA Kit |
| Catalog No. | CFTK-HMM-0053 |
| Test Species | Human |
| Application | This kit is for the in vitro quantitative analysis of human prothrombin precursor fragment F2 (F2) in serum, plasma, tissue homogenates and related fluids. |
| 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 the coated microtiter wells pre-coated with capture antibody to human thrombospondin precursor fragment F2 (F2), 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. There is a positive correlation between the shade of color and the human thrombospondin precursor fragment F2 (F2) 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 | 8, 4, 2, 1, 0.5, 0.25 nmol/L |
| 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 | 0.25 nmol/L-8 nmol/L |
| Sensitivity | < 0.1 nmol/L |
| 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 II, also known as prothrombin, is a key glycoprotein in the human blood coagulation system, encoded by the F2 gene. As a zymogen, it plays a central role in the common pathway of blood coagulation—when activated, it is converted into thrombin, which further catalyzes the conversion of fibrinogen to fibrin, ultimately forming a fibrin clot to stop bleeding. The Human Plasminogen Precursor Fragment F2 (F2) is a specific cleavage product generated during the activation process of prothrombin, and its level in biological samples (such as serum, plasma, and tissue homogenates) is closely associated with the activation status of the coagulation system.
In recent years, with the deepening of research in the fields of hematology, cardiovascular diseases, and thrombophilic disorders, the detection of F2 has become increasingly important. For example:
In the study of thrombotic diseases (such as deep vein thrombosis, pulmonary embolism, and myocardial infarction), abnormal increases in F2 levels often indicate excessive activation of the coagulation system, which can be used as a potential diagnostic marker or a indicator for evaluating disease progression.
In the research of hemorrhagic disorders (such as congenital prothrombin deficiency), reduced F2 levels may reflect impaired prothrombin synthesis or abnormal activation, providing a basis for exploring the pathogenesis of such diseases.
In basic medical research (such as the development of anticoagulant drugs and the study of coagulation regulatory mechanisms), accurate quantification of F2 is essential to assess the effects of experimental interventions on the coagulation system.
Broad Sample Compatibility: The kit is applicable to multiple types of human biological samples, including serum, plasma (with EDTA or heparin as anticoagulants), tissue homogenates, and cell culture supernatants. This flexibility allows researchers to use the kit for different experimental models (such as in vitro cell experiments, animal tissue studies, and clinical sample research), without the need to purchase additional kits for different sample types.
Wide Detection Range with High Sensitivity: It has a detection range of 0.25 nmol/L to 8 nmol/L, covering the common physiological and pathological F2 concentration ranges in most research scenarios. Meanwhile, its sensitivity is less than 0.1 nmol/L, which can accurately detect low-concentration F2 in samples (such as samples from early-stage coagulation activation or mild coagulation disorders), avoiding false-negative results caused by insufficient sensitivity.
Excellent Specificity to Avoid Cross-Interference: The kit uses highly specific capture antibodies and HRP-labeled detection antibodies targeting human F2. It does not cross-react with other soluble structural analogs (such as other coagulation factors, plasminogen fragments, or common proteins in biological samples). This ensures that the detected signal accurately reflects the actual F2 concentration, eliminating the interference of non-target substances on experimental results.
Good Reproducibility for Stable Experimental Data: The intra-plate coefficient of variation (CV) of the kit is less than 10%, and the inter-plate CV is less than 15%. This means that whether multiple samples are tested on the same microplate or repeated tests are performed on different microplates, the results can maintain high consistency. It provides a stable data foundation for researchers to conduct repeated experiments and verify research conclusions.
Convenient and Time-Saving Operation Process: The entire experimental procedure (from sample addition to OD value measurement) can be completed within approximately 4 hours, and the steps are simple and easy to standardize (such as pre-coated microtiter wells, ready-to-use detection antibodies, and clear washing and incubation guidelines). Even researchers with limited ELISA operation experience can quickly master the method, reducing the time cost of experimental training and operation.
Reliable Technical Principle Ensures Result Accuracy: The kit adopts the double-antibody one-step sandwich ELISA technology. The pre-coated capture antibody first specifically binds to F2 in the sample, and then the HRP-labeled detection antibody binds to another epitope of F2, forming a "capture antibody-F2-detection antibody" sandwich complex. The TMB substrate is catalyzed by HRP to produce a color reaction, and the OD value is positively correlated with the F2 concentration. This technology has been widely verified in the field of protein quantification, with strong specificity and accuracy, and is far superior to single-antibody detection methods in reducing non-specific binding.
Strict Quality Control for Stable Performance: During the production process of the kit, each batch of reagents (including antibodies, substrates, and standard products) undergoes strict quality inspection, such as testing the binding activity of antibodies, the stability of substrates, and the accuracy of standard concentrations. The shelf life of the kit is 6 months under proper storage conditions (2-8°C), and the activity loss rate is less than 5% within the expiration date. This ensures that researchers can use the kit with confidence within the valid period, without worrying about performance degradation affecting experimental results.
Cost-Effective Packaging Options Meet Different Needs: The kit provides two packaging specifications (48T and 96T). For small-scale experiments (such as preliminary verification of experimental ideas or a small number of sample tests), the 48T specification can avoid reagent waste; for large-scale experiments (such as batch detection of clinical samples or high-throughput screening), the 96T specification is more cost-effective. This flexible packaging design helps researchers control experimental costs while meeting different experimental scales.
Comprehensive Supporting Materials Simplify Experimental Preparation: The kit is equipped with a detailed datasheet, which includes not only detailed operation steps but also sample processing methods (such as serum centrifugation conditions, tissue homogenization ratios, and precautions for avoiding hemolysis), reagent preparation guidelines (such as dilution methods for 20× Wash Buffer), and result calculation methods (such as drawing standard curves and substituting sample OD values for concentration calculation). In addition, the datasheet also lists the required instruments and consumables (such as enzyme labelers, pipettes, and tips) to help researchers prepare all experimental materials in advance and avoid delays caused by missing materials.
For research use only, not for clinical use.
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