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| Product Name | Chicken Prothrombin (PT) ELISA Kit |
| Catalog No. | CFTK-HMM-0097 |
| Test Species | Chicken |
| Application | This kit is for the in vitro quantitative analysis of chicken prothrombin (PT) 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). Into the microtiter wells pre-coated with chicken prothrombin (PT) 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 chicken prothrombinogen (PT) in the sample. The absorbance (OD) was measured at 450 nm using an enzyme meter and the sample concentration was 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 ng/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 | 0.25 ng/mL-8 ng/mL |
| Sensitivity | < 0.1 ng/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. |
Prothrombin (PT), also known as coagulation factor II, is a key glycoprotein in the extrinsic and common pathways of the chicken coagulation cascade. Synthesized primarily in the liver, it plays an irreplaceable role in maintaining normal hemostasis in chickens—when vascular damage occurs, PT is activated to thrombin, which further converts fibrinogen to fibrin, ultimately forming a blood clot to prevent excessive bleeding. Abnormal PT levels in chickens are closely associated with a range of health and physiological issues, making its quantitative detection critical for poultry research and related fields.
Significance in Poultry Health Research: In modern poultry farming and veterinary research, monitoring chicken PT levels helps assess liver function (since impaired liver function reduces PT synthesis), diagnose coagulation disorders (such as vitamin K deficiency, which inhibits PT activation), and evaluate the impact of pathogens (e.g., certain viruses that damage liver cells, leading to decreased PT production). For example, in studies on avian fatty liver syndrome, researchers often measure PT concentrations to determine the extent of liver damage and its effect on coagulation function.
Demand in Biomedical Research: Chicken models are widely used in comparative physiology and biomedical studies due to their evolutionary proximity to other vertebrates and ease of breeding. PT, as a conserved coagulation factor, serves as a key indicator in studies on coagulation system evolution, cross-species coagulation mechanism comparisons, and the development of animal models for human coagulation disorders (e.g., hemophilia). Accurate quantitative detection of chicken PT is therefore essential to ensure the reliability and reproducibility of such research data.
Limitations of Traditional Detection Methods: Conventional methods for PT detection (such as clotting time assays) have drawbacks like low sensitivity, poor specificity, and reliance on large sample volumes—issues that are particularly problematic when working with limited chicken samples (e.g., juvenile chicken blood or small tissue homogenate volumes). ELISA kits, by contrast, address these limitations with high sensitivity, specificity, and throughput, making them the preferred tool for modern chicken PT research. Our Chicken Prothrombin (PT) ELISA Kit is developed to meet this growing demand, providing researchers with a reliable, efficient solution for PT quantitative analysis.
Broad Sample Compatibility: The kit is validated for use with multiple chicken sample types, including serum, plasma (EDTA or heparin-anticoagulated), tissue homogenates, and cell culture supernatants. This flexibility eliminates the need for researchers to purchase separate kits for different sample types, simplifying experimental workflows and reducing costs.
High Sensitivity and Wide Detection Range: With a sensitivity of <0.1 ng/mL and a detection range of 0.25 ng/mL–8 ng/mL, the kit can accurately detect both low and moderate PT concentrations in chicken samples. This covers the typical physiological range of chicken PT (as confirmed by peer-reviewed research) and ensures that even slight fluctuations in PT levels (e.g., early-stage liver damage-induced decreases) are detected.
Excellent Specificity: The kit uses highly specific capture and detection antibodies that do not cross-react with other soluble structural analogs (such as other chicken coagulation factors like factor I or factor VII) or non-target proteins in biological samples. This avoids false-positive or false-negative results, ensuring the accuracy of experimental data.
Good Reproducibility: Intra-plate coefficient of variation (CV) is <10% and inter-plate CV is <15%, meeting international ELISA kit quality standards. This consistency ensures that results are reliable across different experiments, batches, and operators—critical for long-term research projects or multi-laboratory collaborations.
User-Friendly Protocol: The kit includes pre-coated microtiter wells and ready-to-use reagents (with simple dilution steps for wash buffer only), and the entire assay can be completed in approximately 2.5 hours (including incubation and washing steps). Detailed, step-by-step instructions are provided, making the kit suitable for both experienced researchers and laboratory technicians new to ELISA assays.
Stable Storage and Long Shelf Life: All reagents can be stored at 2–8°C, eliminating the need for expensive ultra-low temperature freezers. The 6-month shelf life from the date of manufacture ensures that researchers can stock the kit without concerns about rapid expiration, reducing the risk of reagent waste.
Time and Labor Efficiency: Compared to traditional clotting assays (which require constant monitoring of clot formation and take 3–4 hours per batch), our ELISA kit streamlines the process with pre-coated plates and standardized incubation steps. Researchers can process up to 96 samples simultaneously, significantly increasing throughput—ideal for large-scale studies (e.g., screening PT levels in a flock of chickens or multiple tissue samples from a chicken model).
Cost-Effective: The kit is available in 48T and 96T formats, allowing researchers to choose the appropriate size based on their sample volume needs (e.g., 48T for small pilot studies, 96T for large experiments). This avoids over-purchasing and reduces per-sample testing costs. Additionally, the compatibility with multiple sample types eliminates the need for separate kits, further lowering overall research expenses.
Reliable Performance Backed by Quality Control: Each batch of the kit undergoes rigorous quality control testing, including checks for sensitivity, specificity, reproducibility, and reagent stability. A certificate of analysis (CoA) is available upon request, providing researchers with documentation of the kit’s performance—essential for meeting the data integrity requirements of peer-reviewed publications and institutional research standards.
Minimal Sample Requirement: The assay only requires 50 µL of sample per well, making it suitable for scenarios where sample volume is limited (e.g., blood samples from newly hatched chicks, which yield small amounts of serum/plasma, or rare tissue samples). This reduces the need for repeated sampling from the same animal, aligning with ethical guidelines for animal research.
Flexible Application Scenarios: Beyond basic PT concentration measurement, the kit can be used in diverse research contexts, including chicken liver function assessment, coagulation disorder diagnosis in poultry models, evaluation of the effects of feed additives (e.g., vitamin K supplements) on PT synthesis, and cross-species coagulation mechanism studies. This versatility makes it a valuable tool for researchers in poultry science, veterinary medicine, and comparative physiology.
For research use only, not for clinical use.
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