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| Product Name | Chicken Antithrombin III (AT-III) ELISA Kit |
| Catalog No. | CFTK-HMM-0099 |
| Test Species | Chicken |
| Application | This kit is used for the in vitro quantitative analysis of chicken antithrombin III (AT-III) in serum, plasma, tissue homogenates and related liquid 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 microwells with chicken antithrombin III (AT-III) 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 antithrombin III (AT-III) 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 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 | 50 ng/mL-1600 ng/mL |
| Sensitivity | < 10 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. |
Antithrombin III (AT-III) is a crucial serine protease inhibitor synthesized primarily in the liver, playing a central role in maintaining the delicate balance of the coagulation and fibrinolytic systems in vertebrates—including chickens, which are widely used as model organisms in veterinary medicine, agricultural biotechnology, and comparative physiology research. For chicken-specific studies, understanding AT-III levels is not only essential for exploring basic biological mechanisms but also holds significant practical value across multiple fields.
Key Roles of Chicken AT-III in Research and Industry
Veterinary Health Monitoring: Poultry flocks (such as broilers, layers, and breeding chickens) are often exposed to stressors like environmental changes, pathogen infections (e.g., avian influenza, Newcastle disease), and dietary imbalances. These stressors can disrupt the chicken’s coagulation system, leading to abnormal AT-III expression. For example, studies have shown that chickens infected with pathogenic bacteria may experience reduced AT-III levels, increasing the risk of thrombotic disorders or excessive bleeding. Monitoring chicken AT-III concentrations via reliable detection tools helps veterinarians and flock managers early identify subclinical health issues, preventing large-scale mortality and economic losses.
Agricultural Biotechnology and Breeding: In modern poultry breeding, traits like disease resistance, growth rate, and reproductive performance are core selection criteria. Emerging research indicates a potential correlation between AT-III activity and chicken robustness—individuals with stable AT-III levels may exhibit stronger resistance to coagulation-related diseases. By quantifying AT-III in breeding populations, researchers can incorporate this biochemical marker into genetic selection programs, accelerating the development of healthier, more productive chicken breeds.
Comparative Physiology and Biomedical Research: Chickens share evolutionary and physiological similarities with other vertebrates, making them ideal models for studying coagulation system evolution and human-related diseases. For instance, research on chicken AT-III can provide insights into the conservation of protease inhibitor functions across species, aiding in the development of animal models for human thrombophilic disorders (e.g., congenital AT-III deficiency). Additionally, chicken embryos are widely used in developmental biology; analyzing AT-III expression during embryonic development helps clarify the ontogeny of the coagulation system.
Challenges in Chicken AT-III Detection and the Need for Specialized Kits
Traditional methods for detecting AT-III (e.g., functional assays using chromogenic substrates, immunoturbidimetry) face limitations when applied to chicken samples:
Species Specificity Issues: Many commercial AT-III detection kits are designed for humans or common laboratory animals (e.g., mice, rats) and lack cross-reactivity with chicken AT-III, leading to inaccurate or unreadable results.
Sample Complexity: Chicken serum, plasma, and tissue homogenates often contain high levels of lipids, nucleoproteins, and other interfering substances. Non-specialized kits may exhibit high background noise, reducing detection sensitivity and precision.
Low Throughput: Functional assays typically require manual pipetting, long incubation times, and specialized equipment (e.g., spectrophotometers with specific wavelength ranges), making them inefficient for processing large numbers of samples— a common need in poultry research and industry.
Against this backdrop, the Chicken Antithrombin III (AT-III) ELISA Kit was developed to address these pain points. By leveraging chicken-specific antibodies and a optimized sandwich ELISA format, the kit ensures high specificity, sensitivity, and throughput, making it a reliable tool for both basic research and applied scenarios in poultry science.
Broad Sample Compatibility: The kit is validated for use with multiple chicken sample types, including serum, plasma (EDTA or heparin anticoagulated), tissue homogenates (e.g., liver, kidney), and cell culture supernatants. This eliminates the need for researchers to purchase separate kits for different sample matrices, reducing experimental costs and complexity.
Wide Detection Range with Excellent Lineararity: Covering a concentration range of 50 ng/mL to 1600 ng/mL, the kit can accurately quantify both low and high levels of chicken AT-III without the need for excessive sample dilution. The standard curve exhibits strong linearity (typically R² > 0.99), ensuring reliable interpolation of sample concentrations.
High Sensitivity for Trace Detection: With a detection limit of < 10 ng/mL, the kit can detect low-abundance AT-III in samples such as chicken embryonic plasma or cell culture supernatants—scenarios where traditional methods may fail to capture meaningful signals. This sensitivity is critical for studies investigating subtle changes in AT-III levels (e.g., during early pathogen infection or developmental stages).
Rapid Assay Workflow: The entire assay process (from sample preparation to result readout) can be completed in approximately 3 hours, including a 60-minute primary incubation and a 15-minute substrate reaction. This is significantly faster than traditional functional assays (which often take 4–6 hours) and enables high-throughput sample processing (up to 96 samples per plate).
Stable Reagents with Long Shelf Life: All kit components (including pre-coated microwells, HRP-labeled detection antibody, and substrates) maintain stability for 6 months when stored at 2–8°C. The pre-coated plates are sealed in aluminum foil pouches with desiccants to prevent moisture-induced degradation, ensuring consistent performance across batches.
User-Friendly Protocol Design: The kit includes detailed, step-by-step instructions with clear guidelines for sample processing (e.g., centrifugation speeds, protease inhibitor recommendations) and reagent preparation (e.g., 20× wash buffer dilution). Even researchers with limited ELISA experience can easily follow the protocol, minimizing human error.
Superior Species Specificity to Avoid Cross-Reactivity: The kit uses capture and detection antibodies specifically raised against chicken AT-III, which have been rigorously tested to ensure no cross-reactivity with other chicken serine protease inhibitors (e.g., α1-antitrypsin, heparin cofactor II) or AT-III from other species (e.g., human, mouse, turkey). This eliminates false-positive or false-negative results caused by non-specific binding, ensuring data accuracy.
High Precision for Reproducible Results: The kit exhibits excellent repeatability, with intra-plate coefficient of variation (CV) < 10% and inter-plate CV < 15%. This level of precision is critical for longitudinal studies (e.g., monitoring AT-III levels in the same chicken over time) or multi-laboratory collaborations, where consistent results across experiments and sites are essential.
Minimal Sample Requirement: Only 50 µL of sample is needed per well, which is particularly beneficial for precious samples (e.g., chicken embryo serum, small tissue homogenates from juvenile chickens) where sample volume is limited. This reduces the need to sacrifice additional animals or collect excessive samples, aligning with ethical guidelines for animal research.
Cost-Effective for Routine and Large-Scale Use: The kit is available in 48T and 96T formats, allowing researchers to choose the appropriate size based on their sample volume. Compared to outsourcing detection services or using single-use assay strips, the kit offers a lower cost per sample, making it ideal for routine monitoring in poultry farms or high-throughput research projects.
For research use only, not for clinical use.
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