- Home
- IVD
- By Technology Types
- By Diseases Types
- By Product Types
- Research
- Resource
- Distributors
- Company
| Product Name | Human Citrullinated Antithrombin (cAT) ELISA Kit |
| Catalog No. | CFTK-HMM-0103 |
| Test Species | Human |
| Application | This kit is used for the in vitro quantitative analysis of human citrullinated antithrombin (cAT) 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 citrullinated antithrombin (cAT) 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 citrullinated antithrombin (cAT) 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 | 120, 60, 30, 15, 7.5, 3.75 U/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 | 3.75 U/mL-120 U/mL |
| Sensitivity | < 0.1 U/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. |
Citrullinated Antithrombin (cAT) is a post-translationally modified form of Antithrombin (AT), a key serine protease inhibitor in the human coagulation system. Unlike the native AT, which primarily regulates blood clotting by inhibiting thrombin and factor Xa, cAT emerges when arginine residues in AT are converted to citrulline via the enzymatic action of peptidylarginine deiminases (PADs). This citrullination process alters the structural conformation and biological activity of AT, leading to reduced anticoagulant function—a change closely linked to multiple pathological conditions.
In clinical and research settings, the detection of cAT has gained significant attention due to its critical role as a biomarker for several diseases. For instance:
Thrombotic Disorders: Reduced cAT activity (resulting from impaired anticoagulant function) is associated with an increased risk of venous thromboembolism (VTE) and arterial thrombosis. Studies have shown that patients with deep vein thrombosis (DVT) or pulmonary embolism (PE) often exhibit abnormal cAT levels, making it a potential indicator for thrombosis risk assessment.
Inflammatory Diseases: Chronic inflammation, such as that seen in rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE), upregulates PAD activity, leading to elevated cAT production. This makes cAT a valuable tool for monitoring inflammation severity and disease progression in autoimmune conditions.
Cardiovascular Diseases: Atherosclerosis, myocardial infarction (MI), and stroke are linked to dysregulated coagulation and inflammation. cAT levels have been correlated with the degree of vascular damage and coagulation activation in these diseases, providing insights into disease pathogenesis and treatment response.
The demand for reliable cAT detection tools has grown rapidly in recent years, driven by the need to advance research on coagulation disorders, inflammation, and cardiovascular health. Traditional detection methods, such as Western blotting or immunoprecipitation, often suffer from low sensitivity, long assay times, and poor quantitative accuracy. In contrast, ELISA-based kits (like the Human Citrullinated Antithrombin (cAT) ELISA Kit) address these limitations by offering high specificity, sensitivity, and reproducibility—making them indispensable for both basic research and translational studies.
Broad Sample Compatibility: The kit is validated for use with multiple human 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 sources, simplifying experimental workflows.
Wide Detection Range: With a detection range of 3.75 U/mL to 120 U/mL, the kit can accurately measure both low and high cAT concentrations in samples. This covers the typical physiological and pathological cAT levels reported in research studies, reducing the need for excessive sample dilution or concentration steps.
Exceptional Sensitivity: The kit exhibits a sensitivity of <0.1 U/mL, enabling the detection of trace amounts of cAT in samples with low analyte levels. This is critical for early-stage disease research (e.g., preclinical studies on thrombosis or inflammation) where cAT concentrations may be minimally elevated.
High Specificity: It uses highly specific capture and HRP-labeled detection antibodies that exclusively bind to human cAT, with no cross-reactivity to other soluble structural analogs (such as native AT, citrullinated proteins unrelated to AT, or coagulation factors like thrombin). This ensures that assay results reflect true cAT levels, avoiding false positives or inaccurate quantification.
Short Assay Time: The entire assay process (from sample preparation to OD value measurement) can be completed within approximately 3.5 hours. This is significantly faster than traditional methods like Western blotting (which often take 6–8 hours) and supports high-throughput sample analysis for labs handling large sample batches.
Stable Storage Conditions: The kit can be stored at 2–8°C for up to 6 months, eliminating the need for long-term storage in -20°C or -80°C freezers. This reduces reagent degradation risks associated with repeated freeze-thaw cycles and simplifies inventory management for labs with limited cold storage space.
User-Friendly Protocol: The kit includes a detailed, step-by-step instruction manual with clear guidelines for sample processing, reagent preparation, and assay procedures. Even researchers with limited ELISA experience can follow the protocol easily, minimizing experimental errors and ensuring consistent results.
Reliable Reproducibility: The kit maintains strict quality control standards, with an intraplate coefficient of variation (CV) <10% and an interplate CV <15%. This means that results from the same sample (tested in duplicate or triplicate) or across different assay runs will be highly consistent, reducing data variability and enhancing the credibility of research findings.
Cost-Effective Design: Available in both 48T and 96T formats, the kit allows labs to choose the size that matches their sample volume needs. The 48T format is ideal for small-scale experiments (e.g., pilot studies), while the 96T format supports high-throughput testing—both options minimize reagent waste and lower the cost per assay compared to single-use or low-volume kits.
Minimal Sample Requirement: The assay only requires 50 µL of sample per well, making it suitable for precious or limited sample types (e.g., pediatric patient samples, rare tissue homogenates, or cell culture supernatants with low yields). This reduces the need to collect large volumes of samples, which is particularly beneficial for clinical research involving human subjects.
No Specialized Equipment Beyond Standard Lab Tools: The kit only requires common laboratory equipment (e.g., 450 nm microplate reader, 37°C thermostat, standard pipettes) for operation. There is no need for expensive specialized instruments (such as mass spectrometers or flow cytometers), lowering the barrier to entry for labs with limited budget or equipment resources.
Validated for Research and Preclinical Use: The kit has been rigorously tested with a large number of normal human specimens, confirming that normal cAT concentrations fall within its detection range. This validation ensures that it can be reliably used in basic research (e.g., studying cAT’s role in coagulation pathways) and preclinical studies (e.g., evaluating drug effects on cAT levels in animal models), supporting the transition of research findings to clinical applications.
For research use only, not for clinical use.
|
There is no product in your cart. |
Copyright © 2025 Alta DiagenoTech. All rights reserved.
