| Usage | Required experimental equipment: 1. Microplate reader (450nm) 2. High-precision pipettes and pipette tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37°C incubator 4. Distilled or deionized water Sample handling and requirements: The detection range of the kit is not equivalent to the concentration range of the analyte in the sample. Before the experiment, it is recommended to estimate the analyte concentration in the sample based on relevant literature and conduct pilot experiments to determine the actual concentration in the sample. If the analyte concentration in the sample is too high or too low, dilute or concentrate the sample appropriately. If the sample being tested is not listed in the instructions, it is recommended to conduct a pilot experiment to verify the validity of the test. Serum: Collect whole blood in a serum separator tube and place it at room temperature for 2 hours or at 4°C overnight. Then centrifuge at 1000×g for 20 minutes and remove the supernatant. Alternatively, store the supernatant at -20°C or -80°C, but avoid repeated freezing and thawing. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant and centrifuge at 1000×g for 15 minutes at 2-8°C within 30 minutes of collection. Remove the supernatant and test it. Alternatively, store the supernatant at -20°C or -80°C, but avoid repeated freezing and thawing. Tissue homogenate: Rinse the tissue with pre-chilled PBS (0.01M, pH 7.4) to remove residual blood (lysed red blood cells in the homogenate may affect the measurement results). Weigh the tissue and mince it. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1g of tissue sample to 9mL of PBS. The specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and grind thoroughly on ice. To further lyse the tissue cells, the homogenate can be sonicated or repeatedly freeze-thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes and remove the supernatant for analysis. Cell culture supernatant: Centrifuge at 1000×g for 20 minutes and remove the supernatant for analysis. Alternatively, store the supernatant at -20°C or -80°C, but avoid repeated freeze-thaw cycles. Other biological specimens: Centrifuge at 1000×g for 20 minutes and remove the supernatant for analysis. Sample Appearance: The sample should be clear and transparent, and suspended matter should be removed by centrifugation. Sample Storage: Samples collected for testing within one week can be stored at 4°C. If testing cannot be performed promptly, aliquot the sample into single-use aliquots and store at -20°C (for testing within one month) or -80°C (for testing within six months). Avoid repeated freeze-thaw cycles. Hemolysis of the sample can affect the final test results, so hemolyzed samples should not be used for this test. Pre-Test Preparation: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare the standard gradient working solution: Add 1 mL of universal diluent to the lyophilized standard. Let stand for 15 minutes to completely dissolve, then gently mix (concentration: 2000 pg/mL). Then dilute the sample to the following concentrations: 2000 pg/mL, 1000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.25 pg/mL, and 0 pg/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each. Pipette 500uL of the 2000pg/mL standard working solution into the first EP tube and mix thoroughly to make a 1000pg/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves as a blank well; there is no need to pipette liquid from the penultimate tube. See the figure below for details. 3. Preparation of HRP antibody working solution: 15 minutes before use, centrifuge the concentrated HRP antibody at 1000×g for 1 minute. Dilute 100× concentrated HRP antibody to a working concentration of 1× with universal diluent (e.g., 10 μL concentrate + 990 μL universal diluent) and use on the same day. 4. Preparation of 1× Wash Buffer: Dissolve 10ml of 20× Wash Buffer in 190ml of distilled water (Concentrated Wash Buffer removed from the refrigerator may crystallize, which is normal. Allow to stand at room temperature, shake gently, and allow the crystals to completely dissolve before reconstitution).
Procedure:
1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes. Seal the remaining strips in a ziplock bag and return to 4°C.
2. Sample Loading: Add 100µl of sample or standard of varying concentrations to the appropriate wells. Add 100µL of Universal Diluent to the blank wells. Cover with sealant and incubate at 37°C for 1 hour. (Recommendation: Dilute the sample to be tested at least 1-fold with Universal Diluent before loading into the plate. This minimizes matrix effects on the test results. When calculating sample concentration, multiply by the dilution factor. It is recommended to run replicates for all samples and standards.) 3. Wash: Discard the liquid and add 300 μL of 1x wash buffer to each well. Let stand for 1 minute, shake off the wash buffer, and pat dry on absorbent paper. Repeat this process three times (a microplate washer can also be used). 4. Add HRP Antibody: After washing, add 100 μL of HRP Antibody Working Solution directly to each well. Cover with a film sealer and incubate at 37°C for 1 hour. 5. Wash: Discard the liquid and wash the plate five times according to the procedure in step 3. 6. Add Substrate: Add 90 μL of substrate (TMB) to each well. Cover with a film sealer and incubate at 37°C in the dark for 15 minutes. 7. Add Stop Solution: Remove the ELISA plate and add 50 μL of Stop Solution directly to each well. Immediately measure the OD value of each well at 450 nm. Calculation of Experimental Results: Interpretation of Results: 1. Calculate the average OD value of the replicate standard and sample wells and subtract the OD value of the blank well as a correction value. Plot the standard curve of the four-parameter logistic function on double-logarithmic graph paper, with concentration as the horizontal axis and OD value as the vertical axis. (Omit the blank well values when plotting.) 2. If the sample OD value is above the upper limit of the standard curve, dilute the sample appropriately and retest. Multiply the sample concentration by the corresponding dilution factor.
 Kit Performance
1. Repeatability: The intra-plate coefficient of variation is less than 10%, and the inter-plate coefficient of variation is less than 10%.
2. Recovery rate: Three different concentration levels of human TARC;CCL17 were added to the selected healthy human serum, plasma and cell culture supernatant, and the recovery rate was calculated.| Sample type | Range | Average recovery | | Serum (n=8) | 84-101 | 96 | | Plasma(n=8) | 93-105 | 103 | | Cell culture supernatant (n=8) | | | Dilution ratio | Recovery rate (%) | Serum | Plasma | Cell culture supernatant | | 1：2 | Range (%) | 84-96 | 88-96 | 90-110 | | Average recovery rate (%) | 92 | 93 | 96 | | 1：4 | Range (%) | 89-103 | 87-108 | 105-115 | | Average recovery rate (%) | 94 | 98 | 108 |
| Sensitivity | 15.65pg/mL | | Theory | This kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Samples, standards, and HRP-labeled detection antibodies are sequentially added to microwells pre-coated with a capture antibody against human thymus activation-regulated chemokine (TARC; CCL17). After incubation and washing, the sample is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of human thymus activation-regulated chemokine (TARC; CCL17) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | | Source | Human | | Synonym | Human Thymus Activation Regulated Chemokine ELISA Kit,Human TARC ELISA Kit | | Detection Type | Double antibody sandwich method | | Composition | | Name | 96T Configuration | Remarks | | Pre-coated 96-well enzyme plate | 8 holes×12 strips | None | | Standard | 2 bottles | Dilution according to the instructions | Universal diluent | 2×20mL | None | Concentrated HRP detection antibody (100×) | 120uL | | 20×washing solution | 2×10mL | Dilution according to the instructions | 20×washing solution | 2×10mL | Dilution according to the instructions | Substrate (TMB) | 10mL | None | Stop solution | 6mL | None | Sealing film | 4 sheets | None | Instructions | 1 portion | none |
| | Background | Thymic activation-regulated chemokine (TARC; CCL17) is a powerful chemokine produced by the thymus and antigen-presenting cells such as dendritic cells, macrophages, and monocytes. CCL17 plays a complex role in cancer. However, in other cancers, such as melanoma, increased CCL17 is associated with improved outcomes. CCL17 was the first CC chemokine discovered to interact with T cells with high affinity. It has a strong chemoattractant effect on T helper cells and T regulatory cells because both express CCR4. | | General Notes | 1. Strictly adhere to the specified incubation time and temperature to ensure accurate results. All reagents must be at room temperature (20-25°C) before use. Refrigerate reagents immediately after use.
2. Improper plate washing may result in inaccurate results. Ensure that all liquid in the wells is aspirated thoroughly before adding substrate. Do not allow the wells to dry out during incubation.
3. Remove any residual liquid and fingerprints from the bottom of the plate, as this will affect the OD value.
4. The substrate developer solution should be colorless or very light in color. Do not use substrate solution that has turned blue.
5. Avoid cross-contamination of reagents and specimens to prevent erroneous results.
6. Avoid direct exposure to strong light during storage and incubation.
7. Do not expose any reagents to bleaching solvents or the strong fumes emitted by bleaching solvents. Any bleaching agent will destroy the biological activity of the reagents in the kit.
8. Do not use expired products, and do not mix components with different product numbers and batches.
9. Recombinant proteins from sources other than the kit may not be compatible with the antibodies in this kit and will not be recognized.
10. If there is a possibility of disease transmission, all samples should be managed properly and samples and testing devices should be handled according to prescribed procedures. | | Storage Temp. | If the unopened kit is stored at 4°C, the shelf life is 6 months. | | Test Range | 31.25-2000 pg/mL |
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AntBio is a biotechnology group company dedicated to serving life sciences, aiming to help scientists accelerate research and improve work efficiency. AntBio provides comprehensive and high-quality reagent tools for basic research, drug development, and diagnosis, including research grade antibodies, proteins, biochemical reagents, and assay kits. These research tools are widely used in different segments of life science research. The group company currently consists of three brands, Absin, Starter-Bio and UA-Bio.
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