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Description
Human sCD40L ELISA KitProduct Specification Usage Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High precision pipette and gun tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37 constant temperature box 4. Distilled water or deionized water Sample processing and requirements: Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4 overnight, then centrifuge at 1000g for 20
Product Specification
| Usage |
Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision pipette and gun tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37℃ constant temperature box 4. Distilled water or deionized water Sample processing and requirements: Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge the specimen at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C, but avoid repeated freezing and thawing. Tissue homogenization: Rinse the tissue with pre-chilled PBS (0.01M, pH 7.4) to remove residual blood (lysed red blood cells in the homogenate will 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., 1 g of tissue sample to 9 mL 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 tissue cells, the homogenate can be sonicated or repeatedly frozen and thawed. Finally, centrifuge the homogenate at 5000 × g for 5-10 minutes, and the supernatant can be assayed. Cell culture supernatant: Centrifuge at 1000×g for 20 minutes, take the supernatant for detection, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. Other biological fluids: Centrifuge at 1000xg for 20 minutes, remove the supernatant, and 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 it stand for 15 minutes to completely dissolve, then gently mix (concentration is 10 ng/mL). Then dilute to the following concentrations: 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, 0.15625 ng/mL, and 0 ng/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each. Pipette 500uL of the 10ng/mL standard working solution into the first EP tube and mix thoroughly to make a 5ng/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 biotinylated detection antibody working solution: Centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute 15 minutes before use. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration with universal diluent (e.g., 10uL concentrate + 990uL universal diluent). Prepare and use immediately. 4. Prepare the enzyme conjugate working solution: 15 minutes before use, centrifuge the 100× concentrated enzyme conjugate at 1000×g for 1 minute. Dilute the 100× concentrated HRP enzyme conjugate to a 1× working concentration with universal diluent (e.g., 10 μL of concentrate + 990 μL of universal diluent). Prepare immediately. 5. Prepare the 1× wash solution: Dispense 10 mL of 20× wash solution into 190 mL of distilled water (concentrated wash solution removed from the refrigerator may crystallize; this is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing). 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 addition: Add 100 μL of sample or standard of varying concentrations to the corresponding wells. Add 100 μL of universal diluent to the blank wells. Cover with a film and incubate at 37°C for 60 minutes. (Recommendation: Dilute the sample to be tested at least 1-fold with universal diluent before adding it to the ELISA plate. This will reduce the impact of matrix effects on the test results. The sample concentration should be multiplied by the corresponding dilution factor when calculating the final sample concentration. It is recommended to run replicates for all test samples and standards.) 3. Add Biotinylated Antibody: Remove the ELISA plate and discard the liquid without washing. Add 100 μL of Biotinylated Antibody Working Solution directly to each well. Cover with a film and incubate at 37°C for 60 minutes. 4. Wash: Discard the liquid and add 300 μL of 1x Wash Solution to each well. Let stand for 1 minute, shake off the wash solution, and pat dry on absorbent paper. Repeat this process three times (a plate washer can also be used). 5. Add Enzyme Conjugate Working Solution: Add 100 μL of Enzyme Conjugate Working Solution to each well. Cover with a film and incubate at 37°C for 30 minutes. 6. Washing: Discard the liquid and wash the plate five times as in step 4. 7. Adding substrate: Add 90 μL of substrate (TMB) to each well, cover with a sealing film, and incubate at 37°C in the dark for 15 minutes. 8. Adding 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 a wavelength of 450 nm. Calculating experimental results: 1. Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as a correction factor. 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. 2. If the sample OD value is higher than the upper limit of the standard curve, dilute the sample appropriately and retest. Multiply the sample concentration by the corresponding dilution factor. |
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| Theory | This kit utilizes a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with a capture antibody against soluble CD40 ligand (sCD40L). After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by peroxidase (HRP) catalysis and to yellow by acid. The intensity of the color is positively correlated with the amount of soluble CD40 ligand (sCD40L) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Soluble CD40 ligandELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Soluble CD40 ligand (sCD40L) is contained within platelet granules, and its presence in the blood is therefore a marker of platelet activation. By interacting with CD40 present on endothelial and smooth muscle cells, sCD40L may trigger the release of inflammatory mediators, leading to increased activity of matrix metalloproteinases and activation of the coagulation cascade. | |||||||||||||||||||||||||||||||||
| 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. |
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| Storage Temp. | If the unopened kit is stored at 4°C, the shelf life is 6 months. | |||||||||||||||||||||||||||||||||
| Test Range | 0.156-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenates, cell culture supernatants, and other biological fluids |
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4.7 ★★★★★
Based on 20 reviews
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Product Reviews
★★★★★ 1
Not for tough chewers at all!!!
Size: 10.5 inch Rubber Flex Tread, Size: 10.5 inch Rubber Flex Tread
5 minutes in and it's destroyed. Very disappointing.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 21, 2026
★★★★★ 5
Great chew toy
Size: 10.5 inch Rubber Flex Tread
This is my second time buying the Tonka rings. My dog loves this chew toy. It does take him a bit to destroy them. I give this toy to him as a reward otherwise it be destroyed in 3 days. I have a pitty. So other dogs it should last longer. They are shin whackers and hurt when they connect. So I make sure to watch the dog and toy around small kids and elderly to be safe. They are heavy and made well.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on October 29, 2025
★★★★★ 5
Finally a toy we can leave out!
Size: 10.5 inch Rubber Flex Tread, Size: 10.5 inch Rubber Flex Tread
There are very few toys that we can leave out for our 11 month old golden. Which is a shame because as a small puppy, we taught him to bring us a toy to initiate play instead of nipping. Ropes or stuffies never lasted more than a few minutes so they always had to be taken away. One time he brought us a benenbone to play tug. So I tried this as a last resort. Finally something we can keep out for him! It’s not completely indestructible. He has figured out how to pull bits of tread off. But he chewed hard on it for a solid 20 minutes and it’s still intact with the exception of a few small tread pieces. Can’t say that for 99% of the other dog toys on the market!
UPDATE: OK, so it's definitely not indestructible. After a few days of dedicated gnawing, he has almost chewed through the yellow ring so we can't leave him too unsupervised with it. I'm still going to leave it as 5 stars since it's the best tug toy that I have found so far and we have tried a lot! I may buy another one and just make sure I don't let him freely chew on it for any length of time.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on April 1, 2023
★★★★★ 3
Really wanted this toy to be better...
Size: 10.5 inch Rubber Flex Tread
I got this toy because I have 3 large dogs that all like to pull things. I can't always play with them even though they follow me everywhere wanting to play, so I saw this toy and thought it would be good because it would give them something they can tug away at with each other. I saw the thick rubber and thought it would last awhile, but I was wrong. My German Shepherd and Pyrenees mix chewed though the first tire rather quick, and before I realized, I was finding chunks of tire around the house and yard. Thankfully, none of them tried to eat it. It seems like it could be a good toy for an aggressive chewer IF they are smaller and not as strong as these large dogs are, but it did not hold up long to mine.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on November 23, 2024
★★★★★ 5
Great and entertaining dog toy
Color: 4 Color For XS/S 2.2", Size: Small
These are probably my dog’s favorite toys currently. He loves the squeaking and they function well as a ball for fetching as well.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 28, 2026
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