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Description
Human™ ELISA KitProduct Specification 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. 37C incubator 4. Distilled or deionized water Sample handling and requirements: Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge at 1000g for 15 minutes at 2 8C within 30 minutes of collection. Remove the supernatant for testing or store at
Product Specification
| 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: Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge at 1000×g for 15 minutes at 2-8°C within 30 minutes of collection. Remove the supernatant for testing or store at -20°C or -80°C, but avoid repeated freezing and thawing. Pre-test preparation: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare a gradient standard working solution: Add 1 mL of universal diluent to the lyophilized standard. Let stand for 15 minutes to completely dissolve, then gently mix (concentration 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 500 μL of universal diluent to each tube. Pipette 500 μL of the 10 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 5 ng/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 uses 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 thrombomodulin (TM) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase and then to yellow by acid. The intensity of the color is positively correlated with the amount of thrombomodulin (TM) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Thrombomodulin ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Thrombomodulin (TM) is an integral membrane protein expressed on the surface of endothelial cells that acts as a cofactor for thrombin. It reduces blood coagulation by converting thrombin from a prothrombin to an antithrombin. TM is also expressed in human mesothelial cells, monocytes, and subsets of dendritic cells. In humans, thrombomodulin is encoded by the THBD gene. The protein has a molecular mass of 74 kDa and consists of a single chain with six tandem repeats of an EGF-like structure, a serine/threonine-rich spacer, and a transmembrane domain. Thrombomodulin is also a glycoprotein on the surface of endothelial cells that, in addition to binding thrombin, regulates the inactivation of C3b by factor I. | |||||||||||||||||||||||||||||||||
| 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 | 0.15-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | plasma |
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4.6 ★★★★★
Based on 11 reviews
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Product Reviews
★★★★★ 5
Best long-lasting dog chew
If you want something to occupy your dog that's harmless, this is the one. He doesn't have to chase it, so it doesn't get lost under the cabinets, and he's been chewing it for hours and barely made a mark on it. When he's chewing on this, he's not whining or asking to sit on my lap, and he can stay nearby as he enjoys this toy.
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Reviewed in the United States on June 12, 2026
★★★★★ 4
Not perfect for every dog, but still worth a shot
I bought the bacon-flavored Benebone with high hopes after reading the reviews. Our 45lb mutt loves to chew on bones (and thankfully that's ALL she loves to chew on!) but we've had trouble finding anything that she can't destroy in one sitting. We've heard that antlers are the longest lasting but can be damaging to the dog's teeth, so we wanted to try something with a little less potential for disaster.
The Benebone wishbone fared okay. I do have to say that she loved the bone, as did our 14lb schnoodle. The first day she had it, they took turns gnawing on it for hours. Unfortunately, the large one's jaws are quite strong, and so by the end of day 1 there were some pretty significant teething marks on each of the ends, and it was starting to look pretty gnarly. I put it up for a little bit, but they've had other nylon bones that looked worse so I gave it back to them the next day.
On day 2, the little one was chewing on it so vigorously (and I think the bone was gnarly enough) that her gums started bleeding. I noticed the blood on the bone so I took it away again.
On day 3, I gave the bone back to the large one to chew on. It wasn't too long, though, until I was hearing some pretty loud snaps and I found that the large pup was actually starting to get larger pieces off of the ends of the wishbone. This had me worried, so I threw the wishbone away.
Relative to other chew toys that the large one likes (she'll only chew on bones and edibles, for some reason - nothing with any sort of rubbery material), this did actually have a decent life span, but we're still looking for something better.
I give the product 3 stars because the dogs clearly loved the bone, and I'm sure that it's perfect for some dogs. For example, if it was just our schnoodle chewing on it, it would have lasted much longer and I would probably be posting a 5-star review here. In the end, though, we did buy the wishbone for the larger dog, and it failed to hold up for her - for the price, I would have wanted something that lasts longer - hence the two star deduction.
UPDATE 8/10/2014:
Not long after I posted this review, Benebone contacted me in the comments to offer a refund and hinted at the potential for a larger bone sometime in the future. I sent in our order number and they processed the refund quickly. I've added back an extra star, because Benebone's stellar customer service has made the product worth the try. I would definitely do business with Benebone again, and I'm looking forward to trying (or rather, having my pup try) a larger version of the bone if/when it surfaces!
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Reviewed in the United States on August 7, 2014
★★★★★ 5
I Import These to Mexico That's How Good They Are
I have purchased this bone at least 10 times and will keep buying it indefinitely. I have 8 rescue dogs, all extreme chewers , we're talking dogs that destroy absolutely everything. This is the only bone I've found that holds their interest and keeps them coming back for more. I've tried every type of bone on the market. This one wins!
My dogs range from 5 lbs to 100 lbs and every single one of them is equally obsessed. I now order 5 at a time because I know they'll all be used. The price point is excellent for what you get, and after constant use across 8 dogs, I haven't noticed any negative effects on their health or teeth.
I love these so much that I actually import them to Mexico, that should tell you everything. Every time someone visits and sees how occupied and happy my dogs are, they immediately ask where they can get them too.
Benebone, you have a customer for life!!!!!!
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Reviewed in the United States on April 28, 2026
★★★★★ 5
Aggressive Chewers' Delight!
Super strong and apparently flavorful, my aggressive chewing puppy loves this! Goes at it for ten minutes at a time, several times per day - woohoo!
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Reviewed in the United States on June 3, 2026
★★★★★ 5
Dogs love them and they are very safe for them to chew!
These bones are indispensable if you have a chewing and destructive puppy! (Or any dog, really) ours love them and never get tired of chewing - so far we have had them for a month and no one is tired
of them or more importantly has managed to eat any of them. They are a great buy and keep dogs really occupied when they may get bored and feel like causing trouble or chewing something!
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Reviewed in the United States on May 1, 2026
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