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Description
Human MDA5 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 preparation and requirements: 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 and
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 preparation and requirements: 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 and mince the tissue. 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 tissue cells, the homogenate can be sonicated or repeatedly frozen and thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes, and collect the supernatant for analysis. Cell Lysis Buffer: Adherent cells should be gently washed with pre-chilled PBS, then trypsinized and harvested by centrifugation at 1000×g for 5 minutes. Suspension cells can be harvested directly by centrifugation. Collected cells should be washed three times with pre-chilled PBS and resuspended in 150-200 μL of PBS per 1×10^6 cells (it is recommended to add protease inhibitors to the PBS; if the cell count is very low, reduce the PBS volume appropriately). Disrupt the cells by repeated freezing and thawing or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and collect the supernatant for analysis. 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 20 ng/mL). Then dilute to the following concentrations: 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, and 0 ng/mL. Serial dilution method: Take 7 EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 20 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 10 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves directly as a blank well; there is no need to aspirate the liquid from the penultimate tube. See the figure below for details. 3. Preparation of Biotinylated Antibody Working Solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration using universal diluent (e.g., 10µL concentrate + 990µL universal diluent). Prepare immediately before use. 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 melanoma differentiation-associated protein 5 (MDA5) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase and to yellow by acid. The intensity of the color is positively correlated with the amount of melanoma differentiation-associated protein 5 (MDA5) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Melanoma differentiation-associated protein 5 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | MDA5 (melanoma differentiation-associated protein 5) is a RIG-I receptor dsRNA helicase encoded by the IFIH1 gene. MDA5 is part of the RIG-I receptor (RLR) family, which also includes RIG-I and LGP2, and functions as a pattern recognition receptor capable of detecting viruses. MDA5 is generally believed to recognize double-stranded RNA (dsRNA) longer than 2000 nt. However, it has been demonstrated that while MDA5 can detect and bind to cytoplasmic dsRNA, it can also be activated by high-molecular-weight RNA complexes composed of both ssRNA and dsRNA. | |||||||||||||||||||||||||||||||||
| 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 | 0.31-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, and other biological fluids |
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4.9 ★★★★★
Based on 8 reviews
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Product Reviews
★★★★★ 1
Paid for new, received used.
Size: 9.5 Wide, Color: Cognac, Size: 9.5 Wide, Color: Cognac
Arrived dirty as if someone whore it for more than a few days. Maybe I was mistaken, but I saw nothing about “previously used” on the Amazon page. Returned it.
I would suggest that the company use quality control when it comes to shipping. The shoes seem find otherwise. I ordered 9.5 and it was just a bit tight. If I reorder I will go for size 10.
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Reviewed in the United States on June 11, 2026
★★★★★ 5
Overall Great.
Size: 10.5 Wide, Color: Black
Great product. Great value. Great style. Overall great.
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Reviewed in the United States on March 3, 2026
★★★★★ 3
Decent Shoes with Misleading Description (Not Leather)
Size: 12 Wide, Color: Black, Size: 12 Wide, Color: Black
I only look for leather when I’m purchasing dress shoes. Under the description “details” on this item it lists the shoe as leather upper and inner lining. A photo shows “genuine Napa leather lining”. If you keep reading (which I have now that I’ve received the shoes) it shows “faux leather” in the small print and that is not “leather”. I run a small leather business and this irks me because it’s clearly false advertising. The details also show the product is made in Brazil but when they arrived today, the shoe tag shows “Made in China” and “Man Made upper and lower”. I don’t hate the shoes. They look nice. What I know is they won’t hold up over time like leather, which is why the description should be accurate and not misleading.
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Reviewed in the United States on March 28, 2026
★★★★★ 5
Great Wallet
I'm pretty particular when it comes to things that I need to carry at all times when I'm out. When looking for a new wallet back in 2014, I initially went with the oft recommended Saddleback Leather Medium Bifold. Initially, that wallet seemed to be both durable and well made. Unfortunately, after several months in the back pocket, two things became clear: first, the leather was so taught that it was breaking my credit cards in half at the pinch point of the credit card slot. Second, what I took for durability in the thickness of the leather quickly turned into nuisance as I ended up with an inch thick brick that ruined my seating comfort. I called Saddleback regarding the first point and they told me that it was a known issue and if I didn't like it I could send the wallet back, which I did. That left me at a crossroads with a new requirement: I wanted a leather wallet that was thin enough to not be uncomfortable, held 6 credit/id cards and a handful of bills. I searched around until I came across Bellroy. The travel wallet seemed to check all the boxes and added some features I never knew I needed. After 6 years of use, I finally decided that the original travel wallet I bought in 2014 had run its useful life, down to holes in the leather and all. I liked it so much, I bought another one. There are some subtle differences which I will highlight below.
1. Fits in Your Pocket
This is ostensibly the best feature of the wallet. It is large in the two dimensions you shouldn't care about and thin where it counts. I have not found a pair of pants that I would wear that it doesn't fit in. With my passport, 6 credit/id cards, and a dozen bills it is roughly 3/4 inches thick. Fabulous.
2. The Right Pen
I had no idea how useful the pen would be. I find occasion to pull it out at least once a week. I used to carry the wonderful Fisher Space Pen, but no longer do as this fills the role in a much for optimized way. Bellroy is kind enough to include an ink refill with every wallet and I still have the refill from 2014. That's not to delegitimize the previous point; the kind of things I write are forms and other short memos. I wouldn't use the pen to write a novel. The new wallet looks to have a pen that Bellroy have either white labelled or made themselves. I haven't yet written with it, but if it is at least as good as the Walkie it replaced, things should be fine.
3. Added Security
The new wallet takes the split billfold and adds a flap that conveniently hides away the back section from prying eyes. In theory this seems like a useful feature. In practice, the flap is not quite long enough to allow for more than one or two bills without the cavity being visible when the wallet is opened. The new wallet also has RFID shielding which I don't recall being a feature of the old wallet. It sounds like a useful feature now that at least one of my credit cards has a contactless payment feature.
4. Leather
It's a thin wallet so you should expect the leather to be thin as well. I got 6 years out of the first wallet and I'm not a guy who is easy on wallets. It has been in my pocket on rooftops doing solar installations, in machine shops, and on every kind of seat imaginable. My old wallet has been fully submerged in water more than once. With all that punishment, it outlasted the warranty by two times. None of the stitching has failed and the only real damage is two small holes in the outer leatherwork. If you are a business traveler, this should go out of style before it is rendered unusable from wear.
In short, this is a great wallet for everyday use. I approve to the extent that I didn't look for a new choice when my old one wore out.
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Reviewed in the United States on March 15, 2020
★★★★★ 5
Very handy
Color: Sienna
I purchased it because I still use paper boarding passes. With luggage claim paper, your passport and ID card, things could get quite messy if you try to put everything in your bag or backpack’s compartment.
This one organizes all those nicely. Because of its size it accommodates multiple boarding passes and luggage claim papers easily. (They used to get crumpled in international flights, and It annoyed me.). Yet it is compact enough to carry in your pocket.
One negative is its price. It is expensive. But it is a quality product. Nice leather and well made.
If you are willing to spend some $, this will spare you from searching your boarding pass and passport frantically at boarding. Highly recommend.
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Reviewed in the United States on July 4, 2025