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Description
Human COL2a1 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 specimens using EDTA or heparin as anticoagulants and centrifuge them at 1000×g for 15 minutes at 2-8℃ within 30 minutes of collection. The supernatant can be tested or stored at -20℃ or -80℃, but repeated freezing and thawing should be avoided. 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 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 capture antibody against Collagen Type II Alpha 1 (COL2a1). 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 Collagen Type II Alpha 1 (COL2a1) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Collagen Type II Alpha 1 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Type II collagen alpha 1 (COL2a1) is the alpha-1 chain of type II collagen, a fibrillar collagen found in cartilage and the vitreous humor of the eye. Mutations in this gene are associated with achondroplasia, early-onset familial osteoarthritis, SED congenital, Langer-Saldino achondroplasia, Kniest dysplasia, Stickler syndrome type I, and Strudwick spondyloepiphyseal dysplasia. Furthermore, defects in processing chondroitin, a calcium-binding protein and the C-peptide of this collagen molecule, are also associated with achondroplasia. Type II collagen provides structure and strength to connective tissue and is primarily found in cartilage, the jelly-like material that fills the eyeball (vitreous humor), the inner ear, and the central portion of the intervertebral disc (nucleus pulposus) between vertebrae in the spine. Three pro-alpha1(II) chains are intertwined to form a three-stranded, rope-like procollagen molecule. These procollagen molecules must be processed by enzymes within the cell. | |||||||||||||||||||||||||||||||||
| 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 | Serum, plasma, and other biological fluids |
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4.5 ★★★★★
Based on 775 reviews
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Product Reviews
★★★★★ 5
Great to stop Doggie boredom/barking
Size: Extra Small, Style: Bristle Bone
For tiny dogs, these are great ways to entertain the pups and get them to clean their own teeth; I have had one for 5 years and the dogs never were able to harm the white parts. I gave it to a pup that was homeless and I didn't have the heart to take it back later - It is quiet as a mouse and fits perfectly in tiny dog mouths. You do have to have their raw hide inserts but they're easy to find, not expensive, and again is great for boredom time for the dogs.
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Reviewed in the United States on June 9, 2026
★★★★★ 4
Favorite chew toy
Size: Medium, Style: Bristle Bone
Pros: this is our dog’s favorite toy. She loves to eat but isn’t much for chewing. At 2 years old, her teeth were already looking pretty bad because she swallows everything whole. That makes most chew toys dangerous. This is the first toy that she chews aggressively. At first we had to give it to her in short spurts because her gums bled. That’s stopped, her teeth and gums look healthier, and she carries it around with or without the inserts. She loves it.
The con: the bristles come apart after aggressive chewing. She swallowed some and I caught the other side coming off.
So whenever I see this on sale, I buy several new ones. I don’t try to make it last and I check it often for damage.
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Reviewed in the United States on May 17, 2026
★★★★★ 5
Great Product
Size: Medium, Style: Jack
Great product. Kept our chewer busy for a long time
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Reviewed in the United States on June 3, 2026
★★★★★ 3
Dog treat and chew bine
Size: Medium, Style: Bouncy Bone
This was an ok purchase for my little dog. After eating the treats. She definitely liked this treat. She is 2 years old and loves her treats. Disappointed, in that it does not last as long as they said it would.
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Reviewed in the United States on May 10, 2026
★★★★★ 5
Boxer Approved!
Size: Small, Style: Bristle Bone
One of my boxer girls (the fawn one in the video, 6 yrs old) hardly ever chews on things, my other boxer girl (the sealed brindle, 11 mos old) chews constantly and we're buying new nylabone chew toys 1-2 times a month for her. I wanted something to encourage my older girl to chew more for dental health and serotonin secretion, I know the benefits of chewing far outweigh the risks for dogs so I've been trying all kinds of things but hooves, ears and real bones get stinky and the girls stop caring for them after a few chew sessions, and my fawn baby only sometimes has interest in chew toys of any kind. When I found this with the different textures, materials (hard plastic, nylon bristles, rubber type discs, & jerky treat) I jumped for one, and it's a HUGE HIT!
She loves it, my 11 mo old loves it, and I definitely need backups because they're going to love this one to death in a couple weeks- I'm so happy about that!!
Between this and the new snuffle mat we bought at the same time, which both my girls go crazy for, I've finally found enrichment toys that boost my girls health both mentally and physically and they can BOTH get so much joy from them!!! Big wins for this dog momma, I'm ecstatic and grateful we've hit the spot for our babies!!
Would recommend and will buy again 10/10, seems durable after at least 5 chew sessions giving the girls turns of 10-15 min each, the video shows the wear from those sessions and it's not too rough yet. Their breath smells better after each session, and my older girl who hardly ever chews sometimes gets bloody gums when she does chew, that's gone away now that she's chewing this toy regularly for about 2 weeks, twice a week (working our way up slowly to 3-4 times weekly).
As you can see in the video, they don't even need the jerky treat anymore, they've fallen in love with the toy so much! But it helps encourage them to get their mouth working in different ways around the whole bone and not just one part, so I'll keep getting the treats for better overall coverage of the whole mouth getting cleaned. The size is a little small for my big boxer girls but not too small, they won't swallow any parts but they are mindful chewers, not too heavy-more like medium chewers, and they actually always choose the smaller toys over the big ones so it's perfect to pique their interest! Easy to take apart and clean, put in the treats and hand over to the dogs, they don't unscrew the end of the bone even if it gets a little loose during a chew sesh. We wash it with water after each use and set it out to dry, never gets stinky so far. Good value and great use for the money!
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Reviewed in the United States on June 20, 2024