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Description
Human GPC3 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: 1. 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: 1. 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. 2. 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. 3. 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. 4. 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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| Sensitivity | 0.06 ng/mL | |||||||||||||||||||||||||||||||||
| 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 glypican 3 (GPC3) 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 glypican 3 (GPC3) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Glypican 3 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Glypican-3 is a protein encoded by the GPC3 gene. The GPC3 gene is located on chromosome X (Xq26). The most common gene (isoform 2) encodes a 70 kDa core protein of 580 amino acids. Three variants have been detected, encoding alternatively spliced forms designated as isoforms 1, 3, and 4. The GPC3 protein core consists of two subunits: the N-terminal subunit is approximately 40 kDa and the C-terminal subunit is approximately 30 kDa. Six glypicans (GPC1-6) have been identified in mammals. Cell surface heparan sulfate proteoglycans consist of a membrane-associated protein core punctated by a variable number of heparan sulfate chains. Members of the glycoprotein-associated integral membrane proteoglycan family (GRIPS) consist of a core protein anchored to the plasma membrane by glycosylphosphatidylinositol linkages. These proteins may play a role in controlling cell division and growth regulation. | |||||||||||||||||||||||||||||||||
| 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, tissue homogenates and other biological fluids |
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4.0 ★★★★★
Based on 14 reviews
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Product Reviews
★★★★★ 5
Second one, first one lasted a good while
We leave it outside in the weather with solid results for our backyard dog, and the dog loves this thing. It's our go to toy for her, ... perfect for kick to fetch, tug of war, hide in the shirt, bump the human leg when it is wet, etc. ;) The first one we used and used and finally it started to show too much weathering, mildew, and finally breaking after like 1.5 years... we have extremely hot and wet weather here. When new though, these balls take a pounding. Our 50 pound dog can play tug of war to the extent we can pick her up while she holds onto the ball with her teeth. The ball doesn't flinch and the dog loves it. Kicking it is fun because you can give it a wallop and it won't go over the fence since the design absorbs a fair bit of the kick energy, but it is still enough for an adult or teen to send it about 1/4 an acre away. What else... oh the dog has a funny habit of finding the one spot that isn't an empty pentagon... as in the plastic is filled in there... and she likes to bit on that part especially during tug of war. I should also mention our dog literally breaks apart and sometimes eats sticks and twigs in the yard, can't be healthy, so a nice diversion is this ball that she will chew on once in a while without destroying it.
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Reviewed in the United States on July 10, 2022
★★★★★ 5
Absolutely love this brand of ball for my dog
I end up replacing these every couple years, yep that's right, years. My border collie will grab this ball in his mouth and then kick another ball around. Don't know why he has to have something in his mouth to play soccer, but he is unique. He managed to tear one of these up when he was in his teething stage, but it took him months, and I took it away as soon as he managed to remove a piece of it. Otherwise, we usually lose these much more often than them getting destroyed through constant chewing, and I suspect heavy weathering outside for an extended period of time. Nowadays he doesn't chew on rubber nearly that often, he has developed his own picky preferences. This ball is his must have for playing around. He loves to drop it down the stairs and play fetch with himself. It's easy for him to grab, it isn't heavy so we don't worry about furnishing, walls, or family members when he is tossing it around. It is much more durable than most, with the exception of really heavy chewers/teething, even then it held up for a long time. As long as you are careful to remove it if they manage to chew through I would even try it out with teething puppies. The lawnmower is also this balls nemesis, it loses every time. If only I could convince the dog that he needs to stop sacrificing toys to the lawnmower :-)
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Reviewed in the United States on January 1, 2025
★★★★★ 5
Will Last!!!
My Saban is a black lab/pittie mix and is a very aggressive chewer with the bulldog jaws. I had bought the large ball for him and to be honest I didn't think it would be as big as it is or as heavy 😂😂😂!!! It's made of a very durable plastic and is holding up extremely well to his chewing on it!!! I'll be honest and say that unless you're using it outside and have some really good muscles in your arm that I would recommend buying this in the smaller size!!!! I normally play with him inside with his toys when the weather isn't good for going outside, but I don't throw this one inside because #1 it's so heavy and big that I can't throw it very good and # if I was to play with it inside I'm afraid the weight of it would break something!!! I would say that this is a great quality and very durable, but I wouldn't buy the large one again!!! I would definitely recommend this brand in a smaller size ball 👌 😊😊
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Reviewed in the United States on October 11, 2023
★★★★★ 4
Great product...wish you had color options
My dogs love these balls, but I wish you could choose the color. This is my dog's favorite toy. He can play tug of war with his siblings, and the ball usually lasts for quite awhile. Considering they are usually pretty destructive puppers, this is a minor miracle.
But, I can't just pick his favorite color and get it delivered. I have to play roulette, and pray that his color is the one that I get when I open the box. If they can allow us to choose the color(s) that we want, then this review will be upgraded to 5 stars.
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Reviewed in the United States on December 17, 2023
★★★★★ 5
A silent toy that still holds a dog’s interest
We bought this large size for our Rottweiler- but it was our 45# dog who fell in love with it. Cooper is 16, has kidney disease, dementia and loss of muscle coordination; so, it was an absolute joy to watch him engage with this toy. His foot went through the ball once, but he figured it out. He pushes it with his nose, and tries to squeak it. He “talks” to it. We haven’t heard that in a long time. It’s light enough that he can lift it - and it rolls and bounces nicely. For him it’s perfect. Our Rottie seems to understand that it’s his toy. Now, that’s love and understanding.
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Reviewed in the United States on May 5, 2026
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