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Description
Human NPHN 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: Urine 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
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: Urine 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: Gently wash adherent cells with pre-chilled PBS, then trypsinize and collect the cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash the collected cells three times with pre-chilled PBS and resuspend 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. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Collect the supernatant for analysis, or store at -20°C or -80°C, 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 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 utilizes a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP enzyme conjugate are sequentially added to microwells pre-coated with a nephrin (NPHN) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of nephrin (NPHN) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Nephrin ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Nephrotic hormone (NPHN) is a protein essential for the proper functioning of the kidney's filtration barrier, which is composed of slit endothelial cells, the glomerular basement membrane, and the epithelial capsule. It is a transmembrane protein and a structural component of the slit diaphragm. It is present in an intricate network at the apical end of the capsule cells, imparting a strong negative charge that repels proteins from passing through Bowman's space. Defects in its gene, NPHS1, are associated with Finnish congenital nephrotic syndrome, resulting in the leakage of large amounts of protein into the urine, a condition known as proteinuria. It is also essential for cardiovascular development. | |||||||||||||||||||||||||||||||||
| 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 | Urine, tissue homogenates, cell lysates, cell culture supernatants, and other biological fluids |
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4.3 ★★★★★
Based on 30 reviews
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Product Reviews
★★★★★ 5
Too cute!!! Comfortable too!
Size: 7, Color: Rosette, Size: 7, Color: Rosette
I was so worried about the platform on this shoes, but let me tell you, I love them, so comfortable and this pink color is too cute! Easy to find shirts to match! The price was well worth it!
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Reviewed in the United States on May 22, 2026
★★★★★ 5
Quality and balance
Color: Grey, Color: Grey
If you are reading this review and looking at this watch, you are most likely someone that appreciates mechanical movements and the rich history behind them and the brands that are known for making quality watches which have a beautiful balance between a timepiece and a piece of jewelry. For me, I have multiple sport watches with many complications and some that cost as much or even more than this Tissot Le Locle but anyone that wears a watch and appreciates the heritage of a mechanical movement must own this watch. The jewelry part of this watch is amazing, not loud, just a refined look making a statement that this is not something cheap. The picture does not do it justice. The dial has an amazing "personality" where under low lighting it looks silver and under normal lighting soft white making a beautiful contrast between the black hands and black roman numerals only to surprise you again when you change the viewing angle and the black numerals change to silver. The black hands will give you no trouble reading the time at a glance and 39mm will prove to be the perfect size for any wrist.
The movement is high quality as expected to be in a Tissot and will be working for many years to come so you can pass this watch down to another generation. As a matter of fact, last year my mother who is 78 found the Tissot watch she had in her youth that was in a box with old family watches and she had not worn for decades. She gave the crown a few spins and the watch came back to life keeping accurate time. This is what you can expect from a Tissot movement.
So if you are buying this for your self, you are in the right place, if you are buying it for someone else, make sure they can appreciate what this is otherwise you are better off spending your money elsewhere.
You should also consider getting quality quick release black and brown leather straps with a classic buckle to make this watch suitable for any attire.
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Reviewed in the United States on March 21, 2026
★★★★★ 5
An Exciting and Beautiful Watch
Color: Grey
Update 2-11-18 More time has passed and my satisfaction with the watch continues to grow; it is gratifying to me to simply look at the watch on my arm, I am so pleased with its appearance and operation that it is a joy to wear and to watch its competent functioning. It is keeping excellent time and shows a loss of only a few seconds each day; it is truly a fine watch.
Update 2-3-18 I have researched the watch and discovered some interesting questions about locating the serial number. In checking the literature sent with the watch I noticed the small tag attached to the watch identified the serial number as a series of nine letters which are repeated on the case of the watch. I also looked up the barcode and the first three numbers indicated Switzerland to be the country of origin and accurately identified this watch specifically. I"m satisfied this is an authentic watch from Tissot.
I know that beauty is in the eye of the beholder but to me this is an elegant and beautiful watch and the nicest I have ever owned; it was delivered this afternoon and impressed me greatly right out of the box. I have reset the time and date and it seems to be operating perfectly. I had felt some concern for its authenticity but after seeing the watch and its packaging my concerns have all disappeared; to me it looks and feels like the excellent quality I would expect from Tissot..
Sadly the Certificate of Authenticity had been removed by the seller and on contacting them I was told they reserved the right to do that if they wanted to. I see in at least one other case shown under comments the seller left the document for the buyer. The watch itself strongly indicates the authenticity I had hoped for from the Tissot brand so I'm not all that troubled by the seller's decision.
The user manual is tiny and limited in information; a better copy is available online as a PDF file; I've downloaded the file and suggest you might want to consider that option as well.
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Reviewed in the United States on February 3, 2018
★★★★★ 4
Jewel case not included!
Color: Grey
I ordered this item and received it without the case shown in the pictures. Contacted Amazon and asked for a replacement - and the customer service over the phone was very understanding about the missing case.
The replacement also showed up without the jewel case.
I love this watch, but the buying experience wasn't great. Also, on certain occasions it has lost time after being worn for several days and then not being worn for one day. Kinda disappointing. Still, gorgeous to look at, feels great on my wrist.
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Reviewed in the United States on July 27, 2022
★★★★★ 5
Good
Color: Grey
Good
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Reviewed in the United States on May 12, 2026