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Description
Human CD40L 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 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. 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. Cell Lysis Buffer: Gently wash adherent cells with ice-cold PBS, then trypsinize and collect cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash collected cells three times with ice-cold 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 freeze-thaw cycles or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and remove the supernatant for analysis. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Remove the supernatant for analysis or store at -20°C or -80°C, avoiding repeated freeze-thaw cycles. 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 Cluster of Differentiation 40 Ligand (CD40L) 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 Cluster of Differentiation 40 Ligand (CD40L) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Cluster Of Differentiation 40 Ligand ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | CD40L is a protein primarily expressed on activated T cells and a member of the TNF superfamily. It binds to CD40 (protein) on antigen-presenting cells (APCs), leading to a variety of effects depending on the target cell type. CD40L has three binding partners: CD40, α5β1 integrin, and αIIbβ3. On TFH cells, it promotes B cell maturation and function by binding to CD40 on the surface of B cells, thereby facilitating cell-to-cell communication. Defects in this gene result in an inability to undergo immunoglobulin class switching and are associated with hyper-IgM syndrome. | |||||||||||||||||||||||||||||||||
| 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 homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.5 ★★★★★
Based on 7 reviews
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Product Reviews
★★★★★ 4
High on Tropes and Satisfaction
Format: Kindle
This is a great Romantasy book full of action, adventure, and everything you look for in this genre.
I won’t lie: it does kinda feel like the author found every common trope from every successful book of this kind and threw them all into this novel. But if it ain’t broke, don’t fix it. Especially in romance, there’s a large audience who has specific expectations, and they want them every time. Nothing wrong with that and many times I’m one of them.
I have no idea what defines a spoiler honestly, so
spoiler alert!!!!!!!
Tropes include:
Only one bed at the inn/bar
Dissatisfaction with life before hunk appears
Lost royalty
The chosen one
Montage of dress up time followed by shocked hunk
Forbidden romance between two from rival peoples
Power that cannot be controlled, simply guided/asked
Gathering intel at the inn/bar
FMC who knows how to fight/use weapons well
There’s probably more but no need to list them all.
Good story and I would recommend!
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Reviewed in the United States on June 14, 2024
★★★★★ 5
LOVED THIS STORY!
Format: Kindle
I never wite reviews, but this book was too good for me to NOT give props and let others readers know it's such a good read. They need to make movies of this one and I can't wait until the next book comes out. The story building was so good and I loved that the main character wasn't a silly useless princess or something. Shes intelligent, strong, and loves her friends. The description of the characters is really good also and its such a good plot. Not your average paranormal romance novel of some princess or the girl working at a diner. LOL! I'm totally team Rykan for her! Fingers crossed for the next book and October 31st can't come soon enough.
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Reviewed in the United States on September 15, 2023
★★★★★ 4
Imperfect characters
Format: Kindle
4.5 stars ⭐
I don't really understand the harsh criticism in some of these reviews (Goodreads). I rarely write reviews myself, but I think this book deserves it.
Look, it absolutely has its issues. I wasn't feeling it in the beginning -- the first few chapters were all tell and no show, literally just telling us all this background in a few paragraphs rather than feeding us bits and pieces at a time. The writing significantly improves after a few chapters, though. And I didn't catch any spelling or grammatical typos often found in new, self published books.
After the rough start, I absolutely flew through the rest of the book and couldn't put it down. Seriously stayed up until 2am with it. So I also don't agree with comments calling it boring. Definitely did not bore me. I thought it ticked all the right boxes: romance, fantasy, a magic system, tragedy, fate, character growth.
A lot of the poor reviews I'm seeing are in regards to the FMC's character. Personally, I like flawed characters and think it's more realistic than reading the POV of someone who always makes the right decisions, is always honest, is always selfless, etc. Humans aren't like that. Readers love when male leads are morally grey, but God forbid a woman isn't perfect.
So I like flawed characters and I like a little bit of tragedy, which the author provides. Ends on a cliffhanger, and I can't wait to read the sequel next month. I really want to learn more about Malachi.
All in all, not a bad start from this author. Give it a chance.
SOMEWHAT SPOILERY:
I LOVE the concept of fated mates that kind of hate each other. Most books with the fated mates trope allow the characters to immediately fall for each other and get together relatively easily. But this is way more interesting ;)
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Reviewed in the United States on October 1, 2023
★★★★★ 5
what just happened
Format: Kindle
I’m not gonna lie. This story had me from the beginning. It’s something about a girl running from an obsessive stalker that happens to be set in a fantasy realm. This story had everything you could want in a great plot. I mean intrigue, politics, betrayal, murder, witches, shades, fairies, shifter, etc. My heart grieved for Dahlia and everything she went through and honestly i don’t know who to root for in this story as all the males are morally gray which I love. All have a hint of evil. Book up this book and you won’t be disappointed. Soo ready for the next book.
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Reviewed in the United States on September 18, 2023
★★★★★ 3
Beta readers, please
Format: Kindle
I haven’t written a review in a while but this one motivated me to because it had potential and with some honest beta readers correcting its nonsense, it could have been pretty good. The writing is awkward at times and the world building was slow and one dimensional until around half way through and then it got much better. The main issues are with the characters, dialogue and plot. The FMC swings from stupidly trusting her “ friends” even when she is repeatedly betrayed to hating those who betrayed her to seemingly forgetting she hated them. It’s a whirlwind that reeks of adolescent drama and doesn’t fit the plot the author was trying, I think, to execute. The ridiculous decisions the character makes are so obviously made to move the plot in the direction the author was trying to go and was so basic in plot writing it was at times like reading a teenager’s writing project. It was hard to connect to the main character and hard to like any of the others. The author has potential but it was not met in this book. I will not be reading the next and really had to push through to even finish this one.
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Reviewed in the United States on December 4, 2023