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Description
Human LTF 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. Cell Lysis Buffer: Gently wash adherent cells with pre-chilled PBS, then trypsinize and collect 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 them in 150-200 μL of PBS per 1×10^6 cells (it is recommended to add protease inhibitors to the PBS; if the level is very low, the PBS volume can be reduced 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. 5. Breast milk cell culture supernatant: Centrifuge at 1000×g for 20 minutes. Remove the supernatant for analysis, or store at -20°C or -80°C, but avoid repeated freeze-thaw cycles. 6. 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 conjugate are sequentially added to microwells pre-coated with a lactoferrin (LTF) 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 lactoferrin (LTF) content in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Lactoferrin ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Lactoferrin, also known as lactoferrin transfer protein (LTF), is a multifunctional protein of the transferrin family. Lactoferrin is a globular glycoprotein with a molecular mass of approximately 80 kDa and is widely present in various secretions, such as milk, saliva, tears, and nasal secretions. Lactoferrin is also present in the secondary granules of PMNs and is secreted by some apical cells. Lactoferrin can be purified from milk or recombinantly produced. Lactoferrin is a component of the human immune system. It possesses antimicrobial (bactericidal and fungicidal) activity and is part of the innate defense, primarily in the mucosa. Lactoferrin interacts with DNA and RNA, polysaccharides, and heparin, and exhibits some of its biological functions in complex with these ligands. | |||||||||||||||||||||||||||||||||
| 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 | Serum, plasma, tissue homogenate, cell lysate, breast milk, cell culture supernatant and other biological fluids |
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4.4 ★★★★★
Based on 73 reviews
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Product Reviews
★★★★★ 5
My dog's favorite toy* (*see durability warnings*)
Style: Variety Pack - Large (3-pack)
This is a plush toy, so *if your dog is a destroyer of things, it'll destroy this toy easily/instantly* & if they're not destroyed right away, I'd definitely expect them to be destroyed eventually just given the type of toy it is- plus much of that depends on the way your dog plays. That said, this plush, multiple squeaker toy has held up really well for the amount of use it gets in our home. Because it contains 3 separate squeakers in it, these toys have been like heaven for my 3 year old, male, Toy Fox Terrier to play with- he freaks out when we squeeze more than 1 squeaker at the same time & I don't do all 3 at once bc I think his head might actually explode LoL 😋! Seriously, ever since he's grown out of puppy-hood, he's much less destructive with his toys now, so we are able to buy toys like these for him, have him go nuts freaking out on them & have them hold up pretty well/for a while before it's time for a replacement, yet since they're not very expensive, it's not terribly hard to replace them if needed (he's still using the last toy in the original 3 pack we got for him). It's great for fetching via a "flinging" motion (they're not heavy enough to actually throw) if you have a smaller dog who doesn't cover much distance too readily, but the best part of this toy is the elongated design of it, because it makes for a perfect "tug of war" type of playtime toy with my little buddy (tugging is a behavior which you may or may not want to encourage your dog to do depending on its disposition). Since my dog thankfully isn't really destructive & doesn't have issues releasing a toy from a tugging interaction anymore, these items have held up pretty well for a plush type toy, but again, if you own a dog known for destroying their toys, or anything else for that matter, these probably aren't going to work out well for you. Given they're pretty cheap to replace, for a throw ready, tug toy which has 3 different places for a dog to chomp on to make it squeak, they've turned out to be perfect for our smaller sized pup & have become a favorite toy of my little guy which he's always ready to play with, at any time, with anyone- he's got it ready to go a lot of the time when we get back home to him after being out. Again, you'll need to go with Kong or something similarly durable if your dog destroys things or is still a puppy who doesn't know better, but for an average amount of wear & tear they've held up really well while remaining at the top of my dog's favorite toy list. So recommended for sure, but just know if you've got a chewy character on your hands, despite all the fun they'd have with these, I'd get a more durable toy instead of this one as these plush toys, while good quality, will not hold up to prolonged abuse (as is the case with most plush toys sold). However, for an "average destruction" level dog, it seems well enough made to last a decent amount of time & they're a source of an enjoyable playtime for our dog & therefore for us as his owners as well! Definitely recommend for it's features, like 3 different squeakers, it's great design for a tugging style of play & it's overall good durability for a plush style toy. 👍
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Reviewed in the United States on July 2, 2019
★★★★★ 5
Better than I expected!
Style: Variety Pack - Large (3-pack)
I have two pitbulls who can destroy any toy! My youngest one likes to chew on my fluffy slippers. She doesn't hurt them, just chews on the fur. I give her a lot of heck for this but she has an affinity for them. I decided to buy her a stuffed dog toy and she loved it! She proudly carried it with her everywhere and would throw it up in the air and catch it. She was as happy as she could be. The problem was that after a few days, my 5-year-old pittie wanted to get in on the action and they turned it into a tug of war. Five minutes later it was in pieces on the floor. I decided to see if there was such a thing as a cloth toy for aggressive chewers. I found these! They weren't that expensive, so I thought, "what the heck", I'll give them a try! They arrived the next day! I took two out of the pack and within two or three days they had killed the squeakers in both of them, and I was thankful! The squeaking was loud and they could not get enough of it. There were three squeakers in each toy! After the squeakers were gone, they seemed to lose interest. So I started doing tug of war with them. Pretty soon they were doing it with each other. I didn't expect them to last a day! However, it has been almost a month and they are still pretty much intact. I mean these dogs are for real and they pull as hard as they can and they still have not ripped them apart. And it's great exercise! There are a few small holes in them but they are not getting bigger. I guess that is how they got the squeakers out! Even the eyes on these toys are still there. I put the third one away for when these two are demolished, but I think I will order another pack of three. That will give me four all together! That should keep them busy for quite a while!
I just want everyone to know that I am pretty limited with my reviews. I don't bother unless I really have something to say, so you can totally believe what I am saying! I don't think you will be upset with this purchase, unless you don't like saliva soaked toys and/or your dog is prone to swallowing it's toys. It is very hard for me to imagine a dog swallowing one of these. From the top of their heads to the tip of their tails they are over a foot long!
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Reviewed in the United States on May 7, 2020
★★★★★ 4
Good quality. Decent price.
Style: Fox, Raccoon, Squirrel
The small size is perfect for my 13# dog, but I was somehow under the impression that one squeaker was in the head while the other was in the tail (and that might be the case for rhe larger size), but one us in the main body, with the other in the tail. The head is flat, and there's no way to push the sqeaker in the body up into the head.
Ordinarily, this wouldn't matter -- and my dog LOVES the one I gave him so far; he's squeaking up a storm! -- but I bought these toys specifically so I could fill the (presumably empty, but in reality not) body between head and tail with chopped up pool noodles so my paraplegic dog could take it into the water treadmill treatment he's starting in a couple weeks. I wanted something that would float and be largely waterproof (no fluff inside in case he drops it in the water), that he could hold in his mouth instead of wearing a muzzle.
I've rated these toys 4 stars because he does love them, they seem well made, and they're pretty affordable. I would buy them again, though not for the same purpose. (He hates rubber toys, so this was my last hope. Lol?)
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Reviewed in the United States on February 21, 2021
★★★★★ 5
Enjoyment and or stuffing to clean up.
Style: Variety Pack - Large (3-pack)
The dogs tore them apart but not as quickly as other toys, they loved them.
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Reviewed in the United States on April 7, 2026
★★★★★ 5
Strong Chewer Friendly Toy
Style: Variety Pack - Large (3-pack)
My dog is a rough chewer and these last a really long time (clearly since this beaver's face is long gone! The no stuffing is a win too for cleaning up afterwards.
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Reviewed in the United States on April 26, 2026
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