Human LTF(Lactoferrin)ELISA Kit (HUES03395)
Human LTF(Lactoferrin)ELISA Kit
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human LTF . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human LTF and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human LTF, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human LTF. The concentration of Human LTF in samples can be calculated by comparing the OD of the samples to the standard curve.
|Detection Range||0.31-20 ng/mL|
|Sample Volume Required Per Well||100uL|
|Sample Type||Serum, plasma and other biological fluids|
This kit recognizes Human LTF in samples. No significant cross-reactivity or interference between Human LTF and analogues was observed.
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human LTF were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human LTF were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||10.00||4.76||4.40||10.00||4.55||4.30|
The recovery of Human LTF spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||89-103||95|
|Cell culture media (n=5)||94-107||100|
Samples were spiked with high concentrations of Human LTF and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
|Serum (n=5)||EDTA plasma (n=5)||Cell culture media (n=5)|
Kit Components & Storage
An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
|Micro ELISA Plate(Dismountable)||8 wells X12 strips||-20'C, 6 months|
|Reference Standard||2 vials|
|Concentrated Biotinylated Detection Ab (100X)||1 vial, 120 uL|
|Concentrated HRP Conjugate (100X)||1 vial, 120 uL||-20'C(shading light), 6 months|
|Reference Standard & Sample Diluent||1 vial, 20 mL||4'C, 6 months|
|Biotinylated Detection Ab Diluent||1 vial, 14 mL|
|HRP Conjugate Diluent||1 vial, 14 mL|
|Concentrated Wash Buffer (25X)||1 vial, 30 mL|
|Substrate Reagent||1 vial, 10 mL||4'C(shading light)|
|Stop Solution||1 vial, 10 mL||4'C|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
Human LTF(Lactoferrin)ELISA Kit (HUES03395) Assay procedure
- 1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- 2. Aliquot 100µl of standard solutions into the standard wells.
- 3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- 4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- 5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- 6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
- 7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- 8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- 9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- 10. Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
- 11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- 12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
Human LTF(Lactoferrin)ELISA Kit (HUES03395) Protein Information
|UniProt Protein Function:||LTF: Transferrins are iron binding transport proteins which can bind two Fe(3+) ions in association with the binding of an anion, usually bicarbonate. Belongs to the transferrin family. 2 isoforms of the human protein are produced by alternative promoter.|
|UniProt Protein Details:|
Protein type:Secreted; Protease; EC 3.4.21.-; Secreted, signal peptide
Chromosomal Location of Human Ortholog: 3p21.31
Cellular Component: cell surface; cytoplasm; extracellular region; extracellular space; nucleus; protein complex; secretory granule; specific granule
Molecular Function:heparin binding; iron ion binding; protein binding; protein serine/threonine kinase activator activity
Biological Process: activation of NF-kappaB transcription factor; antibacterial humoral response; antifungal humoral response; cell redox homeostasis; cellular protein metabolic process; humoral immune response; innate immune response in mucosa; iron assimilation by chelation and transport; negative regulation of apoptosis; negative regulation of ATPase activity; negative regulation of lipopolysaccharide-mediated signaling pathway; negative regulation of viral genome replication; negative regulation of viral reproduction; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of osteoblast differentiation; positive regulation of osteoblast proliferation; positive regulation of toll-like receptor 4 signaling pathway; regulation of cytokine production; regulation of tumor necrosis factor production; retinal homeostasis
|NCBI Summary:||This gene is a member of the transferrin family of genes and its protein product is found in the secondary granules of neutrophils. The protein is a major iron-binding protein in milk and body secretions with an antimicrobial activity, making it an important component of the non-specific immune system. The protein demonstrates a broad spectrum of properties, including regulation of iron homeostasis, host defense against a broad range of microbial infections, anti-inflammatory activity, regulation of cellular growth and differentiation and protection against cancer development and metastasis. Antimicrobial, antiviral, antifungal and antiparasitic activity has been found for this protein and its peptides. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2014]|
|NCBI GenInfo Identifier:||85700158|
|NCBI Gene ID:||4057|
|UniProt Secondary Accession:||P02788,O00756, Q16780, Q16785, Q16786, Q16789, Q5DSM0 Q8IU92, A8K9U8, B2MV13, B7Z4X2, E7EQH5,|
|UniProt Related Accession:||P02788|
|Molecular Weight:||73,161 Da|
|NCBI Full Name:||Lactotransferrin|
|NCBI Synonym Full Names:||lactotransferrin|
|NCBI Official Symbol:||LTF|
|NCBI Official Synonym Symbols:||LF; HLF2; GIG12; HEL110|
|NCBI Protein Information:||lactotransferrin|
|UniProt Protein Name:||Lactotransferrin|
|UniProt Synonym Protein Names:||Growth-inhibiting protein 12; Talalactoferrin|
|UniProt Gene Name:||LTF|
|UniProt Entry Name:||TRFL_HUMAN|