Human LTF(Lactoferrin)ELISA Kit (HUES03395)

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HUES03395
€499

Description

ELISA Kit Technical ManualMSDS

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.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range0.31-20 ng/mL
Sensitivity0.19 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human LTF in samples. No significant cross-reactivity or interference between Human LTF and analogues was observed.

Typical Data

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.

Concentration
(ng/mL)
O.D Average Corrected
202.344
2.344
2.3442.284
101.494
1.554
1.5241.464
50.803
0.793
0.7980.738
2.50.371
0.401
0.3860.326
1.250.249
0.235
0.2420.182
0.630.161
0.143
0.1520.092
0.310.097
0.117
0.1070.047
00.058
0.062
0.06--

Precision

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
Sample123123
n202020202020
Mean (ng/mL)1.002.109.101.002.209.30
Standard deviation0.100.100.400.100.100.40
C V (%)10.004.764.4010.004.554.30

Recovery

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 (%)
Serum (n=5)86-9891
EDTA plasma (n=5)89-10395
Cell culture media (n=5)94-107100

Linearity

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)
1:2Range (%)98-11486-9795-109
Average (%)10591100
1:4Range (%)93-10786-10294-106
Average (%)10093100
1:8Range (%)101-11581-9393-107
Average (%)1078798
1:16Range (%)95-11086-9995-112
Average (%)10292102

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.

Item Specifications Storage
Micro ELISA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 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 Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 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]
UniProt Code:P02788
NCBI GenInfo Identifier:85700158
NCBI Gene ID:4057
NCBI Accession:P02788.6
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
Protein Family:Lactotransferrin
UniProt Gene Name:LTF  
UniProt Entry Name:TRFL_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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