Human HGF (Hepatocyte Growth Factor) CLIA Kit (HUES00081)
Human HGF (Hepatocyte Growth Factor) CLIA Kit
This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human HGF . Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human HGF 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 HGF, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human HGF. The concentration of Human HGF in the samples can be calculated by comparing the RLU of the samples to the standard curve.
|Detection range||31.25-2000 pg/mL|
|Sample type||Serum, plasma and other biological fluids|
|Repeatability||CV < 15%|
This kit recognizes Human HGF in samples. No significant cross-reactivity or interference between Human HGF and analogues was observed.
As the RLU 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 HGF were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human HGF were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||10.19||11.00||10.97||10.78||8.96||7.58|
The recovery of Human HGF 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)||86-99||92|
|Cell culture media (n=5)||91-108||99|
Samples were spiked with high concentrations of Human HGF 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 CLIA 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 A||1 vial, 5 mL||4'C (shading light)|
|Substrate Reagent B||1 vial, 5 mL||4'C (shading light)|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
Human HGF (Hepatocyte Growth Factor) CLIA Kit (HUES00081) 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 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
- 11. Determine the RLU value of each well immediately.
Human HGF (Hepatocyte Growth Factor) CLIA Kit (HUES00081) Protein Information
|UniProt Protein Function:||HGF: HGF is a potent mitogen for mature parenchymal hepatocyte cells, seems to be an hepatotrophic factor, and acts as growth factor for a broad spectrum of tissues and cell types. It has no detectable protease activity. Activating ligand for the receptor tyrosine kinase MET by binding and promoting its dimerization. Defects in HGF are the cause of deafness autosomal recessive type 39 (DFNB39). A form of profound prelingual sensorineural hearing loss. Sensorineural deafness results from damage to the neural receptors of the inner ear, the nerve pathways to the brain, or the area of the brain that receives sound information. Belongs to the peptidase S1 family. Plasminogen subfamily. 6 isoforms of the human protein are produced by alternative splicing.|
|UniProt Protein Details:|
Protein type:Motility/polarity/chemotaxis; Hormone; Cell development/differentiation
Chromosomal Location of Human Ortholog: 7q21.1
Cellular Component: extracellular space; membrane; extracellular region
Molecular Function:identical protein binding; protein binding; growth factor activity; protein heterodimerization activity; serine-type endopeptidase activity; chemoattractant activity
Biological Process: positive regulation of myelination; mitosis; platelet activation; organ regeneration; activation of MAPK activity; myoblast proliferation; hepatocyte growth factor receptor signaling pathway; negative regulation of caspase activity; proteolysis; liver development; positive regulation of phosphoinositide 3-kinase cascade; positive regulation of osteoblast differentiation; positive regulation of peptidyl-tyrosine phosphorylation; positive regulation of angiogenesis; platelet degranulation; positive chemotaxis; epithelial to mesenchymal transition; positive regulation of transcription from RNA polymerase II promoter; blood coagulation; hyaluronan metabolic process; positive regulation of cell migration
Disease: Deafness, Autosomal Recessive 39
|NCBI Summary:||Hepatocyte growth factor regulates cell growth, cell motility, and morphogenesis by activating a tyrosine kinase signaling cascade after binding to the proto-oncogenic c-Met receptor. Hepatocyte growth factor is secreted by mesenchymal cells and acts as a multi-functional cytokine on cells of mainly epithelial origin. Its ability to stimulate mitogenesis, cell motility, and matrix invasion gives it a central role in angiogenesis, tumorogenesis, and tissue regeneration. It is secreted as a single inactive polypeptide and is cleaved by serine proteases into a 69-kDa alpha-chain and 34-kDa beta-chain. A disulfide bond between the alpha and beta chains produces the active, heterodimeric molecule. The protein belongs to the plasminogen subfamily of S1 peptidases but has no detectable protease activity. Alternative splicing of this gene produces multiple transcript variants encoding different isoforms. [provided by RefSeq, Jul 2008]|
|NCBI GenInfo Identifier:||123116|
|NCBI Gene ID:||3082|
|UniProt Secondary Accession:||P14210,Q02935, Q13494, Q14519, Q3KRB2, Q8TCE2, Q9BYL9 Q9BYM0, Q9UDU6, A1L3U6,|
|UniProt Related Accession:||P14210|
|NCBI Full Name:||Hepatocyte growth factor|
|NCBI Synonym Full Names:||hepatocyte growth factor (hepapoietin A; scatter factor)|
|NCBI Official Symbol:||HGF|
|NCBI Official Synonym Symbols:||SF; HGFB; HPTA; F-TCF; DFNB39|
|NCBI Protein Information:||hepatocyte growth factor; hepatopoeitin-A; hepatopoietin-A; lung fibroblast-derived mitogen; fibroblast-derived tumor cytotoxic factor|
|UniProt Protein Name:||Hepatocyte growth factor|
|UniProt Synonym Protein Names:||Hepatopoietin-A; Scatter factor; SF|
|Protein Family:||Hepatocyte growth factor|
|UniProt Gene Name:||HGF|
|UniProt Entry Name:||HGF_HUMAN|