Human CNTF (Ciliary Neurotrophic 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 CNTF . 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 CNTF 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 CNTF, 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 CNTF. The concentration of Human CNTF in the samples can be calculated by comparing the RLU of the samples to the standard curve.
|Detection range||15.63-1000 pg/mL|
|Sample type||Serum, plasma and other biological fluids|
|Repeatability||CV < 15%|
This kit recognizes Human CNTF in samples. No significant cross-reactivity or interference between Human CNTF 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 CNTF were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CNTF were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||11.51||8.19||9.32||12.50||11.92||7.28|
The recovery of Human CNTF 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)||91-104||96|
|Cell culture media (n=5)||98-114||105|
Samples were spiked with high concentrations of Human CNTF 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 CNTF (Ciliary Neurotrophic Factor) CLIA Kit (HUES00038) 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 CNTF (Ciliary Neurotrophic Factor) CLIA Kit (HUES00038) Protein Information
|UniProt Protein Function:||CNTF: CNTF is a survival factor for various neuronal cell types. Seems to prevent the degeneration of motor axons after axotomy. Belongs to the CNTF family.|
|UniProt Protein Details:|
Chromosomal Location of Human Ortholog: 11q12.2
Cellular Component: cytoplasm; extracellular region; extracellular space
Molecular Function:ciliary neurotrophic factor receptor binding; growth factor activity; interleukin-6 receptor binding; protein binding
Biological Process: growth; muscle morphogenesis; negative regulation of neuron apoptosis; negative regulation of photoreceptor cell differentiation; neuron development; positive regulation of cell proliferation; positive regulation of tyrosine phosphorylation of Stat3 protein; regulation of retinal cell programmed cell death; signal transduction
|NCBI Summary:||The protein encoded by this gene is a polypeptide hormone whose actions appear to be restricted to the nervous system where it promotes neurotransmitter synthesis and neurite outgrowth in certain neuronal populations. The protein is a potent survival factor for neurons and oligodendrocytes and may be relevant in reducing tissue destruction during inflammatory attacks. A mutation in this gene, which results in aberrant splicing, leads to ciliary neurotrophic factor deficiency, but this phenotype is not causally related to neurologic disease. A read-through transcript variant composed of the upstream ZFP91 gene and CNTF sequence has been identified, but it is thought to be non-coding. Read-through transcription of ZFP91 and CNTF has also been observed in mouse. [provided by RefSeq, Oct 2010]|
|NCBI GenInfo Identifier:||116585|
|NCBI Gene ID:||1270|
|UniProt Secondary Accession:||P26441,B2RAB2,|
|UniProt Related Accession:||P26441|
|NCBI Full Name:||Ciliary neurotrophic factor|
|NCBI Synonym Full Names:||ciliary neurotrophic factor|
|NCBI Official Symbol:||CNTF|
|NCBI Official Synonym Symbols:||HCNTF|
|NCBI Protein Information:||ciliary neurotrophic factor|
|UniProt Protein Name:||Ciliary neurotrophic factor|
|Protein Family:||Ciliary neurotrophic factor|
|UniProt Gene Name:||CNTF|
|UniProt Entry Name:||CNTF_HUMAN|