Human CNTF (Ciliary Neurotrophic Factor) CLIA Kit (HUES00038)

SKU:
HUES00038
€599

Description

ELISA Kit Technical ManualMSDS

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.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection methodChemiluminescence
Detection range15.63-1000 pg/mL
Sensitivity9.38 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

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

Typical Data

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.

Concentration
(pg/mL)
RLU Average Corrected
100052117
55233
5367553650
50021355
23623
2248922464
25011051
9371
1021110186
1254464
5338
49014876
62.52607
2299
24532428
31.251345
1217
12811256
15.63666
750
708683
024
26
25--

Precision

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
Sample123123
n202020202020
Mean (pg/mL)45.87114.20464.2848.01115.57441.42
Standard deviation5.289.3543.276.0013.7832.14
C V (%)11.518.199.3212.5011.927.28

Recovery

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 (%)
Serum (n=5)102-117108
EDTA plasma (n=5)91-10496
Cell culture media (n=5)98-114105

Linearity

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)
1:2Range (%)95-107102-11690-101
Average (%)10010895
1:4Range (%)100-116101-11691-104
Average (%)10610898
1:8Range (%)95-11083-9699-112
Average (%)10190106
1:16Range (%)102-11484-9694-107
Average (%)1099199

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 CLIA 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 Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 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]
UniProt Code:P26441
NCBI GenInfo Identifier:116585
NCBI Gene ID:1270
NCBI Accession:P26441.1
UniProt Secondary Accession:P26441,B2RAB2,
UniProt Related Accession:P26441
Molecular Weight:
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
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
CLIA
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