Mouse RANk (Receptor Activator of Nuclear Factor Kappa B) CLIA Kit (MOES00353)

SKU:
MOES00353
€649

Description

ELISA Kit Technical ManualMSDS

Mouse RANk (Receptor Activator of Nuclear Factor Kappa B) 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 Mouse RANk . 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 Mouse RANk 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 Mouse RANk, 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 Mouse RANk. The concentration of Mouse RANk in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityMouse
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 Mouse RANk in samples. No significant cross-reactivity or interference between Mouse RANk 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
100051375
55975
5367553650
50021997
22981
2248922464
25010289
10133
1021110186
1254719
5083
49014876
62.52618
2288
24532428
31.251372
1190
12811256
15.63676
740
708683
024
26
25--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse RANk were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse RANk were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)53.73137.63468.8254.92145.53496.13
Standard deviation4.8815.2128.835.8713.5544.40
C V (%)9.0811.056.1510.699.318.95

Recovery

The recovery of Mouse RANk 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)99-114106
EDTA plasma (n=5)85-9690
Cell culture media (n=5)94-10799

Linearity

Samples were spiked with high concentrations of Mouse RANk 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 (%)101-116102-11893-107
Average (%)10810998
1:4Range (%)100-11487-9898-111
Average (%)10692105
1:8Range (%)93-110104-11890-104
Average (%)10011098
1:16Range (%)91-10295-11298-117
Average (%)96103106

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

Mouse RANk (Receptor Activator of Nuclear Factor Kappa B) CLIA Kit (MOES00353) 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 100 µL of standard solutions into the standard wells.
    3. Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100 µL 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 100 µL 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 350 µL 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 100 µL 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 100 µL 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.

Mouse RANk (Receptor Activator of Nuclear Factor Kappa B) CLIA Kit (MOES00353) Protein Information

UniProt Protein Function:TNFSF11: Cytokine that binds to TNFRSF11B/OPG and to TNFRSF11A/RANK. Osteoclast differentiation and activation factor. Augments the ability of dendritic cells to stimulate naive T-cell proliferation. May be an important regulator of interactions between T-cells and dendritic cells and may play a role in the regulation of the T-cell-dependent immune response. May also play an important role in enhanced bone-resorption in humoral hypercalcemia of malignancy. Homotrimer. Up-regulated by T-cell receptor stimulation. Highest in the peripheral lymph nodes, weak in spleen, peripheral blood Leukocytes, bone marrow, heart, placenta, skeletal muscle, stomach and thyroid. Belongs to the tumor necrosis factor family. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Membrane protein, integral

Cellular Component: extracellular space; membrane; cytoplasm; integral to membrane; plasma membrane; extracellular region; intracellular

Molecular Function:protein binding; cytokine activity; tumor necrosis factor receptor superfamily binding; tumor necrosis factor receptor binding

Biological Process: positive regulation of osteoclast differentiation; multicellular organismal development; positive regulation of JNK cascade; osteoclast differentiation; activation of JNK activity; positive regulation of homotypic cell-cell adhesion; positive regulation of corticotropin-releasing hormone secretion; activation of NF-kappaB transcription factor; calcium ion homeostasis; positive regulation of MAP kinase activity; monocyte chemotaxis; regulation of osteoclast differentiation; tumor necrosis factor-mediated signaling pathway; positive regulation of bone resorption; cell differentiation; protein homooligomerization; bone resorption; ossification; positive regulation of I-kappaB kinase/NF-kappaB cascade; cytokine and chemokine mediated signaling pathway; mammary gland epithelial cell proliferation; lymph node development; positive regulation of protein kinase B signaling cascade; organ morphogenesis; immune response; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription factor activity; positive regulation of T cell activation; positive regulation of phosphorylation

UniProt Code:O35235
NCBI GenInfo Identifier:342187063
NCBI Gene ID:21943
NCBI Accession:O35235.2
UniProt Secondary Accession:O35235,O35306, Q3TWY5, Q9JJK8, Q9JJK9, Q9R1Y0,
UniProt Related Accession:O35235
Molecular Weight:22,150 Da
NCBI Full Name:Tumor necrosis factor ligand superfamily member 11
NCBI Synonym Full Names:tumor necrosis factor (ligand) superfamily, member 11
NCBI Official Symbol:Tnfsf11
NCBI Official Synonym Symbols:ODF; OPG; OPGL; RANKL; Ly109l; Trance
NCBI Protein Information:tumor necrosis factor ligand superfamily member 11; OPG ligand; osteoprotegerin ligand; osteoclast differentiation factor; receptor activator of NF-kappaB ligand; TNF-related activation-induced cytokine; receptor activator of nuclear factor kappa B ligand; receptor activator of nuclear factor kappa-B ligand; tumor necrosis factor-related activation-induced cytokine
UniProt Protein Name:Tumor necrosis factor ligand superfamily member 11
UniProt Synonym Protein Names:Osteoclast differentiation factor; ODF; Osteoprotegerin ligand; OPGL; Receptor activator of nuclear factor kappa-B ligand; RANKL; TNF-related activation-induced cytokine; TRANCE; CD_antigen: CD254Cleaved into the following 2 chains:Tumor necrosis factor ligand superfamily member 11, membrane form; Tumor necrosis factor ligand superfamily member 11, soluble form
UniProt Gene Name:Tnfsf11
UniProt Entry Name:TNF11_MOUSE
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Additional Information

Product type:
ELISA
Reactivity:
Mouse
ELISA Type:
CLIA
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