Rat RANkL (Receptor Activator of Nuclear Factor Kappa B Ligand) CLIA Kit (RTES00498)

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RTES00498
£608.91

Description

ELISA Kit Technical ManualMSDS

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

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityRat
Detection methodChemiluminescence
Detection range7.81-500 pg/mL
Sensitivity4.69 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Rat RANkL in samples. No significant cross-reactivity or interference between Rat RANkL 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
50050963
52111
5153751508
25020120
21876
2099820969
1259583
9209
93969367
62.54425
4599
45124483
31.252316
2282
22992270
15.631333
1167
12501221
7.81728
752
740711
028
30
29--

Precision

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

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

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)23.5163.94240.2122.3262.40251.19
Standard deviation2.405.7919.172.487.3126.83
C V (%)10.219.067.9811.1111.7110.68

Recovery

The recovery of Rat RANkL 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)89-10195
EDTA plasma (n=5)94-108100
Cell culture media (n=5)98-110104

Linearity

Samples were spiked with high concentrations of Rat RANkL 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 (%)88-100103-115101-115
Average (%)95109109
1:4Range (%)90-10396-11192-106
Average (%)9610498
1:8Range (%)87-9988-10288-102
Average (%)949394
1:16Range (%)96-111100-11388-101
Average (%)10310695

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

Rat RANkL (Receptor Activator of Nuclear Factor Kappa B Ligand) CLIA Kit (RTES00498) 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.

Rat RANkL (Receptor Activator of Nuclear Factor Kappa B Ligand) CLIA Kit (RTES00498) Protein Information

UniProt Protein Function: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. Induces osteoclastogenesis by activating multiple signaling pathways in osteoclast precursor cells, chief among which is induction of long lasting oscillations in the intracellular concentration of Ca (2+) resulting in the activation of NFATC1, which translocates to the nucleus and induces osteoclast-specific gene transcription to allow differentiation of osteoclasts. During osteoclast differentiation, in a TMEM64 and ATP2A2-dependent manner induces activation of CREB1 and mitochondrial ROS generation necessary for proper osteoclast generation.
NCBI Summary:induces osteoclast formation and bone resoprtion; plays a role in regulation of calcium homeostasis and osteoclast function [RGD, Feb 2006]
UniProt Code:Q9ESE2
NCBI GenInfo Identifier:21362971
NCBI Gene ID:117516
NCBI Accession:Q9ESE2.1
UniProt Secondary Accession:Q9ESE2,Q91ZI9,
UniProt Related Accession:Q9ESE2
Molecular Weight:35,370 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:RANKL  
NCBI Protein Information:tumor necrosis factor ligand superfamily member 11; ODF; OPGL; TRANCE; osteoprotegerin ligand; osteoclast differentiation factor; TNF-related activation-induced cytokine; receptor activator of nuclear factor kappa B ligand; receptor activator of nuclear factor kappa-B ligand
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_RAT
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Additional Information

Product type:
ELISA
Reactivity:
Rat
ELISA Type:
CLIA
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