Human RANkL (Receptor Activator of Nuclear Factor Kappa B Ligand) CLIA Kit (HUES00796)

SKU:
HUES00796
€599

Description

ELISA Kit Technical ManualMSDS

Human 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 Human 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 Human 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 Human 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 Human RANkL. The concentration of Human RANkL 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 range31.25-2000 pg/mL
Sensitivity18.75 pg/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Human RANkL in samples. No significant cross-reactivity or interference between Human 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
200053431
58011
5572155693
100021922
26026
2397423946
50011339
10725
1103211004
2504995
5593
52945266
1252736
2480
26082580
62.51359
1263
13111283
31.25636
712
674646
027
29
28--

Precision

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

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

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (pg/mL)107.75176.51997.84104.15184.36971.82
Standard deviation11.4316.1589.6111.9313.79111.08
C V (%)10.619.158.9811.457.4811.43

Recovery

The recovery of Human 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)88-10395
EDTA plasma (n=5)88-10095
Cell culture media (n=5)92-10498

Linearity

Samples were spiked with high concentrations of Human 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 (%)91-102104-11592-105
Average (%)9610998
1:4Range (%)97-10998-11588-101
Average (%)10310594
1:8Range (%)91-10591-106100-115
Average (%)9897107
1:16Range (%)89-10496-111100-114
Average (%)95102108

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 RANkL (Receptor Activator of Nuclear Factor Kappa B Ligand) CLIA Kit (HUES00796) 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 RANkL (Receptor Activator of Nuclear Factor Kappa B Ligand) CLIA Kit (HUES00796) 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

Chromosomal Location of Human Ortholog: 13q14

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

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

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

Disease: Osteopetrosis, Autosomal Recessive 2

NCBI Summary:This gene encodes a member of the tumor necrosis factor (TNF) cytokine family which is a ligand for osteoprotegerin and functions as a key factor for osteoclast differentiation and activation. This protein was shown to be a dentritic cell survival factor and is involved in the regulation of T cell-dependent immune response. T cell activation was reported to induce expression of this gene and lead to an increase of osteoclastogenesis and bone loss. This protein was shown to activate antiapoptotic kinase AKT/PKB through a signaling complex involving SRC kinase and tumor necrosis factor receptor-associated factor (TRAF) 6, which indicated this protein may have a role in the regulation of cell apoptosis. Targeted disruption of the related gene in mice led to severe osteopetrosis and a lack of osteoclasts. The deficient mice exhibited defects in early differentiation of T and B lymphocytes, and failed to form lobulo-alveolar mammary structures during pregnancy. Two alternatively spliced transcript variants have been found. [provided by RefSeq, Jul 2008]
UniProt Code:O14788
NCBI GenInfo Identifier:12643360
NCBI Gene ID:8600
NCBI Accession:O14788.1
UniProt Secondary Accession:O14788,O14723, Q96Q17, Q9P2Q3,
UniProt Related Accession:O14788
Molecular Weight:30,523 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; OPGL; sOdf; CD254; OPTB2; RANKL; TRANCE; hRANKL2
NCBI Protein Information:tumor necrosis factor ligand superfamily member 11; 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_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
CLIA
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