Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	0.16-10 ng/mL
Sensitivity:	0.10 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human RANk in samples. No significant cross-reactivity or interference between Human RANk and analogues was observed.

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human RANk. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human 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 Human RANk, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human RANk. The concentration of Human RANk in samples can be calculated by comparing the OD of the samples to the standard curve.

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

As the OD 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 (ng/mL)	O.D	Average	Corrected
10	2.263 2.293	2.278	2.207
5	1.53 1.548	1.539	1.468
2.5	0.881 0.853	0.867	0.796
1.25	0.424 0.46	0.442	0.371
0.63	0.246 0.226	0.236	0.165
0.32	0.176 0.154	0.165	0.094
0.16	0.117 0.123	0.12	0.049
0	0.066 0.076	0.071	--

Precision

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

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

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/mL)	0.55	0.98	3.38	0.58	0.96	3.28
Standard deviation	0.03	0.05	0.16	0.04	0.05	0.11
C V (%)	5.45	5.10	4.73	6.90	5.21	3.35

Recovery

The recovery of Human 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)	86-101	93
EDTA plasma (n=5)	91-107	98
Cell culture media (n=5)	93-107	99

Linearity

Samples were spiked with high concentrations of Human 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)
1:2	Range (%)	93-108	93-104	94-106
1:2	Average (%)	100	98	99
1:4	Range (%)	89-104	85-95	84-95
1:4	Average (%)	95	90	89
1:8	Range (%)	86-101	81-91	88-101
1:8	Average (%)	92	86	95
1:16	Range (%)	90-106	82-93	84-97
1:16	Average (%)	97	87	91

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 ELISA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-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 (25×)	1 vial, 30 mL
Substrate Reagent	1 vial, 10 mL	4°C(shading light)
Stop Solution	1 vial, 10 mL	4°C
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

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.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
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.
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.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.