Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	62.50-4000 pg/mL
Sensitivity:	37.50 pg/mL
Sample volume:	100µL
Sample type:	Tissue homogenates,cell lysates and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human ER beta in samples. No significant cross-reactivity or interference between Human ER beta and analogues was observed.

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 ER beta. 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 ER beta 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 ER beta, 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 ER beta. The concentration of Human ER beta in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	ER-beta: a nuclear hormone receptor and transcription factor. Binds and activated by estrogen. Regulates gene expression and affects cellular proliferation and differentiation in target tissues. Binds estrogens with an affinity similar to that of ER-alpha, and activates expression of reporter genes containing estrogen response elements (ERE) in an estrogen-dependent manner. Eight alternatively-spliced isoforms have been described. Isoform beta-cx lacks ligand binding ability and has no or only very low ERE binding activity resulting in the loss of ligand-dependent transactivation ability.
UniProt Protein Details:	Protein type:DNA-binding; Nuclear receptor Chromosomal Location of Human Ortholog: 14q23. 2 Cellular Component: nucleoplasm; mitochondrion; extracellular region; nucleus Molecular Function:receptor antagonist activity; protein binding; ligand-dependent nuclear receptor activity; estrogen receptor activity; enzyme binding; DNA binding; zinc ion binding; transcription coactivator activity; estrogen response element binding; steroid hormone receptor activity; transcription factor activity; steroid binding Biological Process: transcription initiation from RNA polymerase II promoter; estrogen receptor signaling pathway; induction of apoptosis by hormones; neuron migration; vagina development; negative regulation of transcription from RNA polymerase II promoter; uterus development; signal transduction; cell-cell signaling; ovarian follicle development; regulation of transcription, DNA-dependent; steroid hormone mediated signaling; gene expression; positive regulation of transcription factor activity; negative regulation of cell growth; brain development; negative regulation of epithelial cell proliferation
NCBI Summary:	This gene encodes a member of the family of estrogen receptors and superfamily of nuclear receptor transcription factors. The gene product contains an N-terminal DNA binding domain and C-terminal ligand binding domain and is localized to the nucleus, cytoplasm, and mitochondria. Upon binding to 17beta-estradiol or related ligands, the encoded protein forms homo- or hetero-dimers that interact with specific DNA sequences to activate transcription. Some isoforms dominantly inhibit the activity of other estrogen receptor family members. Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been fully characterized. [provided by RefSeq, Jul 2008]
UniProt Code:	Q92731
NCBI GenInfo Identifier:	6166154
NCBI Gene ID:	2100
NCBI Accession:	Q92731. 2
UniProt Related Accession:	Q92731
Molecular Weight:	Predicted: 53 kDa
NCBI Full Name:	Estrogen receptor beta
NCBI Synonym Full Names:	estrogen receptor 2 (ER beta)
NCBI Official Symbol:	ESR2
NCBI Official Synonym Symbols:	Erb; ESRB; ESTRB; NR3A2; ER-BETA; ESR-BETA
NCBI Protein Information:	estrogen receptor beta; estrogen receptor beta 4; nuclear receptor subfamily 3 group A member 2
UniProt Protein Name:	5p152
UniProt Synonym Protein Names:	5p152
Protein Family:	Estrogen receptor
UniProt Gene Name:	ESR2
UniProt Entry Name:	Q7LCB3_HUMAN

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
4000	58749 59413	59081	59046
2000	25435 30283	27859	27824
1000	14435 12533	13484	13449
500	6510 6700	6605	6570
250	3312 3174	3243	3208
125	1671 1491	1581	1546
62.50	747 763	755	720
0	34 36	35	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human ER beta 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 (pg/mL)	192.80	550.74	1973.20	195.81	515.29	1865.39
Standard deviation	16.45	41.86	160.82	17.23	45.04	164.53
C V (%)	8.53	7.60	8.15	8.80	8.74	8.82

Recovery

The recovery of Human ER beta 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)	89-103	95
EDTA plasma (n=5)	86-98	91
Cell culture media (n=5)	96-108	102

Linearity

Samples were spiked with high concentrations of Human ER beta 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 (%)	98-110	97-110	84-95
1:2	Average (%)	104	103	90
1:4	Range (%)	86-101	100-113	90-106
1:4	Average (%)	92	106	97
1:8	Range (%)	97-109	92-107	99-113
1:8	Average (%)	103	100	106
1:16	Range (%)	96-109	91-107	103-118
1:16	Average (%)	101	97	109

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 ×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 A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
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 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.
Determine the RLU value of each well immediately.