Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	0.63-40 ng/mL
Sensitivity:	0.38 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human VDR in samples. No significant cross-reactivity or interference between Human VDR 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 VDR. 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 VDR 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 VDR, 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 VDR. The concentration of Human VDR in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	VDR: Nuclear hormone receptor. Transcription factor that mediates the action of vitamin D3 by controlling the expression of hormone sensitive genes. Regulates transcription of hormone sensitive genes via its association with the WINAC complex, a chromatin-remodeling complex. Recruited to promoters via its interaction with the WINAC complex subunit BAZ1B/WSTF, which mediates the interaction with acetylated histones, an essential step for VDR-promoter association. Plays a central role in calcium homeostasis. Defects in VDR are the cause of rickets vitamin D- dependent type 2A (VDDR2A). A disorder of vitamin D metabolism resulting in severe rickets, hypocalcemia and secondary hyperparathyroidism. Most patients have total alopecia in addition to rickets. Belongs to the nuclear hormone receptor family. NR1 subfamily.
UniProt Protein Details:	Protein type:DNA-binding; Transcription factor; Nuclear receptor Chromosomal Location of Human Ortholog: 12q13. 11 Cellular Component: nucleoplasm; nucleus; receptor complex Molecular Function:retinoid X receptor binding; protein binding; DNA binding; zinc ion binding; sequence-specific DNA binding; steroid hormone receptor activity; vitamin D3 receptor activity; transcription factor activity Biological Process: lactation; transcription initiation from RNA polymerase II promoter; positive regulation of apoptosis involved in mammary gland involution; cell morphogenesis; decidualization; negative regulation of transcription from RNA polymerase II promoter; signal transduction; intestinal absorption; cellular calcium ion homeostasis; negative regulation of cell proliferation; calcium ion transport; steroid hormone mediated signaling; positive regulation of transcription from RNA polymerase II promoter; gene expression; negative regulation of transcription, DNA-dependent; skeletal development; positive regulation of keratinocyte differentiation Disease: Vitamin D-dependent Rickets, Type 2a; Osteoporosis
NCBI Summary:	This gene encodes the nuclear hormone receptor for vitamin D3. This receptor also functions as a receptor for the secondary bile acid lithocholic acid. The receptor belongs to the family of trans-acting transcriptional regulatory factors and shows sequence similarity to the steroid and thyroid hormone receptors. Downstream targets of this nuclear hormone receptor are principally involved in mineral metabolism though the receptor regulates a variety of other metabolic pathways, such as those involved in the immune response and cancer. Mutations in this gene are associated with type II vitamin D-resistant rickets. A single nucleotide polymorphism in the initiation codon results in an alternate translation start site three codons downstream. Alternative splicing results in multiple transcript variants encoding different proteins. [provided by RefSeq, Feb 2011]
UniProt Code:	P11473
NCBI GenInfo Identifier:	137617
NCBI Gene ID:	7421
NCBI Accession:	P11473. 1
UniProt Secondary Accession:	P11473,Q5PSV3, B2R5Q1, G3V1V9,
UniProt Related Accession:	P11473
Molecular Weight:
NCBI Full Name:	Vitamin D3 receptor
NCBI Synonym Full Names:	vitamin D (1,25- dihydroxyvitamin D3) receptor
NCBI Official Symbol:	VDR
NCBI Official Synonym Symbols:	NR1I1; PPP1R163
NCBI Protein Information:	vitamin D3 receptor; 1,25-dihydroxyvitamin D3 receptor; vitamin D nuclear receptor variant 1; nuclear receptor subfamily 1 group I member 1; protein phosphatase 1, regulatory subunit 163
UniProt Protein Name:	Vitamin D3 receptor
UniProt Synonym Protein Names:	1,25-dihydroxyvitamin D3 receptor; Nuclear receptor subfamily 1 group I member 1
Protein Family:	Vitamin D3 receptor
UniProt Gene Name:	VDR
UniProt Entry Name:	VDR_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
40	2.535 2.565	2.55	2.469
20	1.732 1.756	1.744	1.663
10	0.948 0.914	0.931	0.85
5	0.533 0.539	0.536	0.455
2.5	0.309 0.283	0.296	0.215
1.25	0.196 0.172	0.184	0.103
0.63	0.127 0.141	0.134	0.053
0	0.077 0.085	0.081	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human VDR 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)	1.85	5.89	18.83	1.82	6.22	17.53
Standard deviation	0.10	0.35	0.69	0.10	0.35	0.82
C V (%)	5.41	5.94	3.66	5.49	5.63	4.68

Recovery

The recovery of Human VDR 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)	85-99	91
EDTA plasma (n=5)	86-96	91
Cell culture media (n=5)	88-102	95

Linearity

Samples were spiked with high concentrations of Human VDR 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 (%)	91-104	91-104	96-114
1:2	Average (%)	97	97	104
1:4	Range (%)	91-103	81-94	88-98
1:4	Average (%)	96	86	93
1:8	Range (%)	88-100	80-93	82-96
1:8	Average (%)	95	86	89
1:16	Range (%)	88-99	82-97	81-93
1:16	Average (%)	94	89	87

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.