Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	78.13-5000 pg/mL
Sensitivity:	46.88 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human AhR in samples. No significant cross-reactivity or interference between Human AhR 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 AhR. 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 AhR 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 AhR, 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 AhR. The concentration of Human AhR in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	AHR: a nuclear receptor for aryl hydrocarbons involved in xenobiotic metabolism, cell cycle regulation, and development in response to both endogenous and environmental signals. AhR was initially identified as a receptor for dioxins, which are environmental pollutants generated by waste incineration and other industrial processes. AhR ligands include polycyclic aromatic hydrocarbons, including the carcinogen benzo(a)pyrene and other components of cigarette smoke. Naturally occurring AhR ligands include flavonoids, which are aromatic plant secondary compounds commonly found in vegetables and fruits. Cytoplasmic aryl hydrocarbon receptors are found in protein complexes with heat shock proteins. Upon ligand binding, AhR dissociates from heat shock proteins and translocate to the nucleus where it dimerizes with AhR nuclear translocator (ARNT, HIF-1b). The AhR/ARNT heterodimer binds to nuclear xenobiotic response elements to control the expression of genes associated with xenobiotic metabolism, including several cytochrome P450 genes. AhR is ubiquitously expressed and is thought to play a role in regulation of cell proliferation and differentiation, cytokine expression, and xenobiotic metabolism. Research studies link AhR activity with the control of regulatory T-cell and T-helper 17 cell differentiation, regulation of the inflammatory response, and the onset of lung cancer.
UniProt Protein Details:	Protein type:DNA-binding; Transcription factor; Nuclear receptor Chromosomal Location of Human Ortholog: 7p15 Cellular Component: nucleoplasm; transcription factor complex; cytoplasm; nucleus Molecular Function:protein dimerization activity; RNA polymerase II transcription factor activity, enhancer binding; ligand-dependent nuclear receptor activity; protein binding; signal transducer activity; DNA binding; sequence-specific DNA binding; protein heterodimerization activity; Hsp90 protein binding; transcription factor activity; transcription factor binding Biological Process: transcription from RNA polymerase II promoter; prostate gland development; blood vessel development; intracellular receptor-mediated signaling pathway; apoptosis; response to toxin; positive regulation of transcription, DNA-dependent; negative regulation of transcription from RNA polymerase II promoter; cell cycle; regulation of transcription from RNA polymerase II promoter; response to xenobiotic stimulus; regulation of transcription, DNA-dependent; regulation of gene expression; xenobiotic metabolic process; regulation of B cell proliferation; positive regulation of transcription from RNA polymerase II promoter; circadian regulation of gene expression; negative regulation of transcription, DNA-dependent; positive regulation of transcriptional preinitiation complex assembly
NCBI Summary:	This gene encodes a ligand-activated transcription factor involved in the regulation of biological responses to planar aromatic hydrocarbons. This receptor has been shown to regulate xenobiotic-metabolizing enzymes such as cytochrome P450. Its ligands included a variety of aromatic hydrocarbons. [provided by RefSeq, Jul 2008]
UniProt Code:	P35869
NCBI GenInfo Identifier:	3041653
NCBI Gene ID:	196
NCBI Accession:	P35869. 2
UniProt Secondary Accession:	P35869,Q13728, Q13803, Q13804, A4D130,
UniProt Related Accession:	P35869
Molecular Weight:	848
NCBI Full Name:	Aryl hydrocarbon receptor
NCBI Synonym Full Names:	aryl hydrocarbon receptor
NCBI Official Symbol:	AHR
NCBI Official Synonym Symbols:	bHLHe76
NCBI Protein Information:	aryl hydrocarbon receptor; AH-receptor; ah receptor; aromatic hydrocarbon receptor; class E basic helix-loop-helix protein 76
UniProt Protein Name:	Aryl hydrocarbon receptor
UniProt Synonym Protein Names:	Class E basic helix-loop-helix protein 76; bHLHe76
Protein Family:	Aldehyde reductase
UniProt Gene Name:	AHR
UniProt Entry Name:	AHR_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
5000	61076 61436	61256	61233
2500	31700 37008	34354	34331
1250	18281 18057	18169	18146
625	9333 9451	9392	9369
312.5	4997 4669	4833	4810
156.25	2696 2326	2511	2488
78.13	1247 1431	1339	1316
0	23 23	23	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human AhR 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)	260.65	737.59	2444.15	256.59	763.10	2647.18
Standard deviation	33.62	82.54	187.22	26.76	66.16	294.90
C V (%)	12.90	11.19	7.66	10.43	8.67	11.14

Recovery

The recovery of Human AhR 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)	99-111	104
EDTA plasma (n=5)	100-116	107
Cell culture media (n=5)	98-112	105

Linearity

Samples were spiked with high concentrations of Human AhR 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 (%)	99-115	85-98	86-99
1:2	Average (%)	106	91	91
1:4	Range (%)	100-117	95-105	95-111
1:4	Average (%)	107	100	103
1:8	Range (%)	98-115	103-120	98-113
1:8	Average (%)	106	110	106
1:16	Range (%)	91-105	94-108	84-98
1:16	Average (%)	98	101	90

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.