Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	31.25-2000 pg/mL
Sensitivity:	18.75 pg/mL
Sample volume:	100µL
Sample type:	Tissue homogenates
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human CYP1A1 in samples. No significant cross-reactivity or interference between Human CYP1A1 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 CYP1A1. 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 CYP1A1 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 CYP1A1, 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 CYP1A1. The concentration of Human CYP1A1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	CYP1A1: Cytochromes P450 are a group of heme-thiolate monooxygenases. In liver microsomes, this enzyme is involved in an NADPH-dependent electron transport pathway. It oxidizes a variety of structurally unrelated compounds, including steroids, fatty acids, and xenobiotics. Belongs to the cytochrome P450 family.
UniProt Protein Details:	Protein type:Cofactor and Vitamin Metabolism - retinol; Amino Acid Metabolism - tryptophan; Oxidoreductase; EC 1. 14. 14. 1; Xenobiotic Metabolism - metabolism by cytochrome P450 Chromosomal Location of Human Ortholog: 15q24. 1 Cellular Component: endoplasmic reticulum membrane; mitochondrion Molecular Function:oxidoreductase activity, acting on diphenols and related substances as donors; flavonoid 3'-monooxygenase activity; enzyme binding; iron ion binding; heme binding; oxidoreductase activity; steroid hydroxylase activity; oxygen binding; demethylase activity Biological Process: coumarin metabolic process; response to nematode; response to arsenic; response to lipopolysaccharide; cellular lipid metabolic process; response to vitamin A; response to antibiotic; embryonic development ending in birth or egg hatching; flavonoid metabolic process; porphyrin metabolic process; response to wounding; arachidonic acid metabolic process; drug metabolic process; aging; response to food; response to drug; insecticide metabolic process; camera-type eye development; response to iron(III) ion; response to virus; epoxygenase P450 pathway; response to herbicide; dibenzo-p-dioxin catabolic process; vitamin D metabolic process; cell proliferation; response to hyperoxia; gut development; xenobiotic metabolic process; maternal process involved in parturition; response to hypoxia; 9-cis-retinoic acid biosynthetic process; amine metabolic process; hydrogen peroxide biosynthetic process
NCBI Summary:	This gene, CYP1A1, encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This protein localizes to the endoplasmic reticulum and its expression is induced by some polycyclic aromatic hydrocarbons (PAHs), some of which are found in cigarette smoke. The enzyme's endogenous substrate is unknown; however, it is able to metabolize some PAHs to carcinogenic intermediates. The gene has been associated with lung cancer risk. A related family member, CYP1A2, is located approximately 25 kb away from CYP1A1 on chromosome 15. [provided by RefSeq, Jul 2008]
UniProt Code:	P04798
NCBI GenInfo Identifier:	117139
NCBI Gene ID:	1543
NCBI Accession:	P04798. 1
UniProt Secondary Accession:	P04798,Q53G18, A4F3V9, A4F3W0,
UniProt Related Accession:	P04798
Molecular Weight:	58kDa
NCBI Full Name:	Cytochrome P450 1A1
NCBI Synonym Full Names:	cytochrome P450, family 1, subfamily A, polypeptide 1
NCBI Official Symbol:	CYP1A1
NCBI Official Synonym Symbols:	AHH; AHRR; CP11; CYP1; P1-450; P450-C; P450DX
NCBI Protein Information:	cytochrome P450 1A1; CYPIA1; cytochrome P450-C; cytochrome P450-P1; cytochrome P450 form 6; xenobiotic monooxygenase; aryl hydrocarbon hydroxylase; flavoprotein-linked monooxygenase; cytochrome P1-450, dioxin-inducible; cytochrome P450, subfamily I (aromatic compound-inducible), polypeptide 1
UniProt Protein Name:	Cytochrome P450 1A1
UniProt Synonym Protein Names:	CYPIA1; Cytochrome P450 form 6; Cytochrome P450-C; Cytochrome P450-P1
Protein Family:	Cytochrome
UniProt Gene Name:	CYP1A1
UniProt Entry Name:	CP1A1_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
2000	55663 55779	55721	55693
1000	22269 25679	23974	23946
500	11626 10438	11032	11004
250	5022 5566	5294	5266
125	2686 2530	2608	2580
62.5	1350 1272	1311	1283
31.25	643 705	674	646
0	28 28	28	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CYP1A1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CYP1A1 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)	110.25	231.78	842.62	119.28	221.63	804.21
Standard deviation	13.72	23.02	70.53	11.10	18.42	69.32
C V (%)	12.44	9.93	8.37	9.31	8.31	8.62

Recovery

The recovery of Human CYP1A1 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)	100-112	107
EDTA plasma (n=5)	90-108	98
Cell culture media (n=5)	90-106	98

Linearity

Samples were spiked with high concentrations of Human CYP1A1 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 (%)	96-112	95-106	96-109
1:2	Average (%)	103	100	101
1:4	Range (%)	92-104	95-108	100-112
1:4	Average (%)	99	100	106
1:8	Range (%)	92-106	96-108	98-111
1:8	Average (%)	97	102	104
1:16	Range (%)	88-99	95-108	93-106
1:16	Average (%)	94	100	100

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.