Rat CYP2E1 (Cytochrome P450, family 2, subfamily E, polypeptide 1) CLIA Kit (RTES00170)

(No reviews yet) Write a Review
SKU:
RTES00170
€649

Description

ELISA Kit Technical ManualMSDS

Rat CYP2E1 (Cytochrome P450, family 2, subfamily E, polypeptide 1) CLIA Kit

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 Rat CYP2E1 . 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 Rat CYP2E1 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 Rat CYP2E1, 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 Rat CYP2E1. The concentration of Rat CYP2E1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityRat
Detection methodChemiluminescence
Detection range0.63-40 ng/mL
Sensitivity0.38 ng/mL
Sample volume100uL
Sample typeSerum, plasma and other biological fluids
RepeatabilityCV < 15%

Specificity

This kit recognizes Rat CYP2E1 in samples. No significant cross-reactivity or interference between Rat CYP2E1 and analogues was observed.

Typical Data

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
(ng/mL)
RLU Average Corrected
4034648
38428
3653836508
2013833
14799
1431614286
106837
5709
62736243
52916
3120
30182988
2.51633
1531
15821552
1.25936
888
912882
0.63549
629
589559
030
30
30--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat CYP2E1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat CYP2E1 were tested on 3 different plates, 20 replicates in each plate.

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)2.154.4514.262.024.8914.88
Standard deviation0.260.361.170.180.371.28
C V (%)12.098.098.208.917.578.60

Recovery

The recovery of Rat CYP2E1 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)86-10093
EDTA plasma (n=5)100-116106
Cell culture media (n=5)102-114108

Linearity

Samples were spiked with high concentrations of Rat CYP2E1 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)
1:2Range (%)97-11392-10399-111
Average (%)10598105
1:4Range (%)86-10091-10293-105
Average (%)929699
1:8Range (%)92-109101-11386-99
Average (%)10010793
1:16Range (%)91-10585-9996-108
Average (%)9691101

Kit Components & Storage

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 X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Rat CYP2E1 (Cytochrome P450, family 2, subfamily E, polypeptide 1) CLIA Kit (RTES00170) Assay procedure

    1. 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.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids.) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. 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.
    7. 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.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. 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.
    11. Determine the RLU value of each well immediately.

Rat CYP2E1 (Cytochrome P450, family 2, subfamily E, polypeptide 1) CLIA Kit (RTES00170) Protein Information

UniProt Protein Function:CYP2E1: Metabolizes several precarcinogens, drugs, and solvents to reactive metabolites. Inactivates a number of drugs and xenobiotics and also bioactivates many xenobiotic substrates to their hepatotoxic or carcinogenic forms. Belongs to the cytochrome P450 family.
UniProt Protein Details:

Protein type:Xenobiotic Metabolism - metabolism by cytochrome P450; Oxidoreductase; Xenobiotic Metabolism - drug metabolism - cytochrome P450; Mitochondrial; EC 1.14.13.n7; Lipid Metabolism - linoleic acid; Lipid Metabolism - arachidonic acid; Endoplasmic reticulum

Cellular Component: endoplasmic reticulum; Golgi membrane; intracellular membrane-bound organelle; intrinsic to endoplasmic reticulum membrane; mitochondrion

Molecular Function:arachidonic acid epoxygenase activity; enzyme binding; FMN binding; heme binding; iron ion binding; monooxygenase activity; oxidoreductase activity; oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NADH or NADPH as one donor, and incorporation of one atom of oxygen; oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen; steroid hydroxylase activity

Biological Process: drug metabolic process; epoxygenase P450 pathway; heterocycle metabolic process; monoterpenoid metabolic process; response to drug; response to ethanol; response to organic nitrogen; response to ozone; steroid metabolic process; triacylglycerol metabolic process; xenobiotic metabolic process

NCBI Summary:facilitates hepatocyte sensitization to tumor necrosis factor-alpha (TNF-alpha)-mediated cell death; may play a role in xenobiotic metabolism [RGD, Feb 2006]
UniProt Code:P05182
NCBI GenInfo Identifier:1352193
NCBI Gene ID:25086
NCBI Accession:P05182.4
UniProt Related Accession:P05182
Molecular Weight:56,627 Da
NCBI Full Name:Cytochrome P450 2E1
NCBI Synonym Full Names:cytochrome P450, family 2, subfamily e, polypeptide 1
NCBI Official Symbol:Cyp2e1  
NCBI Official Synonym Symbols:Cyp2e  
NCBI Protein Information:cytochrome P450 2E1
UniProt Protein Name:Cytochrome P450 2E1
UniProt Synonym Protein Names:4-nitrophenol 2-hydroxylase (EC:1.14.13.n7); CYPIIE1; Cytochrome P450-J; Cytochrome P450RLM6
Protein Family:Cytochrome
UniProt Gene Name:Cyp2e1  
UniProt Entry Name:CP2E1_RAT
View AllClose

Additional Information

Reactivity:
Rat
ELISA Type:
CLIA
View AllClose