Human CYP1A2 (Cytochrome P450, family 1, subfamily A, polypeptide 2) ELISA Kit (HUES01583)

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HUES01583
€539

Description

ELISA Kit Technical ManualMSDS

Human CYP1A2 (Cytochrome P450, family 1, subfamily A, polypeptide 2) ELISA Kit

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 CYP1A2 . 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 CYP1A2 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 CYP1A2, 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 CYP1A2. The concentration of Human CYP1A2 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range0.16-10 ng/mL
Sensitivity0.10 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human CYP1A2 in samples. No significant cross-reactivity or interference between Human CYP1A2 and analogues was observed.

Typical Data

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
102.433
2.459
2.4462.39
51.519
1.535
1.5271.471
2.50.908
0.902
0.9050.849
1.250.422
0.446
0.4340.378
0.630.255
0.245
0.250.194
0.320.159
0.151
0.1550.099
0.160.1
0.114
0.1070.051
00.049
0.063
0.056--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CYP1A2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CYP1A2 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.531.364.070.511.333.68
Standard deviation0.040.070.190.030.070.17
C V (%)7.555.154.675.885.264.62

Recovery

The recovery of Human CYP1A2 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)91-10698
EDTA plasma (n=5)87-10293
Cell culture media (n=5)90-10196

Linearity

Samples were spiked with high concentrations of Human CYP1A2 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 (%)85-9689-10491-103
Average (%)909598
1:4Range (%)88-10284-9884-97
Average (%)939089
1:8Range (%)91-10385-9983-95
Average (%)969290
1:16Range (%)87-10287-9889-102
Average (%)939394

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 ELISA 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 Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human CYP1A2 (Cytochrome P450, family 1, subfamily A, polypeptide 2) ELISA Kit (HUES01583) 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 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.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human CYP1A2 (Cytochrome P450, family 1, subfamily A, polypeptide 2) ELISA Kit (HUES01583) Protein Information

UniProt Protein Function: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. Most active in catalyzing 2-hydroxylation. Caffeine is metabolized primarily by cytochrome CYP1A2 in the liver through an initial N3-demethylation. Also acts in the metabolism of aflatoxin B1 and acetaminophen. Participates in the bioactivation of carcinogenic aromatic and heterocyclic amines. Catalizes the N-hydroxylation of heterocyclic amines and the O-deethylation of phenacetin.
NCBI Summary:This gene 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. The protein encoded by this gene 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. Other xenobiotic substrates for this enzyme include caffeine, aflatoxin B1, and acetaminophen. The transcript from this gene contains four Alu sequences flanked by direct repeats in the 3' untranslated region. [provided by RefSeq, Jul 2008]
UniProt Code:P05177
NCBI GenInfo Identifier:1476413392
NCBI Gene ID:1544
NCBI Accession:P05177.4
UniProt Secondary Accession:P05177,Q16754, Q6NWU3, Q6NWU5, Q9BXX7, Q9UK49,
UniProt Related Accession:P05177
Molecular Weight:57 KD
NCBI Full Name:Cytochrome P450 1A2
NCBI Synonym Full Names:cytochrome P450 family 1 subfamily A member 2
NCBI Official Symbol:CYP1A2  
NCBI Official Synonym Symbols:CP12; CYPIA2; P3-450; P450(PA)  
NCBI Protein Information:cytochrome P450 1A2
UniProt Protein Name:Cytochrome P450 1A2
UniProt Synonym Protein Names:CYPIA2; Cholesterol 25-hydroxylase
Protein Family:Cytochrome
UniProt Gene Name:CYP1A2  
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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