Human CYP3A4/Cytochrome P450 3A4 ELISA Kit (HUFI00563)

Product Name:	Human CYP3A4/Cytochrome P450 3A4 ELISA Kit
Product Code:	HUFI00563
Size:	96 Assays
Alias:	CYP3A4, Cytochrome P450 3A4, CYP3A3 CYPIIIA3, CYPIIIA4, Nifedipine, 8-cineole 2-exo-monooxygenase, Albendazole monooxygenase, Albendazole sulfoxidase, Cytochrome P450 3A3, Cytochrome P450 HLp, Cytochrome P450 NF-25, Cytochrome P450-PCN1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human CYP3A4 concentrations in serum plasma and other biological fluids.
Sensitivity:	46.875pg/ml
Range:	78.125-5000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human CYP3A4 and the recovery rates were calculated by comparing the measured value to the expected amount of Human CYP3A4 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	86-104	97
EDTA plasma(n=5)	89-104	98
UFH plasma(n=5)	89-104	98

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CYP3A4 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	85-104%	86-102%	87-99%
EDTA plasma(n=5)	82-97%	84-94%	82-97%
UFH plasma(n=5)	85-100%	84-100%	83-98%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P08684

UniProt Protein Function:	CYP3A4: Cytochromes P450 are a group of heme-thiolate monooxygenases. In liver microsomes, this enzyme is involved in an NADPH-dependent electron transport pathway. It performs a variety of oxidation reactions (e.g. caffeine 8-oxidation, omeprazole sulphoxidation, midazolam 1'-hydroxylation and midazolam 4- hydroxylation) of structurally unrelated compounds, including steroids, fatty acids, and xenobiotics. Acts as a 1,8-cineole 2- exo-monooxygenase. The enzyme also hydroxylates etoposide. Belongs to the cytochrome P450 family.
UniProt Protein Details:	Protein type:EC 1.14.14.1; Xenobiotic Metabolism - drug metabolism - cytochrome P450; Cell surface; Oxidoreductase; Membrane protein, integral; Xenobiotic Metabolism - metabolism by cytochrome P450; Xenobiotic Metabolism - drug metabolism - other enzymes; Lipid Metabolism - linoleic acid; Cofactor and Vitamin Metabolism - retinol Chromosomal Location of Human Ortholog: 7q21.1 Cellular Component: endoplasmic reticulum membrane; intracellular membrane-bound organelle; cytoplasm; integral to membrane Molecular Function:quinine 3-monooxygenase activity; albendazole monooxygenase activity; taurochenodeoxycholate 6alpha-hydroxylase activity; 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; oxidoreductase activity; oxygen binding; vitamin D3 25-hydroxylase activity; testosterone 6-beta-hydroxylase activity; steroid binding; enzyme binding; iron ion binding; heme binding; steroid hydroxylase activity; monooxygenase activity Biological Process: steroid metabolic process; drug catabolic process; xenobiotic metabolic process; androgen metabolic process; exogenous drug catabolic process; monoterpenoid metabolic process; alkaloid catabolic process; heterocycle metabolic process; lipid metabolic process; drug metabolic process; vitamin D metabolic process; steroid catabolic process
NCBI Summary:	This gene encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases that 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 glucocorticoids and some pharmacological agents. This enzyme is involved in the metabolism of approximately half the drugs in use today, including acetaminophen, codeine, cyclosporin A, diazepam and erythromycin. The enzyme also metabolizes some steroids and carcinogens. This gene is part of a cluster of cytochrome P450 genes on chromosome 7q21.1. Previously another CYP3A gene, CYP3A3, was thought to exist; however, it is now thought that this sequence represents a transcript variant of CYP3A4. Alternatively spliced transcript variants encoding different isoforms have been identified. [provided by RefSeq, Feb 2011]
UniProt Code:	P08684
NCBI GenInfo Identifier:	116241312
NCBI Gene ID:	1576
NCBI Accession:	P08684.4
UniProt Secondary Accession:	P08684,P05184, Q16757, Q9UK50,
UniProt Related Accession:	P08684
Molecular Weight:	503
NCBI Full Name:	Cytochrome P450 3A4
NCBI Synonym Full Names:	cytochrome P450, family 3, subfamily A, polypeptide 4
NCBI Official Symbol:	CYP3A4
NCBI Official Synonym Symbols:	HLP; CP33; CP34; CYP3A; NF-25; CYP3A3; P450C3; CYPIIIA3; CYPIIIA4; P450PCN1
NCBI Protein Information:	cytochrome P450 3A4; nifedipine oxidase; cytochrome P450 3A3; cytochrome P450 HLp; cytochrome P450-PCN1; cytochrome P450 NF-25; albendazole sulfoxidase; quinine 3-monooxygenase; albendazole monooxygenase; P450-III, steroid inducible; glucocorticoid-inducible P450; 1,8-cineole 2-exo-monooxygenase; taurochenodeoxycholate 6-alpha-hydroxylase; cytochrome P450, subfamily IIIA (niphedipine oxidase), polypeptide 3; cytochrome P450, subfamily IIIA (niphedipine oxidase), polypeptide 4
UniProt Protein Name:	Cytochrome P450 3A4
UniProt Synonym Protein Names:	1,8-cineole 2-exo-monooxygenase (EC:1.14.13.157); Albendazole monooxygenase (EC:1.14.13.32); Albendazole sulfoxidase; CYPIIIA3; CYPIIIA4; Cytochrome P450 3A3; Cytochrome P450 HLp; Cytochrome P450 NF-25; Cytochrome P450-PCN1; Nifedipine oxidase; Quinine 3-monooxygenase (EC:1.14.13.67); Taurochenodeoxycholate 6-alpha-hydroxylase (EC:1.14.13.97)
Protein Family:	Cytochrome
UniProt Gene Name:	CYP3A4
UniProt Entry Name:	CP3A4_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 Âµl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 Âµl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.