Human Cytochrome P450 11A1 (CYP11A1) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to CYP11A1. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to CYP11A1. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After the TMB substrate solution is added, only those wells that contain CYP11A1, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of CYP11A1 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

For Use with serum, plasma, and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

A cytochrome P450 monooxygenase that catalyzes the side-chain hydroxylation and cleavage of cholesterol to pregnenolone, the precursor of most steroid hormones (PubMed:21636783). Catalyzes three sequential oxidation reactions of cholesterol, namely the hydroxylation at C22 followed with the hydroxylation at C20 to yield 20R,22R-hydroxycholesterol that is further cleaved between C20 and C22 to yield the C21-steroid pregnenolone and 4-methylpentanal (PubMed:21636783). Mechanistically, uses molecular oxygen inserting one oxygen atom into a substrate and reducing the second into a water molecule. Two electrons are provided by NADPH via a two-protein mitochondrial transfer system comprising flavoprotein FDXR (adrenodoxin/ferredoxin reductase) and nonheme iron-sulfur protein FDX1 or FDX2 (adrenodoxin/ferredoxin) (PubMed:21636783).

GENE ID	1583
SWISS PROT	P05108
SYNONYMS	CYP11A; P450SCC; Cholesterol desmolase; Cholesterol Side Chain Cleavage Enzyme; Cytochrome P450 Family 11 Subfamily A Polypeptide 1

Materials Supplied

Kit Components	96 Wells Quantity/Size
Pre-coated, ready-to-use 96-well strip plate	1 plate
Plate sealer for 96 wells	2
Standard	2 tubes
Diluent buffer	1 bottle
Detection Reagent A	1 bottle
Detection Reagent B	1 bottle
TMB Substrate	1 tube
Stop Solution	1 tube
Wash Buffer (30 ℅ concentrate)	1 tube
Product data sheet	1 copy

Storage

The TMB Substrate, Wash Buffer (30X concentrate), and the Stop Solution should be stored at 4°C upon receipt, while the other items should be stored at -20°C.

Performance Characteristics

REPEATABILITY	Intra-assay Precision (Precision within an assay): 3 samples with low, middle, and high-level CYP11A1 were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level CYP11A1 were tested on 3 different plates, with 8 replicates in each plate. CV(%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%
SENSITIVITY	The minimum detectable dose was 0.133ng/mL.
ASSAY RANGE	0.312-20ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of CYP11A1. No significant cross-reactivity or interference between CYP11A1 and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between CYP11A1 and all analogs, therefore, cross reactivity may still exist.