Human Cytochrome P450 27B1 (CYP27B1) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to CYP27B1. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to CYP27B1. 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 CYP27B1, 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 CYP27B1 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 involved in vitamin D metabolism and in calcium and phosphorus homeostasis. Catalyzes the rate-limiting step in the activation of vitamin D in the kidney, namely the hydroxylation of 25-hydroxyvitamin D3/calcidiol at the C1alpha-position to form the hormonally active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3/calcitriol that acts via the vitamin D receptor (VDR) (PubMed:10518789, PubMed:9486994, PubMed:22862690, PubMed:10566658, PubMed:12050193). Has 1alpha-hydroxylase activity on vitamin D intermediates of the CYP24A1-mediated inactivation pathway (PubMed:10518789, PubMed:22862690). Converts 24R,25-dihydroxyvitamin D3/secalciferol to 1-alpha,24,25-trihydroxyvitamin D3, an active ligand of VDR. Also active on 25-hydroxyvitamin D2 (PubMed:10518789). Mechanistically, uses molecular oxygen inserting one oxygen atom into a substrate, and reducing the second into a water molecule, with two electrons provided by NADPH via FDXR/adrenodoxin reductase and FDX1/adrenodoxin (PubMed:22862690).

GENE ID	1594
SWISS PROT	O15528
SYNONYMS	VDD1, PDDR; CYP1, P450c1; 25-Hydroxyvitamin D3 1-Alpha-Hydroxylase; Cytochrome P450 Family 27 Subfamily B Polypeptide 1; Calcidiol 1-monooxygenase; VD3 1A hydroxylase

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 CYP27B1 were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level CYP27B1 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 0.29ng/mL.
ASSAY RANGE	0.78-50ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of CYP27B1. No significant cross-reactivity or interference between CYP27B1 and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between CYP27B1 and all analogs, therefore, cross reactivity may still exist.