Human Cytochrome P450 24A1 (CYP24A1) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to CYP24A1. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to CYP24A1. 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 CYP24A1, 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 CYP24A1 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 with a key role in vitamin D catabolism and calcium homeostasis. Via C24- and C23-oxidation pathways, catalyzes the inactivation of both the vitamin D precursor calcidiol (25-hydroxyvitamin D(3)) and the active hormone calcitriol (1-alpha,25-dihydroxyvitamin D(3)) (PubMed:24893882, PubMed:15574355, PubMed:8679605, PubMed:11012668, PubMed:16617161, PubMed:29461981). With initial hydroxylation at C-24 (via C24-oxidation pathway), performs a sequential 6-step oxidation of calcitriol leading to the formation of the biliary metabolite calcitroic acid (PubMed:24893882, PubMed:15574355). With initial hydroxylation at C-23 (via C23-oxidation pathway), catalyzes sequential oxidation of calcidiol leading to the formation of 25(OH)D3-26,23-lactone as end product (PubMed:11012668, PubMed:8679605). Preferentially hydroxylates at C-25 other vitamin D active metabolites, such as CYP11A1-derived secosteroids 20S-hydroxycholecalciferol and 20S,23-dihydroxycholecalciferol (PubMed:25727742). 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:8679605).

GENE ID	1591
SWISS PROT	Q07973
SYNONYMS	CP24; CYP24; P450-CC24; Vitamin D 24-Hydroxylase; 1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial ; Cytochrome P450 Family 24 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 CYP24A1 were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level CYP24A1 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 0.053ng/mL.
ASSAY RANGE	0.156-10ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of CYP24A1. No significant cross-reactivity or interference between CYP24A1 and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between CYP24A1 and all analogs, therefore, cross reactivity may still exist.