Human Cholesterol-25-Hydroxylase (CH25H) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to CH25H. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to CH25H. 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 CH25H, 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 CH25H 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

Catalyzes the formation of 25-hydroxycholesterol from cholesterol, leading to repress cholesterol biosynthetic enzymes (PubMed:9852097). Plays a key role in cell positioning and movement in lymphoid tissues: 25-hydroxycholesterol is an intermediate in biosynthesis of 7-alpha,25-dihydroxycholesterol (7-alpha,25-OHC), an oxysterol that acts as a ligand for the G protein-coupled receptor GPR183/EBI2, a chemotactic receptor for a number of lymphoid cells (By similarity). May play an important role in regulating lipid metabolism by synthesizing a corepressor that blocks sterol regulatory element binding protein (SREBP) processing (PubMed:9852097). As an interferon-stimulated gene, has broad antiviral activities against a wide range of enveloped viruses, such as vesicular stomatitis virus (VSV) and SARS coronavirus-2 (SARS-CoV-2). Its product, 25-hydroxycholesterol, activates the ER-localized enzyme ACAT to induce internalization of accessible cholesterol on the plasma membrane and restricts SARS-CoV-2 S protein-mediated fusion which inhibits virus replication (PubMed:33239446, PubMed:32944968). In testis, production of 25-hydroxycholesterol by macrophages plays a role in Leydig cell differentiation (By similarity).

GENE ID	9023
SWISS PROT	O95992
SYNONYMS	h25OH; Cholesterol 25-monooxygenase

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 CH25H were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level CH25H 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 0.252ng/mL.
ASSAY RANGE	0.625-40ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of CH25H. No significant cross-reactivity or interference between CH25H and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between CH25H and all analogs, therefore, cross reactivity may still exist.