CH25H ELISA Kit (Human)

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level Cholesterol-25-Hydroxylase (CH25H) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level Cholesterol-25-Hydroxylase (CH25H) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

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. May play an important role in regulating lipid metabolism by synthesizing a corepressor that blocks sterol regulatory element binding protein (SREBP) processing. In testis, production of 25-hydroxycholesterol by macrophages may play a role in Leydig cell differentiation.

The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Cholesterol-25-Hydroxylase (CH25H) . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Cholesterol-25-Hydroxylase (CH25H) . Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Cholesterol-25-Hydroxylase (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 Cholesterol-25-Hydroxylase (CH25H) in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Reconstitution & Storage Instructions Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Antibody Array (AA) Protocol

Matrix Recovery Range (%) Average (%) Serum (n=5) 99-105 102 EDTA Plasma (n=5) 92-103 99 Heparin Plasma (n=5) 83-95 86 Matrix Recovery Range (%) Average (%) Matrix Recovery Range (%) Average (%) Serum (n=5) 99-105 102 Serum (n=5) 99-105 102 EDTA Plasma (n=5) 92-103 99 EDTA Plasma (n=5) 92-103 99 Heparin Plasma (n=5) 83-95 86 Heparin Plasma (n=5) 83-95 86

Component Amount Anti-CH25H Microplate 96 Wells (12 x 8 Well Strips) CH25H Lyophilized Standard 2 x 8 ng 100X Biotinylated CH25H Detector Antibody 120 uL Avidin/HRP Conjugate 120 uL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL TMB Substrate 1 x 9 mL Stop Solution 1 x 6 mL Component Amount Component Amount Anti-CH25H Microplate 96 Wells (12 x 8 Well Strips) Anti-CH25H Microplate 96 Wells (12 x 8 Well Strips) CH25H Lyophilized Standard 2 x 8 ng CH25H Lyophilized Standard 2 x 8 ng 100X Biotinylated CH25H Detector Antibody 120 uL 100X Biotinylated CH25H Detector Antibody 120 uL Avidin/HRP Conjugate 120 uL Avidin/HRP Conjugate 120 uL Standard Diluent 1 x 20 mL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL 30X Wash Buffer 1 x 20 mL TMB Substrate 1 x 9 mL TMB Substrate 1 x 9 mL Stop Solution 1 x 6 mL Stop Solution 1 x 6 mL

This assay has high sensitivity and excellent specificity for detection of Cholesterol-25-Hydroxylase (CH25H) . No significant cross-reactivity or interference between Cholesterol-25-Hydroxylase (CH25H) and analogues was observed.