ASAH2 ELISA Kit (Human)

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level N-Acylsphingosine Amidohydrolase 2 (ASAH2) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level N-Acylsphingosine Amidohydrolase 2 (ASAH2) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

Acylsphingosine deacylase 2; BCDase; HNAC1; LCDase; mitochondrial ceramidase; N-acylsphingosine amidohydrolase (non-lysosomal ceramidase) 2; N-acylsphingosine amidohydrolase 2; NCDase; N-CDase; neutral ceramidase; neutral/alkaline ceramidase; non-lysosomal ceramidase.

Hydrolyzes the sphingolipid ceramide into sphingosine and free fatty acid at an optimal pH of 6.5-8.5 (PubMed:10781606, PubMed:26190575) . Acts as a key regulator of sphingolipid signaling metabolites by generating sphingosine at the cell surface. Acts as a repressor of apoptosis both by reducing C16-ceramide, thereby preventing ceramide-induced apoptosis, and generating sphingosine, a precursor of the antiapoptotic factor sphingosine 1-phosphate. Probably involved in the digestion of dietary sphingolipids in intestine by acting as a key enzyme for the catabolism of dietary sphingolipids and regulating the levels of bioactive sphingolipid metabolites in the intestinal tract.

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 N-Acylsphingosine Amidohydrolase 2 (ASAH2) . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to N-Acylsphingosine Amidohydrolase 2 (ASAH2) . 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 N-Acylsphingosine Amidohydrolase 2 (ASAH2), 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 N-Acylsphingosine Amidohydrolase 2 (ASAH2) 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) 97-105 101 EDTA Plasma (n=5) 82-89 85 Heparin Plasma (n=5) 80-101 85 Matrix Recovery Range (%) Average (%) Matrix Recovery Range (%) Average (%) Serum (n=5) 97-105 101 Serum (n=5) 97-105 101 EDTA Plasma (n=5) 82-89 85 EDTA Plasma (n=5) 82-89 85 Heparin Plasma (n=5) 80-101 85 Heparin Plasma (n=5) 80-101 85

Component Amount Anti-ASAH2 Microplate 96 Wells (12 x 8 Well Strips) ASAH2 Lyophilized Standard 2 x 50 ng 100X Biotinylated ASAH2 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-ASAH2 Microplate 96 Wells (12 x 8 Well Strips) Anti-ASAH2 Microplate 96 Wells (12 x 8 Well Strips) ASAH2 Lyophilized Standard 2 x 50 ng ASAH2 Lyophilized Standard 2 x 50 ng 100X Biotinylated ASAH2 Detector Antibody 120 uL 100X Biotinylated ASAH2 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 N-Acylsphingosine Amidohydrolase 2 (ASAH2) . No significant cross-reactivity or interference between N-Acylsphingosine Amidohydrolase 2 (ASAH2) and analogues was observed.