Rat Neutral Sphingomyelinase (NSMASE) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to NSMASE. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to NSMASE. 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 NSMASE, 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 NSMASE 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 hydrolysis of sphingomyelin to form ceramide and phosphocholine. Ceramide mediates numerous cellular functions, such as apoptosis and growth arrest, and is capable of regulating these 2 cellular events independently. Also hydrolyzes sphingosylphosphocholine. Binds to anionic phospholipids (APLs) such as phosphatidylserine (PS) and phosphatidic acid (PA) that modulate enzymatic activity and subcellular location (By similarity). Regulates the cell cycle by acting as a growth suppressor in confluent cells. Acts as a regulator of postnatal development and participates in bone and dentin mineralization. May be involved in IL-1-beta-induced JNK activation in hepatocytes. May act as a mediator in transcriptional regulation of NOS2/iNOS via the NF-kappa-B activation under inflammatory conditions.

GENE ID	94338
SWISS PROT	O35049
SYNONYMS	SMPD2; N-SMase; ISC1; Neutral Sphingomyelin Phosphodiesterase; Sphingomyelin Phosphodiesterase 2,Neutral Membrane; Lyso-platelet-activating factor-phospholipase C

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 NSMASE were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level NSMASE 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 0.068ng/mL.
ASSAY RANGE	0.156-10ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of NSMASE. No significant cross-reactivity or interference between NSMASE and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between NSMASE and all analogs, therefore, cross reactivity may still exist.