Human Fatty Acid Desaturase 3 (FADS3) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to FADS3. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to FADS3. 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 FADS3, 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 FADS3 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

Mammals have different sphingoid bases that differ in their length and/or pattern of desaturation and hydroxyl groups. The predominant sphingoid base in mammalian ceramides is sphing-4-enine (sphingosine or SPH) which has a trans desaturation at carbon 4. FADS3 is a ceramide desaturase that introduces a cis double bond between carbon 14 and carbon 15 of the SPH-containing ceramides, producing sphinga-4,14-dienine-containing ceramides (SPD ceramides). SPD ceramides occur widely in mammalian tissues and cells. Due to their unusual structure containing a cis double bond, SPD ceramides may have an opposite, negative role in lipid microdomain formation relative to conventional ceramides (PubMed:31916624). FADS3 also acts as a methyl-end fatty acyl coenzyme A (CoA) desaturase that introduces a cis double bond between the preexisting double bond and the terminal methyl group of the fatty acyl chain. Desaturates (11E)-octadecenoate (trans-vaccenoate, the predominant trans fatty acid in human milk) at carbon 13 to generate (11E,13Z)-octadecadienoate (also known as conjugated linoleic acid 11E,13Z-CLA).

GENE ID	3995
SWISS PROT	Q9Y5Q0
SYNONYMS	CYB5RP; Delta-9-Desaturase; Cytochrome b5-related protein

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 FADS3 were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level FADS3 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 0.082ng/mL.
ASSAY RANGE	0.312-20ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of FADS3. No significant cross-reactivity or interference between FADS3 and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between FADS3 and all analogs, therefore, cross reactivity may still exist.