Human Phospholipase A2, Calcium Independent (iPLA2) ELISA Kit

Principle of the Assay

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 Human iPLA2. Standards or samples are added to the appropriate microtiter plate wells then with a biotin-conjugated antibody specific to Human iPLA2. 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 Human iPLA2, 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 Human iPLA2 in the samples is then determined by comparing the OD of the samples to the standard curve.

For use with: serum, plasma, and other biological fluids.

For Research Use Only. Not for use in diagnostic procedures.

Target Information

Calcium-independent phospholipase involved in phospholipid remodeling with implications in cellular membrane homeostasis, mitochondrial integrity and signal transduction. Hydrolyzes the ester bond of the fatty acyl group attached at sn-1 or sn-2 position of phospholipids (phospholipase A1 and A2 activity respectively), producing lysophospholipids that are used in deacylation-reacylation cycles (PubMed:9417066, PubMed:10092647, PubMed:10336645, PubMed:20886109). Hydrolyzes both saturated and unsaturated long fatty acyl chains in various glycerophospholipid classes such as phosphatidylcholines, phosphatidylethanolamines and phosphatidates, with a preference for hydrolysis at sn-2 position (PubMed:10092647, PubMed:10336645, PubMed:20886109). Can further hydrolyze lysophospholipids carrying saturated fatty acyl chains (lysophospholipase activity) (PubMed:20886109). Upon oxidative stress, contributes to remodeling of mitochondrial phospholipids in pancreatic beta cells, in a repair mechanism to reduce oxidized lipid content (PubMed:23533611). Preferentially hydrolyzes oxidized polyunsaturated fatty acyl chains from cardiolipins, yielding monolysocardiolipins that can be reacylated with unoxidized fatty acyls to regenerate native cardiolipin species (By similarity). Hydrolyzes oxidized glycerophosphoethanolamines present in pancreatic islets, releasing oxidized polyunsaturated fatty acids such as hydroxyeicosatetraenoates (HETEs) (By similarity). Has thioesterase activity toward fatty-acyl CoA releasing CoA-SH known to facilitate fatty acid transport and beta-oxidation in mitochondria particularly in skeletal muscle (PubMed:20886109). Plays a role in regulation of membrane dynamics and homeostasis. Selectively hydrolyzes sn-2 arachidonoyl group in plasmalogen phospholipids, structural components of lipid rafts and myelin (By similarity). Regulates F-actin polymerization at the pseudopods, which is required for both speed and directionality of MCP1/CCL2-induced monocyte chemotaxis (PubMed:18208975). Targets membrane phospholipids to produce potent lipid signaling messengers. Generates lysophosphatidate (LPA, 1-acyl-glycerol-3-phosphate), which acts via G-protein receptors in various cell types (By similarity). Has phospholipase A2 activity toward platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), likely playing a role in inactivation of this potent pro-inflammatory signaling lipid (By similarity). In response to glucose, amplifies calcium influx in pancreatic beta cells to promote INS secretion (By similarity).

GENE ID	8398
SWISS PROT	O60733
SYNONYMS	PLA2G6; CaI-PLA2; GVI; INAD1; PNPLA9; Ca2+ Independent PLA2; 85 kDa Calcium-Independent Phospholipase A2; Phospholipase A2,group VI(Cytosolic,Calcium-Independent)

Materials Supplied

Kit Components	96 Wells Quantity/Size
Pre-Coated Microplate	48T: 6 strips x 8 wells; 96T: 12 strips x 8 wells
Standard (Lyophilized)	48T: 1 vial; 96T: 2 vials
Biotinylated Antibody (100×)	48T: 60 μL; 96T: 120 μL
Streptavidin-HRP (100×)	48T: 60 μL; 96T: 120 μL
Standard/Sample Diluent Buffer	48T: 10 mL; 96T: 20 mL
Biotinylated Antibody Diluent	48T: 6 mL; 96T: 12 mL
HRP Diluent	48T: 6 mL; 96T: 12 mL
Wash Buffer (25×)	48T: 10 mL; 96T: 20 mL
TMB Substrate Solution	48T: 6 mL; 96T: 10 mL
Stop Reagent	48T: 3 mL; 96T: 6 mL
Plate Covers	48T: 1 piece; 96T: 2 pieces

Storage

Storage

*If the kit is opened, Store the whole kit at 4°C. If the kit is not used up in 1 week. Store the Pre-Coated Microplate, Standard ,Biotinylated Antibody and Streptavidin-HRP at -20°C, the rest reagents at 4°C, please used up within 6 months. *If the kit is not opened, store the whole kit: 4°C(short time storage, valid for 6 months); -20°C (long-term storage, valid for 1 year). Avoid repeated freeze-thaw cycles.

Performance Characteristics

REPEATABILITY	Intra-assay Precision (Precision within an assay)：CV% < 8% Three samples of known concentration were tested twenty times on 1 plate to assess intra-assay precision. Inter-assay Precision (precision between assays)：CV% < 10% Three samples of known concentration were tested in forty separate assays to assess inter-assay precision.
SENSITIVITY	The minimum detectable dose was 30pg/mL.
ASSAY RANGE	78.13–5,000pg/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for detection of Human iPLA2. No significant cross-reactivity or interference between Human iPLA2 and analogues was observed.