LCAT ELISA Kit (Rat)

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

1-alkyl-2-acetylglycerophosphocholine esterase; Lecithin-cholesterol acyltransferase; PAF acetylhydrolase; phosphatidylcholine-sterol acyltransferase; phospholipid-cholesterol acyltransferase; platelet-activating factor acetylhydrolase.

Central enzyme in the extracellular metabolism of plasma lipoproteins. Synthesized mainly in the liver and secreted into plasma where it converts cholesterol and phosphatidylcholines (lecithins) to cholesteryl esters and lysophosphatidylcholines on the surface of high and low density lipoproteins (HDLs and LDLs) . The cholesterol ester is then transported back to the liver. Also produced in the brain by primary astrocytes, and esterifies free cholesterol on nascent APOE-containing lipoproteins secreted from glia and influences cerebral spinal fluid (CSF) APOE- and APOA1 levels. Together with APOE and the cholesterol transporter ABCA1, plays a key role in the maturation of glial-derived, nascent lipoproteins. Required for remodeling high-density lipoprotein particles into their spherical forms (By similarity) . Has a preference for plasma 16:0-18:2 or 18:O-18:2 phosphatidylcholines (PubMed:8820107, PubMed:14636062) . Catalyzes the hydrolysis of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor or PAF) to 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) (By similarity) . Also catalyzes the transfer of the acetate group from PAF to 1-hexadecanoyl-sn-glycero-3-phosphocholine forming lyso-PAF (By similarity) . Catalyzes the esterification of (24S) -hydroxycholesterol (24 (S) OH-C), also known as cerebrosterol to produce 24 (S) OH-C monoesters (By similarity) .

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 Lecithin Cholesterol Acyltransferase (LCAT) . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Lecithin Cholesterol Acyltransferase (LCAT) . 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 Lecithin Cholesterol Acyltransferase (LCAT), 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 Lecithin Cholesterol Acyltransferase (LCAT) 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) 90-102 97 EDTA Plasma (n=5) 78-96 90 Heparin Plasma (n=5) 94-102 99 Matrix Recovery range (%) Average (%) Matrix Recovery range (%) Average (%) Serum (n=5) 90-102 97 Serum (n=5) 90-102 97 EDTA Plasma (n=5) 78-96 90 EDTA Plasma (n=5) 78-96 90 Heparin Plasma (n=5) 94-102 99 Heparin Plasma (n=5) 94-102 99

Component Amount Anti-LCAT Microplate 96 Wells (12 x 8 Well strips) LCAT Lyophilized Standard 2 100X Biotinylated LCAT Detector Antibody 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 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 Stop Solution 1 x 6 mL TMB Substrate 1 x 9 mL Component Amount Component Amount Anti-LCAT Microplate 96 Wells (12 x 8 Well strips) Anti-LCAT Microplate 96 Wells (12 x 8 Well strips) LCAT Lyophilized Standard 2 LCAT Lyophilized Standard 2 100X Biotinylated LCAT Detector Antibody 1 x 120 uL 100X Biotinylated LCAT Detector Antibody 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 120 uL 100X Avidin-HRP Conjugate 1 x 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 Stop Solution 1 x 6 mL Stop Solution 1 x 6 mL TMB Substrate 1 x 9 mL TMB Substrate 1 x 9 mL

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