Next, stop reagent was added and the in TKI-258 tensity of color that develops was measured at 492 nm by a microplate reader. A standard curve of absorbance values of known CETP standards was plotted as a function of the logarithm of CETP standard concentrations using the GraphPad Prism Software program for windows version 5,03. CETP concentrations in the samples were calculated from their corresponding absorb ance values via the standard curve. Serum CETP activity was measured using an assay kit fol lowing the manufacturers instruction. Briefly, 3 ul of plasma sample was added to the reaction mixture containing a fluorescent self quenched neutral lipid as the donor molecule and an acceptor molecule.
A CETP mediated transfer of the fluorescent neutral Inhibitors,Modulators,Libraries lipid to the ac ceptor molecule resulted in an increase in fluorescence, which was read in a fluorescence plate reader at excitation 465 nm and emission 535 nm. A standard curve was pre pared by using known concentrations of fluorescent neutral lipid standards. CETP activity in the samples was calculated from the corresponding fluores cence values via the standard curve and was expressed as pmolul samplehr. LCAT protein and activity measurement Serum LCAT concentrations were analyzed by a commer cial ELISA test kit according to the manufacturers instructions. Test wells were coated with anti LCAT monoclonal antibody, which binds with LCAT in the sample. After the first incu bation and washes to remove all of the unbound material, HRP labeled anti LCAT was added.
After the second in cubation and subsequent washes, the antibody LCAT enzyme complex was incubated with a substrate solution and terminated with a stop reagent. The intensity of color that develops was measured at 492 nm by a microplate reader. A standard curve of absorbance values of known LCAT standards was plotted as a function of the logarithm Inhibitors,Modulators,Libraries of LCAT standard concentrations using the GraphPad Prism Software program for windows version 5,03. Inhibitors,Modulators,Libraries LCAT concentrations in the samples were calculated from their corresponding ab sorbance values via the standard curve. Plasma LCAT activity was assayed using the Calbiochem Fluorometric LCAT assay kit according to the manufacturers instructions. This assay is based on the hydrolysis of an artificial LCAT substrate that fluoresces at 470 nm, resulting Inhibitors,Modulators,Libraries in a product that fluoresces Inhibitors,Modulators,Libraries at 390 nm.
Aliquotes of 3 ul of plasma were mixed with 1 ul of fluorescent LCAT substrate and 200 ul of LCAT assay Buffer, followed by incubation for 5 hr at nm fluorescence emission intensities. Apolipoprotein A1 and B measurements Serum apoAI levels were determined with the Human ApoAI ELISA kit according to the manufacturers instruc tions. Samples were added to ELISA strip plates selleck compound precoated with apoA1 monoclonal antibody. Captured apoA1 in the samples were detected by adding a biotinylated mAb followed by streptavidin HRP.