Revvity, 64APAPEG, HTRF Human ApoA1 Detection Kit, 500 Assay Points

Apolipoproteins are proteins that bind to lipids to form lipoproteins, whose main function is to transport lipids. Apolipoproteins are important in maintaining the structural integrity and solubility of lipoproteins, and play an important role in lipoprotein receptor recognition and regulation of certain enzymes in lipoprotein metabolism. ApoA1 is the major protein component of high-density lipoprotein (HDL). Deficiency of ApoA1 is associated with HDL deficiencies, including systemic non-neuropathic amyloidosis. ApoA1 may also have a role in protection against Alzheimer's disease. HTRF assays offer many advantages over other technologies: Homogeneous add-and-read format No wash steps Low background Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates Stable signal, providing flexibility in time of readout or size of assays
Assay principle The Apolipoprotein A1 assay is a sandwich immunoassay involving two monoclonal antibodies, one labelled with Eu-Cryptate (Donor) and the second with d2 (Acceptor). The intensity of the signal is proportional to the concentration of apolipoprotein present in the sample. Assay protocol The ApoA1 assay features a streamlined protocol with only one incubation step following the sample/calibrator and Antibody conjugates dispensing. The antibodies labelled with HTRF donor and acceptor may be pre-mixed and added in a single dispensing step to further streamline the assay procedure. This protocol requires a single 3-hour incubation period at RT.
ApoA1 detection upon retinoid X receptor (RXR) agonist treatment Human hepatoma cells (HepG2) in 80-90% confluence were grown in DMEM (Gibco; 11885, low glucose) supplemented with 2% FBS. 5 µL of HepG2 cells (2,000 & 5,000 per well) and 5 µL of the RXR ligand 9-cis retinoic acid (9-cis-RA) in DMEM, 2% FBS at various concentrations were dispensed into a 384-well low volume black culture plate. After a 48 hour incubation at 37°C in CO2 atmosphere, 10 µL of the two pre-mixed detection reagents were added.