The Apolipoprotein B 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.

The HTRF ApoB standard curve is generated with Diluent 5, provided in the kit.

Intra-assay (n=24)

Each of the 3 samples was measured 24 times, and the % CV was calculated for each sample.

Samples were cell culture supernatants from hepG2 cells.

Inter-assay (n=3)

Each of the samples was measured in 3 independent experiments (3 days), and the % CV was calculated for each sample. 

Samples were cell culture supernatants from HepG2 cells.

The excellent % of recovery obtained from these experiments shows the good dilution linearity of the assay. Samples were serially diluted HepG2 cell supernatants.

Cross reactivities were assessed using recombinant proteins from the Apolipoprotein family. Proteins were tested up to 4,000 ng/mL, and standard curves were generated for each. Signals were plotted on the assay standard curve to interpolate concentrations. Cross-reactivity was calculated as a mean of 6 tested concentrations from 10 to 4,000 ng/mL. The assay is ApoB specific, as other apolipoproteins were not detected using it.

The human cell line HepG2 was used for an ApoB secretion study, using insulin as an inhibitor. HepG2 cells were cultured in 10% FCS supplemented RPMI1640 medium at 37°C, 5% CO2. 50 µL of HepG2 cells in suspension were transferred into a 96-well cell-culture plate at densities from 100 to 25, 000 cells/well. The cells were stimulated for 24h with 100nM of insulin in a final volume of 100 µL.  5 µL of supernatant were then transferred into a white detection plate (384 low volume), and 5µL of diluent were added on top  of the sample. Then 10 µL of the HTRF Human ApoB detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

As expected, insulin treatment decreased the release of secreted ApoB. The insulin effect measured on ApoB secretion was lower using the highest cell density tested (i.e. 100,000 cells/well), indicating that 50 to 25,000 cells/well densities are more appropriate for measuring the secretion of apolipoprotein with this cell model.