Mirrorball enables a mix-and-read, bead-based assay for screening antigens. One application of this is in the screening for antibodies raised against soluble antigens. To demonstrate this capability a bead-based sandwich immunoassay for rabbit IgG was established.
Beads coated with antirabbit Fc antibody were mixed with a range of rabbit IgG concentrations and Cy5-labeled anti-rabbit F(ab´)2 antibody. After a period of incubation, plates were scanned using Mirrorball and bead-bound fluorescence quantified. In addition, beads were identified using laser scatter.
High sensitivity detection of rabbit IgG was achieved with a limit of detection of <1 ng/mL rabbit IgG (Figure 2). A reduction in bead fluorescence was observed at concentrations above 150 ng/mL due to the well-documented “hook effect”. The variability of assay replicates was low (4% CV) suggesting stable measurement of bead fluorescence despite a solution background of 0.67 nM AlexaFluor® 647 antibody conjugate.
Independent recognition of beads using label-free scatter detection permitted detection of beads in all wells, including zero control wells. This capability improves detection sensitivity and eliminates false negatives from wells containing reduced bead number.