Benchmarking Study

Mirrorball^® is ideally suited to the analysis of mix-and-read assays for antibody discovery research. The adoption of such assays for applications such as hybridoma screening was first enabled by the ABI 8200 Cellular Detection System from Applied Biosystems (now discontinued). This system is based on Fluorometric Microvolume Assay Technology (FMAT^®) resulting in the instrument being commonly referred to as an ‘FMAT’.

We have used an IgG quantification assay to compare the performance of Mirrorball and FMAT in a mix-and-read, bead-based format. The schematic of the protocol is presented in Figure 1.

Assays were performed in 384-well microplates and done in duplicate plates to eliminate possible effects of scanning. Both bead fluorescence and count were evaluated on each instrument.

The results are shown in Figure 2 (click on each graph to enlarge).

Figure 2. Quantification of rabbit IgG using a bead-based assay.

The results demonstrate that fluorescence quantitation is similar on both the Mirrorball and FMAT® instruments. However, unlike Mirrorball, FMAT could not detect all beads using fluorescence, as demonstrated by the significant reduction in bead count at levels less than 1 ng/mL rabbit IgG. This indicates that FMAT has a reduced dynamic range relative to Mirrorball and reflects the reliance of the FMAT instrument on bead fluorescence for object recognition [owing to the lack of a scatter channel].

 Figure 1.