Figure 1.

MFI of single antigen bead assays has analytic limitations and cannot be used as a quantitative metric of antibody amount. (A) An ideal test should always be able to distinguish antibody binding (green fluorescent signal) from negative control (white) with a clear threshold and no overlap between the MFI distributions. (B) Decreased density of antigen (Ag) on the surface of the bead will result in MFI measurement that underestimates the amount of the antibody present. (C) In contrast, nonspecific binding to the bead can result in artificially high background and signal MFI, with overestimation of antibody. (D) Interfering substances may prevent the detection of the antibody of interest with lower MFI. (E) Epitopes shared between different beads can dilute the amount of antibody bound to any single bead, with an erroneously low MFI on the given bead of interest.