Interference may prevent binding of the antibody of interest
High titer antibody leading to complement activation and deposition of C1 complex on the bead, IgM antibody, or other serum factors may interfere with binding of the secondary detection reagent, giving false-negative results, and is known as the prozone effect. Serum dilution or treatment with EDTA/dithiothreitol, osmosis, or heat has been shown to reduce this effect but is often variably applied. Interference may also be caused by drugs (eg IVIG) or non specific serum proteins. Dilution does not result in a predictable decline in bead mean fluorescence intensity; therefore, the mean fluorescence intensity of a single bead in an undiluted serum cannot be assumed to quantitatively or reliably represent antibody amount.