Graphical illustration of deriving the NT-PCR neutralizing titer in a normal analytical setting. Instead of performing statistical analysis of multiple runs (as in figure 2) the point at which the slope of the curve shows a sudden decrease to a near-zero value identifies the point of significant increase of virus replication caused by incomplete virus neutralization (if antibody dilution is plotted against percent virus replication) and defines the neutralizing antibody titer. Mathematically, this point can be derived by treating the curve as a composition of two near-linear fragments (colored curves). The intersection (arrows in enlarged insert), which results in the best linear fit (R2) on both modeled linear segments (= highest R2a+R2b) defines the NT-PCR titer. Numbers indicate curves derived from different number of replicates per run: #1 and #2: mean values of two wells; #3: mean value of three wells; #4: mean value of four wells analyzed per dilution step, respectively. Independent of the number of replicates analyzed the resulting neutralization titer was always the same, suggesting that duplicate measurements should suffice for normal clinical or analytical application of the NT-PCR assay.