A: GT1 cells exposed to 22L infected brain homogenate dilutions and assayed for%PrP-res at sequential culture passages. There is a clear discrimination over ~3 logs of infectious input (30–30,000 CE) from p3 onwards. At doses ≥ 3 × 10⁴ CE, the%PrP-res rapidly reaches its GT1 maximal level of 25%, and this maximal PrP-res is maintained in subsequent passages (exponential curve fit of the data with an R = 0.994 shown). A ten-fold input reduction to 3,000 CE converts PrP more slowly; from p1–p7 there is an excellent linear fit of the data (solid black line, y = 69429 + 1.523x; R = 0.975). These points could also fit a 2^nd order polynomial when p0 is included (R = 0.98, dotted curve), but the acute initial rise in PrP-res from p0 to p1 should be considered separately (see Discussion). A linear but slower increase in PrP-res at the next lower dose of 300 CE is also apparent. At an input 30 CE and 3 CE, lower levels of PrP-res over 7 passages are maintained, indicating these GT1 cells are stably infected. However, the%PrP-res did not increase with these lower doses, suggesting an equilibrium is maintained between dividing uninfected cells and agent replication and/or spread. The lowest CE eliciting a low but constant PrP-res is defined as the tissue culture endpoint (here 3 CE) where equally loaded mock infected lanes were used as membrane background (PrP-res bands were not detected). Each plotted point with ± SEM bars represents the average of 2–4 independent experiments (6–12 wells assayed); two points at p1 and one at p7 are averages from one experiment. B: Standard 22L titration plot used as a reference in subsequent rapid assays of 22L infected samples with unknown titers. To make this plot, multiple dilutions of brain were assayed for%PrP-res at p3 (red) and p5 (blue). One tissue culture infectious dose (TC-ID) was experimentally defined as 1% PrP-res (by p3) and 2% PrP-res (by p5). These values were observed with application of 30 CE, and the other points plotted relative to this value, e.g., 3 × 10⁴ CE will have 1,000x that TCID. There is a simple linear-log relationship between %PrP-res and infectivity from 10–2,000 TCID (R > 0.99); the slope is steeper between 2–10 TC-ID (R > 99), and unknowns in this range were confirmed by using a higher input inoculum. This GT1 assay yielded the same infectious dose per gram of 22L brain homogenate as the mouse assay (see text). Points shown with SEMs are from > 3 independent experiments and the data include two different 22L infected brains.

GT1 assays of different 22L infected N2a58 clones and passages for infectivity by Western blot and in-situ PrP-res detection. Top shows scatter plot of p3 versus p5 brain TC-ID determinations (column 1 & 2) as well as the combined brain data (column 3). The average TC-ID is shown by a line and numerically at the top of each column (± SEM). Different batches of the starting 22L infected N2a58 C1 clone (Column 4) and its C2a derivatives are shown. Some of the C2a cells developed variant phenotypes of growth and PrP expression sampled at the passages indicated (see text). Bottom shows an independent assessment of PrP-res by in-situ immunodetection. Mock infected N2a58 cells (A&C) are compared p39 and p31 C2a cells (B&D) and show representative fields at low and higher magnifications respectively. The PrP amyloid (dark red bodies) are obvious in the 22L infected C2a cells and not present in the mock controls. At the higher magnification the number of cells with large PrP amyloid aggregates are easily counted, e.g., 30 of 36 cells in panel D are positive. Thus a high proportion of the C2a cells are infected; positive cells are also an underestimate because only a 4µM section of each cell is sampled.

Western blot of PrP and PrP-res bands in a 22L brain homogenate used for infection and in the GT1 target cells exposed to this inoculum analyzed at early and later passages. The PrP-res band intensity in the infected GT1 cell lanes are considerably higher than in brain CE, even at the earliest passages (G1p1 and G2p2). These high levels are maintained for 7 passages (G2p7 panel). Moreover, at p1 and p2 post-infection the very different brain-specific PrP-res pattern is not visible in the GT1 target cells. Hence the inoculum has been effectively removed in p0, and the GT1 target cells at p1 and p2 are already producing substantial quantities of their own PrP amyloid. The PrP-res in the wells exposed to only 3 CE was not appreciable on film but did frequently show a faithful PrP-res signal of 0.3% with imaging equipment. A CE of 3 was therefore defined as the tissue culture endpoint (TC-ID₅₀) for 22L. PrP-res lanes (PK +) were loaded with 8x the PrP control, except for the duplicate 30 CE lane marked with an * in passage 7 (G2p7); this lane was loaded 19x to show the 1.1% signal on film.