A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions

Abstract

Prions are usually quantified by bioassays based on intracerebral inoculation of mice that are slow, imprecise, and costly. We have isolated neuroblastoma N2a sublines highly susceptible to mouse prions, as evidenced by accumulation of infectivity and the scrapie form of prion protein (PrPSc), and developed quantitative in vitro assays for prion infectivity. In the scrapie cell (SC) assay, susceptible N2a cells are exposed to prion-containing samples for 3 days, grown to confluence, and split 1:10 three times, and the proportion of PrPSc-containing cells is determined with automated counting equipment. In a log/log plot, the dose-response is linear over two logs of prion concentrations. The SC assay is about as sensitive as the mouse bioassay, 10 times faster, >2 orders of magnitude less expensive, and suitable for robotization. SC assays performed in a more time-consuming end point titration format extend the sensitivity and show that infectivity titers measured in tissue culture and in the mouse are similar.

Fig. 1.

Dose–response relationship between the dilution of infected RML homogenate and the number of PrP^Sc-positive colonies determined by the colony assay. Twenty thousand susceptible N2aPD88 cells (a) and nonsusceptible N2aR33 cells (b) were exposed to the indicated dilutions of RML strain (I2424, see Table 1) in OFCS. After 3 days, the cells were split 1:10 and grown for 3 days. After two more splits, 10⁵, 10⁴, or 10³ cells were mixed with noninfected N2aPD88 cells for a total of 10⁵ cells and plated on coverslips. After 4 days, PrP^Sc-positive colonies were identified as described in Materials and Methods. Where countable, the number of positive colonies is indicated at the upper right of the blot. (c) Double-logarithmic plot of RML strain dilution versus number of PrP^Sc-positive colonies. The countable positive colonies were recalculated per 10,000 plated cells.

Fig. 3.

Dose- and time-dependent response of susceptible and nonsusceptible N2a cells to various dilutions of RML homogenate in the SC assay. (a) Representative wells of an ELISPOT plate showing spots given by N2aPD88 cells exposed to the indicated dilutions of RML strain or to medium (control), as described in experiment 1. (b) Double-logarithmic plot of spot number versus RML strain dilution. Experiment 1 (filled symbols): 20,000 susceptible cells (N2aPD88, circles) and nonsusceptible cells (N2aR33, squares; N2aATCC, triangles) were incubated for 3 days with serial 1:10 dilutions of RML strain (I2424) in OFCS, as indicated. The cells were split three times 1:10, aliquots of 25,000 cells were filtered onto membranes of an ELISPOT plate, and PrP^Sc-positive cells were determined by the SC assay. Mean values of quadruplicate measurements ± SD are shown. Background values of noninfected cells (19 ± 4 for N2aPD88, 5 ± 1 for N2aR33, and 9 ± 2 for N2aATCC) were subtracted. N2aPD88 cells are ≈3 logs more susceptible than the nonsusceptible cells. Experiment 2 (open symbols): Serial 1:3.3 dilutions of RML strain (I2424, 10^–1) into uninfected CD1 brain homogenate (10^–1) were diluted 1:1,000 in OFCS, to give a final dilution of total brain homogenate of 10^–4 and the dilution of RML strain indicated. Twenty thousand susceptible (N2aPD88, circles) and nonsusceptible cells (N2aR33, squares) were exposed to the samples and processed as in experiment 1. Mean values of six measurements ± SD are shown. Background values of cells incubated with noninfected CD1 homogenate (16 ± 7 for N2aPD88 and 6 ± 6 for N2aR33) were subtracted. In the double-logarithmic plot the relationship between spot number and RML strain dilution is linear from 10^–7 to 10^–5 (correlation coefficient r = 0.94). The values for the dilutions 10^–7 and 3.3 × 10^–7 are significantly over background (P = 0.006 and P = 0.0007, respectively; Mann–Whitney U test) but not that for 3.3 × 10^–8.(c) Time-dependent increase in the proportion of infected cells. PD88 cells were exposed to a 10^–5 (•) or 10^–6 (○) dilution of RML strain (I2424) in OFCS for 3 days and split 1:10 every third day. The spot number per 25,000 cells was determined at the times indicated. Mean values of triplicate measurements and SD are shown. Spot numbers were corrected for background values of noninfected cells (16 ± 7). The rate of accrual is initially ≈20–30% per day but levels off as the saturation value of detectable infected cells is approached (≈2,000 per 25,000 cells).

Fig. 2.

Clearance of PrP^Sc particles from the initial inoculum after three successive 1:10 splits of infected cells. Twenty thousand susceptible N2aPD88 cells (gray bars) and nonsusceptible NN2a cells (white bars) were cultured for 16 h, plated in a 96-well plate, and exposed for 3 days to a 10^–4 dilution of RML strain (I2424) in OFCS, in the absence (–) or presence (+) of the anti-PrP mAb ICSM 18 (5 μg/ml, added 4 h before infection), as indicated. As controls, N2aPD88 and NN2a cells were incubated with OFCS only. Three days after infection, the cells were suspended, octaplicate aliquots of 25,000 cells were filtered onto the membranes of an ELISPOT plate, and the PrP^Sc-positive signals were quantified by the SC assay. Both susceptible and nonsusceptible cells gave rise to PrP^Sc-positive signals. An anti-PrP antibody that abrogates PrP^Sc accumulation did not diminish the spot number, demonstrating that all spots can be accounted for by PrP^Sc particles from the inoculum. After three 1:10 splits, nonsusceptible and susceptible cells incubated with antibody showed <2% of the spots given by susceptible cells. The numbers for noninfected N2aPD88 cells and NN2a cells, the fifth and sixth bar of each group, respectively, represent the background. Mean values and SD are shown.

Fig. 4.

Serial 1:10 dilutions of RML strain (I2424, 10%) into uninfected brain homogenate (CD1, 10%) were prepared. Twenty thousand susceptible cells (subclone N2aPK1) were incubated for 3 days with 300 μlof10^–7 (a) and 10^–8 (b) dilution of RML strain in OFCS. The cells were split 3 times 1:10 (12 days, white bars), 5 times (18 days, light gray bars), 8 times (27 days, gray bars), and 12 times (39 days, black bars); 25,000 cells were subjected to the SC assay. Mean ± SD of background values of noninfected cells were 11 ± 6, 16 ± 13, 14 ± 5, and 9 ± 2 at 12, 18, 27, and 39 days, respectively. After 39 days of culture, wells with spot numbers exceeding the mean value of the background by 5 SDs were scored as positive. The dilution leading to 50% positive wells was 10^–7.5, as calculated by following ref. . The brain therefore contained 10^7.5/(0.3) = 10⁸ tissue culture LD₅₀ units per g.