SDS–PAGE and Western blotting
PrPSc detection was run on the filtrate from nanofilters by Western blot analysis. 12% NuPAGE BisTris (Invitrogen) gels were used
for electrophoresis, which was performed at 180 V for 1 h in 1 x MES running buffer. Wet blotting was performed at 110 V,
250–500 mA for 1.5 h in 1 x transfer buffer. Subsequently, the membrane was blocked in blocking buffer (TBS–T containing 5%
Topblock, Juro) for at least 1 h at room temperature and then incubated overnight with the PrP-specific monoclonal mouse antibody
POM1 (1:3000 dilution) at 2–8°C. The membrane was washed three times for 10 mineach with fresh TBS–T buffer and then incubated
for 30 min with the secondary rabbit antimouse IgG1 (Zymed) conjugated with HRP (1:10,000 diluted) at room temperature. Finally,
the membrane was again washed three times for at least 10 min each, and the enzymatic reaction initiated by the addition of
Enhanced Chemiluminescence (ECL) Western blotting substrate (Pierce).
Tissue culture infectivity assay
For the infectivity-level detection of filtrate samples, the scrapie cell endpoint assay (SCEPA) was used (25–27). Cells growing
in 96-well plates are exposed to serial dilutions of input and filtrate samples from experiments 1, 2, and 3, and propagated
for several successive cell splits. Dilutions were performed into OFCS so that the final dilution was not less than the equivalent
of 10–4 brain homogenate. Dilutions were done so that the total concentration of brain was 10–4 in each inoculum. Samples were kept at 4 °C until cells were infected.
Wells that received only a single infectious unit become extensively infected, as evidenced by the accumulation of PrPSc in many cells in the well. At an appropriate dilution, only a fraction of the wells receive an infectious unit and score
positive, and the infectious titer of the starting material can be determined by the Poisson distribution and reference to
standard curves.
Cells used for titration of infectivity
A highly prion-susceptible subclone of mouse neuroblastoma cells (N2A) was used to titrate infectivity. This clone was derived
from the original N2A American Type Culture Collection (ATCC) cell line by serial isolation of highly prion-susceptible subclones.
Cells were stored in liquid nitrogen, and fresh vials were thawed for each titration assay.
Culturing of cells
Highly susceptible N2A cells were cultured in Optimem-I medium (Invitrogen) containing 10% fetal calf serum (Perbio or Invitrogen)
and 1000 units/mL penicillin G sodium and 100 μg/mL streptomycin G (OFCS). Cells were split at least every three days or when
confluent at 1:3 to 1:5 dilutions. The medium was exchanged every second day. Cells were grown in an atmosphere of 5% CO2 and saturated humidity at 37°C in a cell-culture incubator.
Preparation of cells for SCEPA
Four days before starting the SCEPA assay, a frozen aliquot (about 3 x 106 cells in 0.5 mL freezing medium) was thawed, centrifuged, and suspended in 10 mL OFCS. Cell suspension was seeded in a 10
cm Petri dish and grown to confluence (3–5 days; change medium after 3 days). At confluence, there were about 6 x 106 cells, enough for 3 assay plates.
Infection of cells with prion samples
Twenty thousand susceptible cells in 200 μL OFCS were plated into a 96-well plate. After 16 h at 37°C, the medium was replaced
by a 0.3-mL assay sample in OFCS. After 3 days, the confluent monolayer (about 105 cells) was gently suspended. A 100-μL aliquot of each sample was transferred into a well of a 96-well plate containing 200-μL
OFCS and grown to confluence. The cells were split 1:3 three more times as above and subsequently 4 times 1:10 (for experiments
1 and 2) and 5 times for experiment 3 and the FSH interference assay. Then the cells were grown to confluence.
After they reached confluence, 25,000 cells from each well were filtered onto the membrane of an ELISPOT plate, treated with
PK (0.5 mg/mL for 90 min at 37 °C), and denatured. Individual infected (PrPSc -positive) cells were detected by immunocytochemistry using alkaline phosphatase–conjugated POM1 mouse anti-PrP. Wells clearly
showing positive spots were identified using light microscopy and compared to mock controls.
Calculation of prion titer
From the proportion of negative to total wells, the number of infectious tissue culture units (TCI) per mL input was calculated
by the Poisson equation P(o)= e
–m; where P(o) is the probability that a well is not infected; m is the number of infectious particles/aliquot inoculum to which the
cells are exposed. Therefore lnP(o)= ln [empty wells/ total wells] = –m and m = ln [total wells/empty wells]. To calculate the number of TCI units/mL in the undiluted original inoculum, the TCI units/aliquot
were multiplied with the dilution factor. TCI units are different from LD50 units: one LD50 unit is the dose that kills half of the animals (or infects half of the wells), whereas the TCI unit is the dose that contains
on average one infectious particle. Usually the TCI is similar to the LD50.
|
