Determination of virus titer by endpoint titration
To determine the virus titer of a sample, serial threefold dilutions were made with cellculture medium. 100 μl aliquots
of each dilution were added to 8 wells of a 96wellMTP with cells (in 100 μl cell culture medium per well). The cells were
cultivated for a specified incubation period. Then, they were inspected microscopically for virusinduced changes in cell
morphology.
LargeVolume Plating (LVP)
The detection limit of a sample depends on its volume incubated with the indicator cells. To improve the detection limit,
a large volume of the sample was analyzed (LVP). Briefly, 200 μl of the minimal diluted sample was added to a defined number
of wells containing the indicator cells in 100 μl cell culture medium. The cells were cultivated for a specified incubation
period. Then, they were inspected microscopically for virusinduced changes in cell morphology.
For LVP determination. If virusinduced changes are observed in some wells of the large volume plating (1550% of all wells) for a sample, the virus
titer is calculated according to the Spearman and Kärber formula. It is assumed that a 1:3 higher concentrated dose compared
to the highest dose analyzed leads to an infection of all parallel cultures.
Only two reaction rates (i.e., number of virus positive wells divided through the number of wells tested per dilution) are
reported: the reaction rate determined by the LVP and the reaction rate of the virtual 1:3 higher concentrated dose (mode
C of calculation).
If virusinduced changes are observed in only a few wells of the LVP (< 15 % of all wells) for a sample, the virus titer is
calculated according to the following formula (mode D of calculation):
D: predilution factor of the sample
n_{p}: number of viruspositive wells
n: number of all wells tested
V_{w}: sample volume per well (0.2 mL)
If no virusinduced changes are observed for a sample, the virus titer is determined by the Poisson distribution at the 95%
confidence limits derived from "Note for guidance on quality of biotechnological products" (5, 6) (mode E of calculation):
p: 0.05
v: tested sample volume in mL
V: process fraction volume in mL
For endpoint titration. The virus titer (TCID_{50}/mL), which causes a positive result in 50% of the tested cultures (TCID_{50}) of an endpoint titration, is calculated according to the method of Spearman and Kärber (mode A of calculation):
Y_{0}: decadic logarithm of highest dilution factor of the sample, which causes the infection of all parallel cultures (= 8 parallel
cultures for mode A and B of calculation)
d: decadic logarithm of dilution step (=log_{10} 3)
P_{i}: observed reaction rate starting at Y_{0}. Observed reaction rate (number of virus positive wells divided through the number of wells tested) per dilution i starting
at Y_{0}
v: decadic logarithm of volume conversion factor (= log10(5) for mode A and B, log_{10} (3 1/3) for mode C of calculation)
Calculation of the standard error:
s_{e}: standard error
P_{i}: observed reaction rate (number of virus positive wells divided through the number of wells tested, per dilution)
n_{i}: number of determinations
d: decadic logarithm of dilution step (=log_{10} 3)
Calculation of the confidence limits:
c: confidence limits
s_{e}: standard error
If the highest dose tested in the endpoint titration does not result in the infection of parallel cultures, the virus titer
for this sample is calculated according to the Spearman and Kärber formula. It is assumed that a 1:3 higher concentrated dose,
compared to the highest dose analyzed, leads to an infection of all parallel cultures (mode B of calculation).
Determination of the reduction
For virus filtration:
Reduction factor of hold:
R: reduction factor
A_{0}: total virus load of load, prefiltered
A_{n}: total virus load of hold, prefiltered
MuLV, PRV, Reo3
