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Samples are taken from the vessels during the inactivation period and
the residual infectivity titre measured by tissue culture techniques.
By plotting this titre logarithmically against time it is possible to
extrapolate and predict the titre at the end of the inactivation period.
The European Pharmacopoeia requirement is that the slope of the inactivation
line is linear and the predicted titre should be less than 1 infectious
virus particle per 10 000 litres of virus harvest.
Following inactivation, a further test is done in which BHK-21 cell monolayers or other susceptible cells are inoculated with antigen harvest representing at least 200 doses equivalent of vaccine and incubated for several days during which they are examined regularly for the presence of virus induced cytopathic effects (CPE). After this time, the old medium is removed and used to inoculate fresh cells and the old cells replenished with fresh medium. Both the old cells and the fresh cells are examined regularly. This process is repeated with a third ‘passage’. Any evidence of CPE attributable to virus is taken to be a failure of the test and the antigen batch is rejected. In another version of the test, the cell monolayers are freeze-thawed at the end of each cycle so as to release any intracellular virus.
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