Division 2

The VNT is based on the determination of the virus neutralising activity of the antibodies of the serum sample, expressed as neutralising 50 % end point.

A constant amount of classical swine fever virus is incubated at 37 °C with diluted serum. For screening purposes, the sera are initially diluted 1/10. When a full titration is necessary two-fold dilutions of serum starting at 1/2 or 1/5 can be prepared. Each dilution is mixed with an equal volume of a virus suspension containing 100 infectious doses (TCID 50).

After incubation the mixture is inoculated onto cell cultures which are incubated for three to five days. After this incubation period the cultures are fixed and any viral replication in the infected cells is detected by an immune labelling system. Either the neutralisation peroxidase-linked antibody (NPLA) or the neutralisation-immunofluorescence (NIF) assays may be used.

The results of the VNT are expressed as the reciprocal of the initial serum dilution at which half the inoculated cell cultures (50 % end point) fail to show viral replication (no specific labelling). A point between two dilution levels is estimated. The final dilution system is based on the actual dilution of serum during the neutralisation reaction, i.e. after addition of virus, but before adding the cell suspension.