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Neutralisation Test
Karber Method
Cytopathic Effect in
Tissue Culture
2D Neutralisation Test-Plate Layout
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The two-dimensional neutralisation test is used to characterise
the relationship between field isolates of a particular serotype and a
vaccine strain known to confer protection against a broad spectrum of
subtypes of that serotype (Rweyemamu
et al., 1978). This is essentially used to predict the most effective
vaccine strain to utilise in the event of an outbreak of FMD.
This test utilises a reference serum raised against a vaccine strain. The titre of this reference serum against 100 TCID50 of the homologous (vaccine) strain is known. The titre against the same dose of a heterologous (field) strain is then determined to indicate how antigenically 'similar' the heterologous virus is to the vaccine (homologous) virus.
Outline method
The basic principles, procedures, equipment and reagents are similar to those of the neutralisation test.
Additional biological reagents are:
21-day post-vaccinal (bovine) reference sera (inactivated at 56ºC for 45-60 min)
Reference virus homologous for the reference serum
Test virus - current outbreak strains of the same serotype as the reference serum and virus
1. Test field strains are passaged on IBRS-2 cells until adapted to give 100% CPE (Cytopathic Effect in Tissue Culture) in 24 h. Passages should be kept to a minimum. When adapted, determine the virus log10 titre by end-point titration.
2. Prepare microtitre plates according to the two-dimensional neutralisation test plate layout; one plate for each test virus neutralisation (VN), one for each test virus titration (VT), and similar for the reference virus and its titration plate.
3. In the VN plate add 100 µl of medium to each well in column 1, and 50 µl to each well in columns 2-12.
4. Add 50 µl of reference sera to wells 2-11 in row A, making an initial serum dilution of 1/2.
5. Dilute this sera two-fold (0.3 log) down the plate (A-H).
6. The weakest of five half-log dilution steps of virus should contain at least ten TCID50, (for example, if the initial titre is 105.5, the half log dilution steps of virus used in the test should be in the region of 102.5 to 104.5, which ensures that the theoretical 2 log dose (103.5 ) is midway between and therefore lies in the middle of the plate). Add 50 µl of each dilution of test virus (starting with the weakest) to the wells in each pair of columns; i.e 104.5 in columns 10 and 11, 104.0 in columns 8 and 9, 103.5 in columns 7 and 6 and so on. The 104.5 dilution is also added as a virus control in column 12.
7. Stack the plates, cover the top one(s) and incubate at either 37ºC for 30 min or one hour at ambient temperature.
8. In the VT plate add 50 µl of medium to each well in columns 2-12. Leave column 1 empty.
9. Add 100 µl of the chosen virus dilution (eg 103.0) to each well in column 1 (this is taken from the 103.0 dilution stock used in the VN test plate) and dilute two-fold (0.3 log) across the plate (2-11).
10. Add 50 µl of medium to all wells, and incubate with the VN test plates.
After incubation, add 50 µl of IBRS-2 cell suspension, at a seeding rate of 1 x 106 cells per ml, to all wells in all plates. Seal each plate with thin plastic backing tabs and incubate at 37ºC for 48-72 h.
Stain each plate with naphthalene black and read.
Antibody titres of the reference serum against the homologous (reference) and heterologous (field) viruses for each virus dose used are calculated using the Karber method. The titre of the reference serum against 100 TCID50 of each virus can then be estimated by regression. The relationship between the field strain and the reference strain is then expressed as an 'r' value as described for the strain characterisation ELISA.
Intrepretation of strain characterisation data is complex and should only be performed by someone with relevant experience. In the case of neutralisation, r values greater than 0.3 indicate that the field strain is sufficiently similar to the vaccine strain that use of the vaccine is likely to confer protection against challenge with the field strain. Conversely, values less than 0.3 suggest that the field strain is so different from the vaccine strain that the vaccine is unlikely to protect. In these cases, either the field strain should be examined against alternative vaccine strains or, rarely, it will be necessary to adapt a suitable field strain to become a new vaccine strain.
Tests should always be repeated more than once. The confidence with which
'r' values can be taken to indicate differences between strains is related
to the number of times that the examination is repeated. In practice,
a minimum of at least three repetitions is advised.
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