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Antigen Titration
FMD Strain Characterisation
ELISA
Karber Titres -Liquid Phase
ELISA
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Outline method for FMD virus characterisation by ELISA
1. Grow the field strain either in BHK or IB-RS2 cells.
2. Harvest and titrate the virus (Antigen Titration) using a panel of antisera raised against vaccine strains of the same serotype. These are selected according to geographical and epidemiological criteria.
3. Select the optimum trapper (coating)/detector combination and the working dilution of the field strain. This should not be less than 1:6.
4. Titrate 21-day post-vaccinal serum of a chosen vaccine strain against the field strain and the homologous vaccine strain. The homologous titre, which should not fluctuate more than two-fold either side of the mean value, and a control titration of reference serum and virus, are included on every test plate. (FMD Strain Characterisation ELISA)
5. To determine the serum titre, calculate the average optical density (OD) of 24 antigen control wells. This represents the maximum OD value for the test, i.e., the 100% control value. Divide this by 2 to determine 50% inhibition value. Score wells positive if the OD is less than or equal to 50% and negative if the OD value is greater than this.
The end-point is defined as the dilution at which half of the wells show 50% inhibition or more (i.e., identify the dilution at which one out of the two duplicate wells has an OD less than 50% of the antigen control). If the end-point falls between two dilutions, it is taken as the mid-point between these dilutions, as estimated by the Karber method. (Karber Titres for Liquid Phase ELISA)
6. Derive an 'r' value, the relationship between a field and a vaccine strain, as:
r = titre of reference serum against field virus
titre of reference serum against reference virus
At least two consistent results are needed for acceptance.
7. Interpretation
For r values derived by ELISA the following guidelines are used for interpretation:
(Kitching et al., 1988)
r value Interpretation
0.4-1.0 Close relationship between field and
vaccine strain. A potent vaccine
containing the vaccine strain is likely to
confer protection.
0.2-0.39 The field strain is antigenically related to
the vaccine strain. The vaccine strain
might be suitable for use if no closer match
can be found provided:
- a potent vaccine is used
- animals are preferably immunised
more than once.
<0.2 The field strain is only distantly related to
the vaccine strain and the vaccine strain
is unlikely to protect against challenge with
the field strain.
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