Page 345 - The Veterinary Laboratory and Field Manual 3rd Edition

314 Susan C. Cork, M. Faizal Abdul Careem and M. Sarjoon Abdul-Cader

• For the negative control, add 0.5 ml phenol may be higher in tests where there is likely to be
saline and 0.5 ml antigen into tube No. 9. a lot of cross reaction, that is, in tests with low
• For the positive control, add antibody (known specificity. However, raising the ‘cut off’ titre can
positive) in 0.5 ml phenol saline and 0.5 ml reduce the sensitivity of the test. For tests which
antigen into tube No. 10. used commercially prepared reagents published
• Incubate the tubes at 37°C in a water bath guidelines on ‘cut off’ titres are available. For
for 24 h. tests using ‘in house’ reagents, the principles
• Read the results. of test development and proper test validation
are outlined in the OIE Manual for Vaccines
Diagnostic Tests and for Terrestrial Animals(see
Results
reference list).
++++: 100% agglutination with clear
upper layer and sedimented aggregate
Immunodiffusion tests
+++: 75% agglutination with 25% turbid
upper layer
When soluble antigen is mixed with specific
++: 50% agglutination with 50% turbid antibody in solution in optimum proportions
upper layer an immune complex (precipitate) is formed
and the reactions can be visualized in an agar
+: 25% agglutination with 75% turbid
upper layer gel. This is the basis for the agar gel immuno-
diffusion test (Figures 6.8a and b) and is used
-: no agglutination. frequently in regional laboratories. The test is
simple to carry out and is fairly sensitive (and
The highest dilution showing 50% agglutination specific) as long as the reagent and the control
is usually considered to be the ‘titre’. serum are not the same. The immunodiffusion
test can be used to measure reactants (that is,
it is semi-quantitative) but it is usually used in
Interpretation
regional laboratories to detect an unknown reac-
This test allows determination of the titre (or tant (antigen or antibody) by comparison with a
degree) of the antibody response. A second known reactant (that is, a qualitative test).
blood sample collected a few weeks later may In the double diffusion Ochtelony test, anti-
show a higher titre which would indicate a ‘ris- gen and antibody are placed in wells cut in agar
ing titre’ and therefore is suggestive of current (Figures 6.8a–c) and allowed to diffuse towards
infection rather than previous exposure to the one another. At the point where they meet in the
antigen. Paired sera should preferably be tested correct proportions a precipitate is formed which
at the same time in the same test. is seen as an opaque line in the gel. Care must be
To facilitate the interpretation of ‘in house’ taken in the interpretation of the precipitation
semi-quantitative and quantitative test results it lines and only when there is a line of conflu-
is necessary to calibrate the test system to deter- ence can a positive result be determined with
mine a ‘cut off’ point. This can be achieved by confidence. Manufacturers of various reagents
using known strong and weakly positive samples will prescribe the optimum conditions required
from known infected animals, and known nega- for the test. Immunodiffusion tests are usually
tive samples, and titrating each of these out to carried out in petri-dishes. The gel used is usu-
assess level of the reaction. The ‘cut off’ titre ally made with a purified agar (for clarity) which

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