Division 4 Test for specific antibodies in HI tests

Virus isolation or molecular detection are likely to be the most sensitive for determining AI virus infections of pigs. However, serological responses in pigs have been detected in the absence of virus isolation or detection. HI tests, using pig sera, require some modifications to the test used for avian sera referred to in Chapter VIII.

Pig sera are notorious for their property of non-specific inhibition in HI tests and therefore each serum sample must be treated with a receptor destroying enzyme (RDE) to prevent this from occurring. The following method must be used:

1. (a)

   To 100 μl of pig antiserum add 400 μl RDE (predetermined working dilution) and mix thoroughly.

2. (b)

   Incubate at 37 ^oC for one hour.

3. (c)

   Then incubate for 30 minutes at 56 ^oC.

4. (d)

   Cool samples at 4 ^oC for a minimum of 15 minutes.

5. (e)

   Add 10 μl of 30 % (v/v packed cells) chick red blood cells and mix vigorously.

6. (f)

   Incubate at 4 ^oC overnight. Alternatively, if it is essential to use samples same day, incubate at 37 ^oC for one hour and centrifuge at 300 x g for five minutes.

The treated serum is then used in HI tests as described for avian sera in paragraph [..], the initial dilution is 1:10. A set of sera of pigs with a known sero-negative status regarding AI must be used to assess the specificity of the HI test for the virus strain to be used (see use of virus strain for serology derived from the outbreak; Chapter VIII). During the outbreak in the Netherlands in 2003 up to 2,6 % non-specific reactors were detected in the HI test using pig sera that were collected independently of the outbreak