Systematic Veterinary Virology

HAEMAGGLUTINATION TEST

• 25 µl of PBS is dispensed into each well of a plastic V-bottomed microtitre plate.
• 25 µl of the virus suspension (i.e. infective or inactivated allantoic fluid) is placed in the first well.
• Twofold dilutions of 25 µl volumes of the virus suspension are made across the plate.
• A further 25 µl of PBS is dispensed to each well.
• 25 µl of 1% (v/v) chicken RBCs is dispensed to each well.
• The solution is mixed by tapping the plate gently. The RBCs are allowed to settle for about 40 minutes at room temperature, i.e. about 20°C, or for 60 minutes at 4°C if ambient temperatures are high, when control RBCs should be settled to a distinct button.
• HA is determined by tilting the plate and observing the presence or absence of tear-shaped streaming of the RBCs. The titration should be read to the highest dilution giving complete HA (no streaming); this represents 1 HA unit (HAU) and can be calculated accurately from the initial range of dilutions.
• Calculation of 4HA units: 4HAU is obtained by dividing 1HAU with four. If 1HAU is 512; then 4HAU is 512/4 = 128. The virus should be diluted to 1:128 before proceeding with HI test.
• Confirmation of correctness of 4HAU virus : The correctness of 4HAU dilution is confirmed by doing a HA test with 4HAU virus. When performed the HA titre should not exceed 2³ (ie. 4HAU, 2HAU & 1HAU). If it exceeds then identification of 1HAU is wrong and suitable modification to the protocol should be done. HI test should not be performed with out confirming the correctness of dilution of 4HAU of virus.