Division Procedure

Dispense 0,025 ml PBS into all wells of a plastic microtitre plate (with V-bottomed wells).

Place 0,025 ml of serum into first well of plate.

Use microtitration diluter to make two-fold dilutions of serum across plate.

Add 0,025 ml of diluted allantoic fluid containing 4 or 8 HAU.

Mix by tapping and place plate at 4 °C for a minimum of 60 minutes or room temperature for a minimum of 30 minutes.

Add 0,025 ml 1 % RBCs to all wells.

Mix by gentle tapping and place at 4 °C.

Plates are read after 30-40 minutes when control RBCs are settled. This is done by tilting and observing the presence or absence of tear-shaped streaming at the same rate as control wells containing RBCs (0,025 ml) and PBCs (0,05 ml) only.

The HI titre is the highest dilution of antiserum causing complete inhibition of 4 or 8 units of virus (an HA titration to confirm the presence of the required HAU should be included in each test).

The validity of the results is dependent on obtaining a titre of less than 2³ for 4 HAU or 2² for 8 HAU with the negative control serum and a titre of within one dilution of the known titre of the positive control serum.