Haemagglutin (HA) Typing and Haemagglutination Inhibition (HI)

Variations in the procedures for HA and HI tests are practised in different laboratories. The following recommended examples apply in the use of V-bottomed microwell plastic plates in which the final volume for both types of test is 0.075 ml. The reagents required for these tests are isotonic PBS (0.1 M), pH 7.0-7.2, and red blood cells (RBCs) taken from a minimum of three SPF or SAN chickens and pooled in an equal volume of Alsever's solution. Cells should be washed three times in PBS before use as a 1% (packed cell v/v) suspension. Positive and negative control antigens and antisera should be run with each test, as appropriate.(OIE Standard Protocol)

Haemagglutination Test

i)    Dispense 0.025 ml of PBS into each well of a plastic V-bottomed microtitre plate.

ii)    Place 0.025 ml of virus suspension (i.e. infective allantoic fluid) in the first well. For accurate determination of the HA content, this should be done from a close range of an initial series of dilutions, i.e. 1/3, 1/4, 1/5, 1/6, etc.

iii)    Make twofold dilutions of 0.025 ml volumes of the virus suspension across the plate.

iv)    Dispense a further 0.025 ml of PBS to each well.

v)    Dispense 0.025 ml of 1% (v/v) chicken RBCs to each well.

vi)    Mix by tapping the plate gently and then allow the RBCs 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, by which time control RBCs should be settled to a distinct button.

vii)    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.

Haemagglutination inhibition test

i)    Dispense 0.025 ml of PBS into each well of a plastic V-bottomed microtitre plate.

ii)    Place 0.025 ml of serum into the first well of the plate.

iii)    Make twofold dilutions of 0.025 ml volumes of the serum across the plate.

iv)    Add 4 HAU of virus/antigen in 0.025 ml to each well and leave for a minimum of 30 minutes at room temperature (i.e. about 20°C) or 60 minutes at 4°C.

 v)    Add 0.025 ml of 1% (v/v) chicken RBCs to each well and after gentle mixing, allow the RBCs 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, by which time control RBCs should be settled to a distinct button.

vi)    The HI titre is the highest dilution of serum causing complete inhibition of 4 HAU of antigen. The agglutination is assessed by tilting the plates. Only those wells in which the RBCs stream at the same rate as the control wells (containing 0.025 ml RBCs and 0.05 ml PBS only) should be considered to show inhibition.

vii)    The validity of results should be assessed against a negative control serum, which should not give a titre >1/4 (>22 or >log2 when expressed as the reciprocal), and a positive control serum for which the titre should be within one dilution of the known titre.

HI titres may be regarded as being positive if there is inhibition at a serum dilution of 1/16 (24 or log2 4 when expressed as the reciprocal) or more against 4 HAU of antigen. Some laboratories prefer to use 8 HAU in HI tests. While this is permissible, it affects the interpretation of results so that a positive titre is 1/8 (23 or log2 3) or more.

Chicken sera rarely give nonspecific positive reactions in this test and any pretreatment of the sera is unnecessary. Sera from species other than chickens may sometimes cause agglutination of chicken RBCs, so this property should first be determined and then removed by adsorption of the serum with chicken RBCs. This is done by adding 0.025 ml of packed chicken RBCs to each 0.5 ml of antisera, shaking gently and leaving for at least 30 minutes; the RBCs are then pelleted by centrifugation at 800 g for 2-5 minutes and the adsorbed sera are decanted. Alternatively, RBCs of the avian species under investigation could be used.