Mechanism: The antigen in HI tests is simply a solution of the antigenic particles which is capable of inducing the reaction of haemagglutination when mixed with a suspension of red blood cells. The presence and concentration of antibody is measured by its ability to inhibit the agglutination at various dilutions.
Reagents:
The antigen is usually prepared by growing a naturally haemagglutinating virus in chick embryo and collecting allantoic fluid.
The only other reagent required for carrying out this test is a suspension of red blood cells. Most HI tests carried out in routine poultry serology use chicken erythrocytes.
Methods:
It is possible to carry out rapid haemagglutination tests and haemagglutination-inhibition tests on a plate, just as for the bacterial agglutination tests described above.
However HA and HI are generally only used in this way to confirm the presence and identity of a haemagglutinating antigen.
Identification and quantification of HI antibody, on the other hand, is nearly always carried out by the equivalent of the slow agglutination test, originally in tubes, now almost always in micro-titre plates.
Picture above-Haemagglutination-inhibition tests in a micro-titre plate. Wells with a non-agglutinated button of red cells are read as HI positive.
Seven different samples of influenza virus, numbered 1 through 7 at the left, were serially diluted as indicated at the top, mixed with chicken red blood cells (RBC), and incubated on ice for 1 to 2 hours.
Wells in the bottom row contain no virus.
Agglutinated RBCs coat wells evenly, in contrast to nonagglutinated cells, which form a distinct button at the bottom of the well. The HA titer, shown at the right, is the last dilution that shows complete hemagglutination activity.
(From Fields Vriology (2007) 5th edition, Knipe,DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.9)