Haemagglutination Inhibition (HI)
Mechanism: The antigen in HI tests is simply a solution of the antigenic particles (usually a virus) which is capable of inducing the reaction of haemagglutination when mixed with a suspension of red blood cells. This agglutination is not an antigen/antibody reaction but, rather is the attachment of viral particles by their receptor sites to more than 1 cell. As more and more cells become attached in this manner agglutination becomes visible. The presence and concentration of antibody is measured by its ability to inhibit the agglutination at various dilutions (Figure 2.6).
Reagents: The antigen is usually prepared by growing a naturally haemagglutinating virus (NDV and Avian Influenza viruses) in chick embryo and collecting allantoic fluid (Beard et al.,1975). Occasionally it may be possible to use re-constituted vaccine as a haemagglutinating antigen. If virological examinations are also carried out in the same laboratory or if the viruses used in antigen production are not vaccinal then it would be important to inactivate the antigen by chemical treatment (though this will reduce the titre of the antigen). Some viruses (e.g. Infectious Bronchitis) though not naturally haemagglutinating can be made to haemagglutinate by treatment with an enzyme treatment. Preparation of such antigens is more complicated since it will normally be required to concentrate virus, usually by ultra-centrifugation. 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. It is usually recommended that the source of the erythrocytes be un-vaccinated chickens. In the UK a license is required under the Animals (Scientific Procedures) Act, 1986 to collect blood as source of erythrocytes. It is this authors experience that age and vaccination history of the donor has no effect on the results (McMullin, 1979). It is important to carry out at least three wash cycles to ensure that the final suspension is free of antibody and other serum proteins. A fourth cycle is advisable if the HI titres in the source birds are 1:128 or higher. After washing the red cells are re-suspended in PBS at a standard concentration. The packed red cells should be re-suspended at least at 0.75%, since lower erythrocyte concentrations tend to be associated with more variable HA values for the antigen and hence affect HI results (McMullin, 1979)
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. Detailed descriptions of these methods may be found in the literature (Allen and Gough, 1974).
Figure 2.6. Haemagglutination-inhibition tests in a micro-titre plate. Wells with a non-agglutinated button of red cells are read as HI positive.
Assays: The commonly-used HI tests in chickens are for Newcastle disease (Paramyxovirus-1), Infectious bronchitis (Coronavirus), and EDS-76 (adeno-virus). HI tests may also be carried out for Avian Influenza. However, since there is poor cross-reactivity between the different haemagglutinin groups, AGP is favoured for routine screening.Various serotypes of IBV have been used in HI tests as an aid in suggesting the likely infecting strain. This use is complicated by a high degree of cross-reactivity.\par Comments: HI tests require inexpensive reagents though they are labour-intensive. The fact that a series of dilutions are separately tested means that the results are highly reproducible.