4. Haemagglutination Inhibition Test
A wide variety of different viruses possess the ability to agglutinate the erythrocytes of mammalian or avian species. The actual animal species whose erythrocytes could be agglutinated depends on the actual virus. Examples of viruses which could haemagglutinate include influenza, parainfluenza, adenoviruses, rubella, alphaviruses, bunyaviruses, flaviviruses and some strains of picornaviruses. Antibodies against the viral protein responsible for haemagglutination can prevent haemagglutination; this is the basis behind the haemagglutination-inhibition test (HAI). The specificity of the HAI test varies with different viruses. With some viruses such as influenza A, the haemagglutination antigen is the same as the antigen responsible for virus adsorption and thus virus neutralization, and therefore the HAI test is highly specific for the different strains of the virus. With other viruses, the HAI test is less specific eg. flaviviruses, where HAI antibodies against one flavivirus may cross-react with other related flaviviruses. HAI tests are more sensitive than complement-fixation tests but are less sensitive than EIAs and RIAs.
The HAI test is simple to perform and requires inexpensive equipment and reagents. Serial dilutions of patient's sera are allowed to react with a fixed dose of viral haemagglutinin, followed by the addition of agglutinable erythrocytes. In the presence of antibody, the ability of the virus to agglutinate the erythrocytes is inhibited. The HAI test may be complicated by the presence of non-specific inhibitors of viral haemagglutination. and naturally occurring agglutinins of the erthrocytes. Therefore, the sera should be treated before use or false positive or negative results may arise. HAI tests are widely used for the diagnosis of rubella and influenza virus infections. The following is a brief description of the HAI test for rubella.
For rubella HAI testing, one day old chick or goose erythrocytes are used. Bovine albumin veronal buffer (BAVB) is used as the diluent. The HAI test should be carried out using 4 haemagglutination units of rubella antigen. The actual concentration of antigen required should be determined before each HAI test by carrying out a rubella antigen titration from 1:2 to 1:1024. One HA unit is defined as the highest dilution of antigen that gives complete haemagglutination of cells.
In the actual HAI test, the patients' sera are diluted in BAVB from 1:8 to 1:1024. Either V-shaped or U-shaped 96 -well microtitre plate may be used. Non-specific inhibitors of viral haemagglutination may be removed by the treatment of sera before testing by kaolin, RDE, potassium periodate (KIO) or by heat inactivation. Non-specific agglutinins for erythrocytes may be removed by the addition of erythrocytes to the sera prior to testing to allow the erythrocytes to absorb the non-specific agglutinins. This procedure may be carried out for each serum before testing or may be carried out for sera which had shown agglutination in the serum control wells (serum and erythrocytes only) in a previous HAI test. 4HA of rubella antigen is then added to each well containing diluted test sera except for the serum control wells. A back titration of rubella antigen should be incorporated into the test from 4 HA units to 0.25 HA units. The plate is then allowed to stand at room temperature for 60 minutes after which either 0.5% goose cells or 0.4% chick cells are added to each well and incubated at 4oC for 60 minutes. The plate is then read.
The erythrocytes only control should show a button at the bottom of the well. The serum controls for each serum should show the absence of agglutination. The haemagglutinin back titration should show agglutination at 4, 2 and 1 HA units. A fourfold or greater rise in HAI antibody between acute and convalescent phase sera is indicative of a recent rubella infection.
The advantages of HAI tests are that they are relatively easy and inexpensive to perform. The disadvantages are that HAI tests are not as sensitive as EIAs or RIAs, the actual reading of results is subjective and the reagents should be fresh or else abnormal agglutination patterns may arise which makes the reading and interpretation of the test very difficult. As a result the HAI test for rubella had been replaced by more sensitive and reliable EIA and RIA tests for rubella IgG in many virus diagnostic laboratory
Enzyme Linked Immunoassay (ELISA)
Virological Methods Slideset