Hemagglutination Inhibition Assay (HIA)

The Hemagglutination assay is used to detect and quantify the concentration of virus required to cause blood agglutination. Viruses are composed of a glycoprotein envelope which are able to bind to salic acid molecules found on the receptor sites of red blood cells (RBC's) When cross links are formed between the two, a lattice is produced which can be seen as agglutination. Usually when RBC's are suspended in saline solution the cells will gradually drop to the bottom of the tube due to gravitational forces. This can then be seen as a red dot. However when viruses are added to the solution interactions between the virus and RBC's may be seen. The RBC's are prevented from falling to the bottom on the tube and instead a shield is formed keeping the cells suspended. This can be seen as a red liquid rather than a single red dot.

The HA assay will use a round bottom shaped microtitre plate with equal volumes of saline added to each well. Following this serial dilutions of virus will be added to each well noting the dilution. Equal volume of RBC suspension is then added to each of the well where it is then left to mix and settle in the plate. After about 30 minutes or so some of the wells should show signs of agglutination (red liquid) whilst others a single red dot. The last well to show a shield can be said to have 1 HA unit of virus. Taking into account the volume and dilution of virus added to each of the wells the titre can be calculated. This is this lowest concentration of virus required to cause blood agglutination, anything lower agglutination will not be seen.

The Procedure

1) Add 50 ul of PBS to each well, using a round bottomed 96 well microtitre plate

2) To the first column of wells add 50 ul of virus sample, mix well.

3) From the first column take 50 ul of solution and transfer to the next column, mix well.

4) Repeat this process for all wells working to the right. For the last well discard 50 ul of the solution to ensure all wells contain the same volume.

5) Add 50 ul of 0.5 % RBC working solution to each well, mix gently. 

6) Leave the plate for 30-60 minutes at room temperature to allow the solution to settle.

7) Negative wells will be seen as a dot, positive wells will be seen as a red liquid.

8) The HA titre may then be calculated. It is the highest dilution of virus that is able to produce agglutination.

Hemagglutination Inhibition Assay (HIA)is a procedure used to identify certain viruses that can cause haemagglutination. Certain viruses such as Rubella, Herpes zoster and Influenza are composed of a protein envelope that is recognised to bind to erythrocytes in mammalian and avian species. This binding causes the formation of a lattice which can be seen as agglutination. The efficiency of haemagglutin binding is dependent upon the type of linkage that connects the RBC to the receptor molecule of the virus, as well as the type of virus and host specie.

The Hemagglutination Inhibition Assay can be used to prevent RBCs from binding to viruses through the addition of specific antibodies. Antibodies for either the hosts RBC or Virus receptor can be added to a patients sera to prevent attachment, with the antibodies binding to the receptor sites instead. Viruses such as Rubella and Infleunza are highly specific to the antibodies used within HAI which makes the use of this method highly effective for diagnosis. Other viruses such as Flaviviruses have been found to cross react to other related viruses which make the use of the HAI test less sensitive and specific. 

The HAI test may be influenced by the presence of naturally occurring agglutinins and non specific inhibitors. To reduce the effect of this the patients sera should be treated beforehand to prevent false positive or negative results. Following this the patients sera undergoes serial dilutions and is added to a microtitre plate.  Virus antigen is then added to the plate wells, if antibodies are not present within the sera or are in a low concentrations agglutination will be seen. The controls do not contain any antigen and no signs of agglutination should be seen. From these dilutions the titre of antibodies in the sera can be calculated when agglutination is shown. 

If a high concentration of antigen is added and agglutination is not observed this can indicate the patient has been recently infected with virus, with antibodies developed to overcome the infection.

The advantages to using HAI tests are that they are relatively easy and inexpensive to perform, they can also be adapted for the detection of different viral infections . However they are considered less sensitive to EIA's or RIA's.  It is also important to ensue samples and reagents used are are fresh otherwise abnormal agglutination patterns may arise. Pretreatment of sera may also cause the accidental removal of antibodies being tested which may influence the results.