Hemagglutination Inhibition Test: Principle, Procedure, Uses

Many viruses (such as influenza) carry a surface protein, hemagglutinin, that binds red blood cells and cross-links them into a lattice. This is hemagglutination, and it is the phenomenon the HAI test switches off. In a microtiter well, agglutinated cells settle as a diffuse irregular film, while unagglutinated cells roll down into a compact button. For hemagglutination as a general method, see agglutination test types.

Principle

Hemagglutination occurs when a hemagglutinating virus (measles is used in this example) is mixed with red blood cells (see image a). But, if the serum of a person infected with measles virus is mixed with RBC and measles virus, there won’t be any agglutination of RBC. This phenomenon is known as hemagglutination inhibition.

This happens because antibodies in that person's serum bind the virus and block its hemagglutinin (positive result). With the hemagglutinin occupied by antibody, the virus can no longer attach to and cross-link the red cells, so no agglutination occurs.

If the patient’s serum does not contain antibodies against surface proteins of the test virus, there will be hemagglutination as surface molecules are free to hemagglutinate RBCs (negative result).

Figure: Hemagglutination and hemagglutination inhibition test

The basis of the HAI assay is that antibodies to that particular virus (for example; measles virus) will prevent the attachment of the virus to RBC. Therefore hemagglutination is inhibited when antibodies are present.

HAI Titer: The highest dilution of serum (Ab) that prevents hemagglutination is called the HAI titer of the serum.

1. If the serum contains no antibodies that react with the measles virus, then hemagglutination will be observed in all wells.
2. Likewise, if antibodies to the measles virus are present, hemagglutination will not be observed until the antibodies are sufficiently diluted.

The HAI test can be complicated by non-specific inhibitors of viral hemagglutination in the serum, and by naturally occurring agglutinins against the red cells. Therefore, the sera should be treated before use or false positive or negative results may arise.

The hemagglutination (HA) test vs the hemagglutination inhibition (HAI) test

These two tests are easy to confuse because they use the same reaction, but they answer opposite questions.

The hemagglutination (HA) test measures the virus. Two-fold dilutions of the virus are mixed with a fixed suspension of red cells. Wells with enough virus show hemagglutination (a diffuse film of cross-linked cells); wells that are too dilute show a compact button.

The highest virus dilution that still causes complete hemagglutination is the HA titer, and it is expressed in hemagglutinating units. One HA unit is the amount of virus that just agglutinates an equal volume of the standardized red-cell suspension. The HA test answers: how much virus is here?

The hemagglutination inhibition (HAI) test measures the antibody. Here the virus dose is fixed, and the patient's serum is titrated against it. Antibody, if present, blocks the virus and prevents agglutination. The HAI test answers: how much antibody is here?

The link between them is the HA test that comes first. Before an HAI test can be run, the virus must be standardized to a defined dose, by convention 4 HA units per 25 µL (WHO standard for influenza). That dose is worked out by an HA titration, and a back-titration is run alongside the HAI test to confirm 4 HA units were actually used. Too much or too little virus, and the HAI titer reads falsely low or high. This is why the procedure below starts with "virus of known HA titer" and why quality control includes a back-titration.

In short: run the HA test to standardize the virus, then run the HAI test to measure the antibody against it.

Materials and Reagents

1. Red cells from an appropriate species. Chicken, turkey, goose, and guinea pig cells are commonly used; the choice depends on the virus. Cells are collected in Alsever's solution or heparin.
2. Diluent (e.g. bovine albumin veronal buffer) at appropriate pH
3. Solutions to remove nonspecific hemagglutinins from serum
4. Infected cultural fluid or standard antigen (e.g preparation of influenza virus) for serology

Procedure

1. Obtain a preparation of virus (e.g. influenza viruses) with known HA titer or determine its HA titer
2. Prepare two-fold dilutions of patient/test serum to be tested e.g. from 1:4 to 1:1024.
3. Add a fixed, standardized amount of virus to every well of a 96-well plate, by convention 4 HA units per 25 µL (determined by a prior HA titration, see the section above), except for the serum control wells.
4. Allow the plate to stand at room temperature for 60 minutes (time varies according to specific requirements).
5. Add red blood cells (RBC) and incubate at 4°C for 30 minutes.
6. Read the wells.

Results/interpretation

Read each well as one of two patterns. A compact, sharply defined button of cells at the bottom of the well means the cells are not agglutinated: the antibody has blocked the virus, so this is a positive (inhibited) well. A diffuse film or hazy shield of cells spread across the well means the cells are agglutinated: no blocking antibody, a negative well. Tilting the plate helps read the endpoint, because a true button flows like a teardrop while an agglutinated film stays put.

The HAI titer is the highest serum dilution that still shows inhibition (the last button before agglutination reappears). For example, an HAI titer of 1:1280 means the serum still blocked hemagglutination when diluted 1280-fold, so it carries a high level of specific antibody.

Two interpretation rules are worth knowing for influenza serology. An HAI titer of 1:40 or higher is generally considered seroprotective. And when paired sera are tested (one sample early in illness, one two to three weeks later), a fourfold or greater rise in titer between them is the standard evidence of recent infection or of seroconversion after vaccination. A single titer is harder to interpret than a documented rise, the same paired-sample principle that applies across serology.

Uses of Hemagglutination-Inhibition Test

1. Hemagglutination inhibition test is widely used for the diagnosis of infection caused by orthomyxoviruses (influenza), paramyxoviruses (measles, mumps), mononucleosis,  arboviruses-togaviruses (including rubella), flaviviruses, and bunyaviruses.
2. Virus in an infected cell culture can be detected by hemagglutination, and the identity of the virus, or of antibodies in a patient's serum, can then be confirmed by the specific inhibition of that hemagglutination.
3. Although influenza viruses can be detected by hemadsorption test, typing of the isolate is done most efficiently by hemagglutination inhibition (HAI).

Quality Control

1. Known positive serum
2. Known negative serum
3. Serum and cells without antigen (to detect nonspecific agglutination)
4. Back titration of hemagglutination activity of the antigen to confirm that 4 hemagglutinating units (HAU) of antigen were used

Where students get confused

No clumping is the positive result. This is the reverse of an ordinary agglutination test. In HAI, the patient's antibody blocks the virus from agglutinating the red cells, so a smooth button of un-agglutinated cells (no lattice) means antibody is present. Clumping means antibody is absent. Whenever a test has "inhibition" in its name, expect this inverted reading.

The HAI titer is the highest dilution that still inhibits. Because antibody is what blocks agglutination, you read the titer as the greatest serum dilution at which hemagglutination is still prevented. Beyond that dilution, there is too little antibody to block the virus, and clumping reappears.

Why sera are pretreated. Serum contains non-specific inhibitors of viral hemagglutination and naturally occurring agglutinins, both of which can mimic or mask antibody. Removing them before testing is what keeps the result specific, which is why the pretreatment step is not optional.

How to remember

Inhibition flips the sign. In a plain hemagglutination test, clumping is the positive signal. Add antibody and the virus can no longer clump the cells, so in HAI the button (no clumping) is positive and the film (clumping) is negative. The antibody's whole job is to stop the reaction, so its presence is read as the absence of clumping.

HA measures the bug, HAI measures the defense. The HA test titrates the virus (how much bug is here); the HAI test titrates the antibody against a fixed dose of virus (how much defense is here). You always run HA first to set the virus dose for HAI.

Key exam facts

References

1. Levinson W (2020). Review of Medical Microbiology and Immunology. 16th edn. McGraw-Hill.
2. Sano K, Ogawa H (2014). Hemagglutination (inhibition) assay. Methods in Molecular Biology. 1200: 47-52.
3. Tille PM (2022). Bailey and Scott's Diagnostic Microbiology. 15th edn. Elsevier.
4. Kaufmann L, Syedbasha M, Vogt D, et al. (2017). An optimized hemagglutination inhibition (HI) assay to quantify influenza-specific antibody titers. Journal of Visualized Experiments. (130): 55833. doi:10.3791/55833