Hemagglutination Inhibition Test: Principle, Procedure, Uses

The nucleic acids of various viruses encode surface proteins (e.g.hemagglutinin (HA) of influenza virus) that agglutinate red blood cells (RBC) of a variety of species. The reaction of viral hemagglutinins with red blood cells results in a lattice of agglutinated cells that settle irregularly in a tube or microtiter well, a process known as hemagglutination. Unagglutinated cells settle in a compact button.


Hemagglutination occurs when measles viruses and red blood cells are mixed (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 arises because antibodies present in the serum of that infected person react with the measles viruses and neutralize them (positive result); as the hemagglutinins are occupied, they cant bind and agglutinate RBCs.

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).

Hemagglutination test and hemagglutination inhibition test
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 may be complicated by the presence of non-specific inhibitors of viral haemagglutination and naturally occurring agglutinins of the erythrocytes. Therefore, the sera should be treated before use or false positive or negative results may arise.

Materials and Reagents

  1. Red cells from an appropriate species (chicken, goose, guinea pig, trypsinized human O) 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


  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 amount of virus to every well of a 96-well plate, equivalent to 4 HA units (varies according to the virus), 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.


The highest dilution of serum (Ab) that prevents hemagglutination is called the HAI titer of the serum. A smooth or jagged shield of cells or an irregular button indicates agglutination. Observation of the movement of the button of red cells when the plate is tilted may help to clarify the endpoint.hi-titer-assay

This virus sample has an HAI titer of 1280, which means that the greatest dilution of antibodies that still blocked hemagglutination from occurring was at 1280 dilution. At this dilution, the antibodies were still capable of recognizing and binding to the antigens on the virus.

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. The presence of the virus in infected cell cultures can be detected by hemagglutination; the identity of the virus or of antibodies in a patient’s serum can be determined by 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 ensure that 4 hemagglutinating viruses (HAU) were tested)


  1. Levinson. (2010). Review of Medical Microbiology and Immunology. McGraw-Hill.
  2. Sano, K., & Ogawa, H. (2014). Hemagglutination (inhibition) assay. Methods in molecular biology (Clifton, N.J.), 1200, 47–52. https://doi.org/10.1007/978-1-4939-1292-6_4 

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

10 thoughts on “Hemagglutination Inhibition Test: Principle, Procedure, Uses

  1. how we can distinguish between the results of no reaction ( when there is no virus or AB) and haemoaglutination results ( when there is Ab ) ??

  2. Please check the results again the dilution given are 1:10 , 1:20 , 1:40 ans so on … rather its should be 1:1 , 1:2 , 1:4 , 1:8 …….. 1:2048

  3. Can we interpret presence of antibodies after vaccination…?.can we know efficiency of vaccine…?

  4. Please, I want to confirm if there is different Elisa kit for influenza D virus IDV which is different from IgG and IgM. I want to work on this virus as part of Postgraduate Diploma project but I don’t know more about it.

    Kindly help me out.

    Gideon O.


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