Hemagglutination Inhibition (HI) Assay Protocol


HI assay is a serological technique used to determine the presence and titer of specific antibodies against a particular virus or other pathogens. The ability of a virus to agglutinate red blood cells can be inhibited by corresponding antibodies, preventing the agglutination of red blood cells. The inhibitory effect is positively correlated with the concentration of antibodies. Therefore, the hemagglutination inhibition test can be used to evaluate the efficacy of antibodies.

HI assay.Fig. 1. HI assay. (Spackman, 2020)


Reagents Instruments and Consumables
  • Viral antigen
  • Red blood cells (RBCs) from a species susceptible to the virus
  • Inactivated viral antigen
  • Antibody positive and negative control serum samples
  • Target serum samples
  • Phosphate-buffered saline (PBS)
  • Sterile ddH2O
  • Microtiter plates or test tubes
  • Microcentrifuge tubes
  • Pipettes and tips
  • Microtiter plate reader
  • Centrifuge
  • Microscope
  • Incubator or water bath


1. Prepare serial dilutions of the target serum samples in PBS, starting from a 1:10 dilution, and transfer them into the wells of the microwell plate.
2. In the first row of the microwell plate, add 100 μL of PBS (negative control), and in the last row, add 100 μL of the positive control serum (known to contain high antibody titers).
3. In the remaining wells of the microwell plate, add 50 μL of the inactivated viral antigen.
4. Add 50 μL sterile ddH2O to each well containing the viral antigen and serum dilutions.
5. Mix the contents of each well gently using a multichannel pipette and cover the microwell plate with a lid or adhesive film.
6. Incubate the microwell plate at 37°C for 1-2 hours to allow the antibodies to bind to the viral antigens.
7. After incubation, add 50 μL of freshly washed RBCs (previously prepared by centrifugation at 1000 rpm for 5 minutes and resuspended in PBS) to each well, using a multichannel pipette. Mix gently.
8. Incubate the microwell plate at room temperature (20-25°C) for 30 minutes to allow hemagglutination to occur.
9. Observe the microwell plate under a low-power microscope or in a light box for hemagglutination. Hemagglutination inhibition will result in a button-like appearance, indicating the absence of specific antibodies against the virus.
10. Determine the highest dilution of the target serum sample that completely inhibits hemagglutination. This dilution is called the HI antibody titer.
11. Record and report the HI antibody titer of the target serum sample.
12. Clean the microwell plate thoroughly with an appropriate disinfectant to prevent cross-contamination.
13. Analyze the results using a microtiter plate reader if desired, to quantify the degree of hemagglutination inhibition.


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  1. Spackman, E.; et al. Hemagglutination inhibition assay. Methods Mol Biol. 2020, 2123: 11-28.
For research use only, not directly for clinical use.
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