Hemagglutination inhibition test

Principle

• Some viruses (e.g. influenza, measles, rubella, mumps, many arboviruses) carry haemagglutinin that binds red cells and cross-links them (haemagglutination).
• Specific antibodies in patient serum bind the haemagglutinin and prevent agglutination: inhibition indicates antibody presence.

Specimen

• Serum, ideally paired acute and convalescent samples 10–14 days apart.

Reagents and equipment

• Standardised virus antigen, red cells of a suitable species (e.g. turkey, chicken, guinea pig or human group O; goose cells for many arboviruses), diluent, V- or U-bottom microtitre plates.
• Serum treatments to remove non-specific inhibitors (e.g. receptor-destroying enzyme for influenza) and natural agglutinins (absorption with the red cells used).

Procedure

• First titrate the virus (HA test) to find the haemagglutinating units; use 4 HA units per well for HAI.
• Make doubling dilutions of treated serum; add standardised virus; incubate (e.g. 30 minutes at room temperature).
• Add red cell suspension; incubate until control cells settle (about 30–60 minutes); read.

Results

• Inhibition (antibody present): red cells form a compact button (or tear-drop streaming when the plate is tilted).
• No inhibition: diffuse mat or lattice of agglutinated cells.
• Titre: highest serum dilution showing complete inhibition.
• A fourfold or greater rise between paired sera indicates recent infection or vaccine response. For influenza, an HAI titre of ≥1:40 is commonly used as a correlate of about 50% protection.

Quality control and pitfalls

• Controls: serum control without virus (non-specific agglutination), red cell control, virus back-titration to confirm 4 HA units, known positive and negative sera.
• Incomplete removal of non-specific inhibitors gives false positives; cross-reactions occur between related viruses (e.g. flaviviruses).
• Red cell species, age of cells and virus dose affect results.

Clinical use

• Influenza surveillance and antigenic characterisation for vaccine strain selection, vaccine immunogenicity studies, and serology for measles, rubella and arboviruses (largely replaced by ELISA in routine labs).