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Influenza Hemmagglutination Inhibition Assay

According to WHO statistics, seasonal influenza viruses cause 3-5 million cases of infection with 290,000-650,000 deaths worldwide each year. There are 4 types of seasonal influenza viruses. Types A (which can infect and be transmitted between birds and some mammals such as pigs and humans) and B (which only infects humans and seals) are responsible for the seasonal epidemic disease. Influenza infection poses a severe threat to public health and the agricultural economy. Because the virus mutates quickly it requires the development and reformulation of new vaccines each year.

Based on a twice a year review of the epidemiological situation the WHO, if necessary, makes recommendations for new vaccine strains.  Vaccine effectiveness is relatively low especially if the vaccine strain does not match the circulating influenza strain. The hemagglutination inhibition (HAI) assay is the commonly used method for quantifying antibody titers of influenza virus and for serological studies evaluating protection.  There are two glycoproteins on the surface of influenza virus, hemagglutinin (HA) and neuranimidase (NA), that are targeted by antibodies and have potential utility as vaccine targets. Due to immunodominance, the antibody response in vaccinated/infected subjects is more robust against HA than against NA. Antibody response against HA is determined using the HAI assay (HAI) is described in the WHO manual. Serum samples from vaccinated/infected subjects are first treated with the Receptor Destroying Enzyme (RDE) to remove non-specific inhibitors to avoid false positives. The RDE is then inactivated and serum samples are adsorbed against (avian or guinea pig) red blood cells (RBC’s), to prevent non-specific agglutinins (i.e. false negatives).  After pre-treatment, serially-diluted serum is pre-incubated with 4 HA units prior to addition of RBC suspension.  Standardization of the RBC’s is a critical factor for consistency of HAI titer reporting by testing laboratories. Following RBC incubation at ambient temperature, hemagglutination reaction is recorded as positive or negative.

HAI assay for serological detection of infection and protection against influenza


HA on the surface of influenza virus or as recombinant protein, agglutinates RBC and holds them in a lattice thereby preventing the RBC from sinking to the bottom of the well (a positive hemagglutination reaction). A positive HAI result occurs when HA-specific antibodies that target the globular head block the HA-induced agglutination of RBC and the RBC sink to the bottom of the well forming a button or a tear shape when the plate is tilted (indicating a negative hemagglutination reaction). The reciprocal of the highest dilution of serum where there is inhibition of hemagglutination is recorded as the HAI titer. According to the CDC, HAI titer of ≥ 40 corresponds to 50% reduction of influenza infection and is considered as correlate of protection. Some laboratories observed that certain virus strains passaged using Madin Darby Canine Kidney Cell Line (MDCK) show reduction in agglutination HA titer. At IBT Bioservices, we source HA recombinant proteins that are agglutination competent. These reagents allow the assay to be performed outside of the biological safety cabinet and speed up data turnaround time for vaccine studies.



  1. World Health Organization. Influenza (seasonal) published 06Nov2018
  1. World Health Organization. (2011). WHO Global Influenza Surveillance Network: Manual for the laboratory diagnosis of virological surveillance of influenza. Geneva: World Health Organization.

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Alpha (UK) – B. 1.1.7 / 501Y.V1

amino acid mutations: del69–70 HV, del144 Y, N501Y, A570D, D614G, P681H, T761I, S982A, D1118H

Beta (South Africa) – B.1.351

amino acid mutations: K417N, E484K, N501Y, D614G, A701V

Gamma (Brazil) – P.1

amino acid mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I

Epsilon (Ca, USA) B.1.427

amino acid mutations: L452R, D614G

SARS-CoV-2 Parental Strain Wild Type (Wuhan)
SARS-CoV-2 D614G Variant

amino acid mutations: D614G

Epsilon (Ca, USA) B.1.429

amino acid mutations: S13I, W152C, L452R, D614G

SARS-CoV-2 Delta Variant

amino acid mutations: L452R, E484Q