The Hemagglutination Inhibition Assay to Detect Serum Antibodies Against a Target Antigen

Begin with a V-shaped bottom multi-well plate and add diluted serum from an influenza-vaccinated human containing influenza-specific antibodies, to the test well.

Introduce viral hemagglutinin — an influenza-specific antigen in the test and control wells.

During incubation, in the test well, influenza-specific antibodies interact with antigens, forming complexes. While in the control well, antigens remain unbound.

Incubate the wells with avian red blood cells or RBCs.

In the control well, free antigens interact with RBC's surface receptors specific to viral hemagglutinin, causing RBCs to clump, leading to hemagglutination, and forming a compact pattern at the well's bottom.

However, in the test well, the presence of antigen-antibody complexes and the absence of free antigens inhibit hemagglutination, settling RBCs at the bottom.

Post-assay, tilt the plate. The control well exhibits a small, compact circular pattern with a hazy appearance — indicative of hemagglutination.

In contrast, the test well shows a tear-like diffused pattern, suggesting hemagglutination inhibition.