This article describes a method for assessing hemagglutination inhibition using a multi-well plate assay. The interaction between influenza-specific antibodies and viral antigens is evaluated to determine the presence of hemagglutination.
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.
Begin by labeling 96-well microtiter plates with the appropriate experimental information. Then, turn the plate in the vertical orientation and use a multichannel pipette to add 25 microliters of PBS to every well except the first well of the bottom back titration row. Add 50 microliters of the freshly prepared antigen solution of interest to the first well of the back titration row, followed by the addition of 25 microliters of RDE-treated serum samples to the first wells of the top 10 rows.
Add 25 microliters of the appropriate anti-serum to the first well of the 11th row as a positive control. Then, transfer 25 microliters from the first well of each row to their successive wells to perform serial two-fold dilutions. Pipette up and down 10 to 15 times for each dilution step, discarding the final 25 microliters from the last wells.
Next, add 25 microliters of the antigen solution to each well of rows 1 through 11, and 25 microliters of PBS only to each well of the back titration row. Tap the plate carefully 10 times on all four sides to mix, and cover the plate for 30 minutes at room temperature. At the end of the incubation, add 50 microliters of red blood cell solution to each well, and mix the plate with more tapping. Then, cover the plate again and incubate the red blood cells according to the species used, as outlined in the table.
After adding the red blood cells to the plate, adhere to the appropriate incubation time for the type of blood used in the assay. A too-short or too-long incubation will lead to an incorrect interpretation of the results.
At the end of the incubation, assess the hemagglutination by tilting the plate 90 degrees for 25 seconds, and mark the results for each well while the plate is still tilted on a printed scheme of the 96-well plate.
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