An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

The overall goal of this hemagglutination inhibition assay is to measure the antibody titers against specific viruses of interest. This method can help answer key questions in the vaccinology field about the vaccine-mediated immunity and protection within different populations and across various age and patient groups. The main advantages of this technique are that it is accurate and that it allows the fast titer determination of the presence of neutralizing antibodies.

Though this method can provide insight into human antibody titers, it can also be applied to other systems, such as antibody titers for mouse serum, also culture supernatants. Begin by labeling 96 well microtiter plates with the appropriate experimental information. Then turn the plate in the vertical orientation and use a multi-channel 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 ten rows. Add 25 microliters of the appropriate antiserum to the first well of the 11th row as a positive control. The 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 one 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 side to mix.

Then 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.

In this experiment, the vaccine-induced antibody response was assessed in 26 healthy volunteers who received an inactivated, trivalent influenza vaccine containing influenza A/H1N1/California/2009, A/H3N2/Texas/2012 and B/Massachusetts/O2/2012 prior to the 24 2015 influenza season. A cross-reactive immune response for A/H3N2/Switzerland/2013 and A/H3N2/Texas/2012 virus strains was observed, with significantly lower geometric mean hemagglutination inhibition titers and induced seroprotection against influenza A/H3N2/Switzerland/2013 compared to influenza A/H3N2/Texas/2012. After vaccination, the antibody titers against both strains increased, even though the A/H3N2/Switzerland/2013 strain was not present in the vaccine.

Viral hemagglutinin demonstrates a species dependent potential for erythrocyte hemagglutination. Of interest, guinea pig blood does not properly hemagglutinate with influenza B and turkey blood has the potential for hemagglutination with high titers and a low cross-reactivity. Once mastered, this technique can be completed in the three hours if it's performed properly.

While attempting this procedure it's important to remember to serum with RDE to inactivate any unspecific inhibitors and unspecific binding during the hemagglutination of the virus. Following this procedure, other methods like ELISA can be performed to answer additional questions about the role of individual pathogen specific immunoglobulins. After its development, this technique paved the way for researchers in the field of viral immunology to further our understanding of vaccine-related immunity in healthy and immunosuppressed patients within different age groups.

After watching this video, you should have a good understanding of how to perform a hemagglutination inhibition assay to quantify influenza strain specific antibody titers on a large scale. Don't forget that working with viral antigens, animal blood, and human serum samples can be extremely hazardous and that precautions such as working in a BSL2 laboratory should always be taken while performing this procedure.