Source: Kaufmann, L. et al., An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers. J. Vis. Exp. (2017)
The video demonstrates a hemagglutination inhibition assay for detecting serum antibodies against specific antigens. Confirmation of hemagglutination inhibition in the test well and hemagglutination in the control well is achieved through distinct visual patterns. This assay plays a crucial role in assessing immune response, vaccine effectiveness, and studying viral infections in virology and immunology.
All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Serum Collection
2. Preparation of Antigens
CAUTION: Five different antigens are used (see Table of Materials). Prepare antigens in a Biosafety Level 2 (BSL-2) laboratory.
3. Preparation of Cholera Filtrate
NOTE: Cholera filtrate is used as a receptor-destroying enzyme (RDE) according to the World Health Organization (WHO) protocol. This removes innate inhibitors from the serum which would interfere with the assay.
4. HA Assay
NOTE: To ensure that the hemagglutination inhibition (HI) assays are comparable between several plates, the same amount of virus particles must be used for each plate. The hemagglutination (HA) assay (also called HA titration) is performed to quantify the virus particles necessary for hemagglutination and is recorded in HA units. A "unit" of hemagglutination is an operational unit dependent on the method used for HA titration and is not a measurement of an absolute amount of virus. Thus, an HA unit is defined as the amount of virus needed to agglutinate an equal volume of a standardized RBC suspension. According to the WHO, the standard amount used for the HI assay is 4 HA units per 25 µL. For an illustration of the principle of the HA assay see Figure 1.
NOTE: The red blood cells (RBCs) used are dependent on the type of influenza virus in the assay (Table 1). Further, for various types of 96-well microtiter plates, the incubation time as well as the appearance of the non-agglutinated cells differ (Table 2).
5. HI Assay
NOTE: The workflow of the protocol has been optimized to allow more efficient handling of multiple samples at the same time, by using polymerase chain reaction (PCR) tube stripes and a thermocycler (see below).
Table 1: Influenza antigens and corresponding species of RBCs. According to the manufacturer's instructions (NIBSC).
Influenza antigen | A/California/7/09 (H1N1) | A/Switzerland/9715293/2013 (H3N2) | A/Texas/50/2012 (H3N2) | B/Brisbane/60/08 | B/Massachusetts/02/2012 |
RBC species | Chicken | Guinea Pig | Guinea Pig | Turkey | Turkey |
Table 2: Assay conditions with different species of RBCs. According to the WHO protocol. (* flows when tilted).
RBC species | Chicken | Turkey | Guinea pig | Human type O |
Concentration of RBCs (v/v) | 0.75% | 0.75% | 1% | 1% |
Type of microtiter plate | V bottom | V bottom | U bottom | U bottom |
Incubation time, RT | 30 min | 30 min | 1 hour | 1 hour |
Appearance of non-agglutinated cells | Button* | Button* | halo | halo |
Figure 1: Principle of hemagglutination and hemagglutination inhibition. No hemagglutination occurs in a negative control situation without viruses and antibodies (left column), and erythrocytes hemagglutinate only in the presence of influenza virus (middle column). However, when the hemagglutinin of the influenza virus is blocked by virus-specific antibodies then no hemagglutination can occur (right column).
Figure 2: Plate design of the HA assay. The HA titration is performed in duplicates. No antigen was added to the control rows. Also, see Figure 4 for the determination of the best antigen concentration.
Figure 3: Agglutination patterns of avian and mammalian RBCs. V-shaped microtiter plates are used when working with avian RBCs. The readout is performed in a tilted plate position, and non-agglutinated RBCs start to run down forming a tear-like shape. U-shaped microtiter plates are used when working with mammalian RBCs. The readout is then performed in a non-tilted position, and non-agglutinated RBCs form a small halo.
Figure 4: Readout of the HA titration with avian RBCs to determine the titer of 4 HA units. The optimal antigen amount required for hemagglutination is measured by the hemagglutination assay (antigen titration assay). The last well where complete hemagglutination occurs is the HA titration endpoint which contains 1 HA unit. Because of the 2-fold dilutions of the antigen, two wells ahead of the HA titration endpoint, the titer corresponds to 4 HA units.
Figure 5: Plate design and workflow of the HI assay. Five timepoints of two people can be measured on one plate. The HI titer ranges from 8 to 1,024. An anti-serum of the used antigen served as a positive control and a back titration was performed to check if the antigen dilution equals 4 HA units. The serial dilution of the serum sample is shown for 2 individual vaccine recipients.
The authors have nothing to disclose.
25 ml Disposable Multichannel Pipette Reservoirs | Integra | 4312 | |
8-well PCR tubes | Brand GMBH | 781332 | For serum aliquots |
96-well microtiter plate, U-shaped | TPP | 92097 | For HI assay when using mammalian RBCs |
96-well microtiter plate, V-shaped | Corning Costar | 3897 | For HI assay when using avian RBCs |
Aqua ad iniect. Steril | Bichsel AG | 1000004 | For preparing influenza antigen and cholera filtrate solutions |
Chicken RBC (10%) | Cedarlane | CLC8800 | 10% suspension of chicken red blood cells in Alsever's solution |
Cholera filtrate | Sigma-Aldrich | C8772 | Used as receptor destroying enzyme (RDE) |
Dulbecco's PBS | Sigma-Aldrich | D8537 | For diluting the serum samples, RBCs and antigens |
Eppendorf Multichannel pipette, 12-channel, 10-100 µl | Sigma-Aldrich | Z683949 | |
Eppendorf Multichannel pipette, 8-channel, 10-100 µl | Sigma-Aldrich | Z683930 | |
Guinea Pig RBC (10%) | Cedarlane | CLC1800 | 10% suspension of guinea pig red blood cells in Alsever's solution |
Influenza Anti-A/California/7/09 HA serum | NIBSC | 14/134 | Used as positive control at the HI assay |
Influenza Anti-A/Switzerland/9715293/2013-like HA serum | NIBSC | 14/272 | Used as positive control at the HI assay |
Influenza Anti-A/Texas/50/2012-Like HA Serum | NIBSC | 13/178 | Used as positive control at the HI assay |
Influenza Anti-B/Brisbane/60/2008-HA serum | NIBSC | 13/254 | Used as positive control at the HI assay |
Influenza Anti-B/Massachusetts/02/2012 HA serum | NIBSC | 13/182 | Used as positive control at the HI assay |
Influenza antigen A/California/7/09 (H1N1)(NYMC-X181) | NIBSC | 12/168 | Inactivated, partially purified A/California/7/09 (H1N1)(NYMC-X181) virus (ca. 46µgHA/ml) |
Influenza antigen A/Switzerland/9715293/2013 (NIB88) | NIBSC | 14/254 | Inactivated, partially purified A/Switzerland/9715293/2013 (NIB88) virus (ca. 55µgHA/ml) |
Influenza antigen A/Texas/50/2012 (H3N2)(NYMCX-223) | NIBSC | 13/112 | Inactivated, partially purified A/Texas/50/2012 (H3N2)(NYMCX-223) virus (ca. 74µgHA/ml) |
Influenza antigen B/Brisbane/60/2008 | NIBSC | 13/234 | Inactivated, partially purified B/Brisbane/60/2008 virus (ca. 42µgHA/ml) |
Influenza antigen B/Massachusetts/02/2012 | NIBSC | 13/134 | Inactivated, partially purified B/Massachusetts/02/2012 virus (ca. 35µgHA/ml) |
Serum-Tubes | S-Monovette, Sardstedt | 01.1601.100 | For serum extraction with clotting activator |
Turkey RBC (10%) | Cedarlane | CLC1180 | 10% suspension of turkey red blood cells in Alsever's solution |
Phosphate Buffered Saline (PBS) | Gibco |