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

Published: November 30, 2023

Abstract

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.

Protocol

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 Collect serum samples from humans at time points of interest. For this study, we collected sera at days 0 (time of influenza vaccination), +7, +30, +60, and +180 after vaccination. To obtain the serum, centrifuge the…

Representative Results

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 occ…

Declarações

The authors have nothing to disclose.

Materials

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

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Citar este artigo
The Hemagglutination Inhibition Assay to Detect Serum Antibodies Against a Target Antigen. J. Vis. Exp. (Pending Publication), e21787, doi: (2023).

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