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

Abstract

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay). As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

Antigen Extraction and B Cell Activation Enable Identification of Rare Membrane Antigen Specific Human B Cells

“…The titer of influenza A/California/04/09 (H1N1) antibody in B cell culture supernatant samples was measured by HI assay according to the World Health Organization (WHO) protocol manual on animal Influenza diagnosis and surveillance (WHO/CDS/CSR/NCS/2002.5 Rev. 1), following our previously described procedure (15). The neat supernatant samples were pre-treated with 3-fold of cholera filtrate (cat no.…”

Influenza A haemagglutinin specific IgG responses in children and adults after seasonal trivalent live attenuated influenza vaccination

“…Based upon the properties of influenza virus to agglutinate the erythrocyte, the HI assay can be used with human sera to measure the inhibitory antibody concentration [210] . There are several methods to quantify viral strain specific antibodies but the HA inhibition assay is commonly used and immensely reliable to detect antibodies to the circulating viruses in sera.…”

Fc-optimized antibodies elicit CD8 immunity to viral respiratory infection

“…Haemagglutination inhibition assay HAI activity was evaluated using previously described protocols 34 . In brief, serum samples (diluted 1:3 in cholera filtrate) or Fc domain variants of antibodies (starting concentration at 100 μg ml −1 followed by 1:3 serial dilutions) and viruses (A/Puerto Rico/8/34 or A/Netherlands/602/09; 10 7 pfu ml −1 ) were incubated in V-bottom 96 microtitre plates for 30 min at room temperature.…”