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

Summary

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

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.

Introduction

Infection with influenza virus is associated with considerable morbidity, mortality, and high healthcare costs^1,2,3,4. In particular, elderly, newborns, pregnant women, and patients with chronic disease are at risk for more severe clinical outcomes. Therefore, vaccination against circulating influenza virus strains is the primary measure to decrease the burden of disease in these high-risk populations. The increase of the individual immune response after vaccination, e.g., influenza-specific antibodies above a protective threshold, ....

Protocol

The study protocols were approved through the local ethical review board (www.EKNZ.ch) and written informed consent was obtained from all participants.

1. Serum Collection

1. 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.
2. To obtain the serum, centrifuge the sample tubes at 1,200 x g for 10 min at room temperature (20 - 25 °C).
   NOTE: Non-centrifuged blood samples should be stored at 4 °C, and for no longer than 24 h.
3. Aliquot the serum into different tubes (cryo-via....

Results

Pre- and post-vaccination induced antibody response against Influenza A H3N2
The vaccine-induced antibody response was assessed in 26 healthy volunteers who received an inactivated trivalent subunit influenza vaccine containing Influenza A/H1N1/California/2009, A/H3N2/Texas/2012, and B/Massachusetts/02/2012 prior to the 2014/2015 influenza season. Figure 6 shows a representative example of 2 vaccine recipients. Interestingly, during tha.......

Discussion

Quantification of pre- and post-vaccination influenza virus specific antibody titers is an important tool necessary for vaccine studies. Based on the surrogate measures of protection against virus infection, such as seroprotection (>1:40) or seroconversion (4-fold titer increase), vaccination strategies can be optimized⁹. Using the provided protocols can determine: (i) the hemagglutination potential of a particular virus, and (ii) the antibody titers for a virus of interest.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
Single Donor Human RBC, Type 0	Innovative Research	IPLA-WB3	Suspension of single donor human red blood cells in Alsever's solution (ca. 26%)
Turkey RBC (10%)	Cedarlane	CLC1180	10% suspension of turkey red blood cells in Alsever's solution
Phosphate Buffered Saline (PBS)	Gibco