Comparison of hemagglutination inhibition assay, an ELISA-based micro-neutralization assay and colorimetric microneutralization assay to detect antibody responses to vaccination against influenza A H1N1 2009 virus

Abstract

The hemagglutination inhibition (HI) assay has been the main method used to investigate immune responses to vaccination against influenza H1N1 (2009) virus. However microneutralization tests (MNT) have been shown to be more sensitive and more specific. In this study, the three methods of choice: (i) the HI assay, (ii) an ELISA-based conventional MNT and (iii) a colorimetric MNT in terms of their ability to detect antibody responses in serum pairs collected from 43 healthy individuals before and 21 days after vaccination were compared. The colorimetric MNT was established yielding intra- and inter-run imprecisions of 7.5% and 12.4%, respectively. Testing of antisera to seasonal influenza viruses demonstrated the assay to be specific for antibodies to influenza H1N1 (2009) virus. A good correlation between the three methods was found, being highest for the ELISA-MNT and the colorimetric MNT (r = 0.714 for geometric mean titers (GMT) and r = 0.695 for titer increases). Similar rates of fourfold titer increases were detected: 95.3% in the ELISA-MNT vs. 93.0% in colorimetric MNT and 95.3% in HI assay. The ELISA-based MNT demonstrated the highest titer range leading to the highest postvaccination GMT and the highest titer increase (>50-fold). The lowest GMTs were measured with the HI assay, while the colormetric MNT detected the highest GMT in prevaccination sera. Taken together, similar seroconversion rates were obtained with the three assays. The ELISA-MNT appeared to be the best method to compare absolute pre- and postvaccination GMTs. The colorimetric MNT, being less labour-intensive than the ELISA-MNT, seems to be a suitable tool in vaccination studies.

Introduction

Clinical trials have investigated immune responses to the monovalent split-virus inactivated vaccine against influenza H1N1 (2009) virus (Liang et al., 2010, Plennevaux et al., 2010, Zhu et al., 2009). In these studies antibody responses were measured mainly by the hemagglutination inhibition (HI) assay because this method can be easily adapted to new influenza viruses. However, it has been shown for seasonal and for avian influenza that microneutralization tests (MNT) are more sensitive and more specific (Frank et al., 1980, Gitelman et al., 1986, Rowe et al., 1999). A previous study for seasonal influenza A indicated that strain-specific IgG antibodies with neutralizing capacity may be present, but these antibodies were not generally detected in homologous HI assays (Remarque et al., 1998). Conventional microneutralization tests are performed in different ways with regard to titer determination, cytopathic effect formation in cell cultures, red blood cells for detection of released virus or usage of the ELISA technique to identify virus infected cells. Especially the ELISA method is time-consuming, difficult to perform under biosafety-level 3 (BSL-3) conditions and requires the need for monoclonal antibodies that are not widely available for new viruses. Recently, a colorimetric MNT has been introduced for seasonal influenza (Lehtoranta et al., 2009). This assay is an adaptation of micro-cytotoxicity tests (van de Water et al., 1993) and is based on the cleavage of a tetrazolium salt by mitochondrial dehydrogenases reflecting the amount of viable and metabolically active cells.

In the present study, the three assays of choice: (i) HI assay, (ii) ELISA-MNT and (iii) colorimetric MNT were compared to detect immune responses in serum pairs collected from 43 healthy individuals before and 21 days after administration of a monovalent vaccine against pandemic influenza H1N1 (2009) virus. The colorimetric MNT was adapted and validated to detect antibodies to pandemic influenza and the method was modified to include less hands-on manipulations.