Journal of Clinical Virology

Volume 46, Issue 4, December 2009, Pages 314-317

Microneutralization assay for the measurement of neutralizing antibodies to human metapneumovirus rights and content



Human metapneumovirus (hMPV) is a newly discovered virus which causes respiratory illness in persons of all ages.


A simple and rapid method to determine neutralizing antibody titers against hMPV is needed to facilitate the development of vaccines and therapeutics for hMPV. Therefore, we sought to adapt the methodology used for RSV microneutralization assay (MNA) to measure neutralizing antibody titers against hMPV.

Study design

Serial 2-fold dilutions of serum were made in 96 well microtiter plates and incubated with ∼50 pfu of hMPV A or B strain for 60 min at room temperature. LLC-MK2 cells were added to the serum–virus mixtures and plates incubated at 35 °C in CO2 for 5 days. Plates were fixed with acetone; air dried, blocked and then developed with monoclonal antibody to the hMPV N protein followed by horse radish peroxidase labeled antibody and substrate. Neutralization titer was defined as the titer of serum that reduced color development by 50% compared to the positive control wells.


Titers measured by MNA correlated well with those determined by standard plaque reduction assay (R = 0.77). Neutralization titers determined by MNA demonstrated excellent inter-assay variability (coefficient of variance = 7%). In addition, there was good correlation of antibody titers from 10 hMPV infected adults measured by MNA using either group A or group B hMPV (R = 0.87).


MNA is a simple and reproducible method for the measurement of serum neutralizing antibody against hMPV.

Section snippets


Human metapneumovirus (hMPV) is an RNA virus in the pneumovirus subfamily of Paramyxoviridae first described in young children with bronchiolitis from the Netherlands.1 Since its discovery in 2001, the virus has been shown to have world wide circulation and to be a common cause of respiratory illness in persons of all ages.2 Similar to a related virus, respiratory syncytial virus (RSV), immunity to hMPV appears to be incomplete and re-infections occur throughout adulthood.3 Correlates of


In this report, we describe the development of a MNA assay for use with trypsin dependent strains of hMPV as a simple and accurate method for the measurement of hMPV neutralizing antibody titers.

Virus culture

LLC-MK2 cells and Vero cells were maintained in MEM, 5% fetal calf serum (FCS). The CAN 97-83 (group A) and CAN 98-75 (group B) strains of hMPV were kindly provided by Dr. Guy Boivin at Laval University Quebec, Canada. Viruses were grown in LLC-MK2 cells with viral growth media (VGM) consisting of 1% bovine serum albumin, 1% HEPES, 0.5% glucose and porcine pancreatic trypsin (2 units/ml). The titer of the viral stock was quantified at 1.3 × 106 using a plaque assay described below.

Plaque assay

Twenty-four well

Plaque reduction neutralization assay

Serum neutralizing antibody was measured using a modification of a plaque assay as described by Williams et al.5 Twenty-four well plates were set with 2 × 105 cells/ml Vero cells to achieve ∼80% confluence the next day. Ten serial 2-fold dilutions of sera starting at 1:50 were incubated at RT for 60 min with an equal volume hMPV to provide approximately 50 plaque forming units (pfu)/200 μL. Two hundred microliters of the serum–virus mixtures were added in duplicate to cell monolayers and allowed to


Twenty acute and convalescent serum pairs were analyzed using the microneutralization assay using CAN 97-83 (MNA-A) and CAN 98-75 (MNA-B) strains of hMPV. Twenty samples (10 acute and convalescent pairs) were run in three assays using CAN 97-83 hMPV performed on different days with excellent inter-assay variability (coefficient of variance = 7%). The mean log 2 titers for MNA-A and MNA-B values were 11.53 ± 1.96 and 11.90 ± 2.10, respectively. The correlation of MNA-A and MNA-B values was high with a


Adapting the MNA methodology used for RSV to hMPV was complicated by the nature of LLC-MK2 cells and the need for trypsin in the growth media of hMPV. In the RSV assay, EDTA is used to disperse Hep-2 cells but was not effective for LLC-MK2 cells and resulted in an poorly adherent irregular monolayer. While the use of bovine pancrease trypsin produced a homogenous single cell suspension of LLC-MK2 cells, a monolayer would not set in the 96 well plates with the trypsin present when added to the


In summary, MNA is a simple and reproducible method for the measurement of serum neutralizing antibody against hMPV. This assay should be useful in studies of disease pathogenesis and facilitate clinical studies of hMPV vaccine candidates.

Conflicts of interest

Dr Falsey consults for Medimmune, GSK, Sanofipasteur, Astrazeneca, and ADMA Biologics and has received honoraria. She also serves on the advisory board of Quidel Inc. Dr Walsh consults for Astrazeneca and ADMA Biologics and has received honoraria. Ms. Formica has no conflicts of interest.


The authors wish to thank Patricia A. Hennessey and Mary Criddle for collection of serum samples and Jamie Biear for technical assistance.

References (16)

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    The neutralization assay based on GFP expression (NA-GFP) eliminated the immunostaining procedures and shortened the time for completion. Another assay, a microneutralization assay, which had been used for the measurement of neutralizing antibodies to RSV, was adapted to HMPV.14 It was performed by adding suspended cells to an antibody-virus mixture followed by an enzyme immunoassay with monoclonal antibody (mAb) to the HMPV N protein, and the titer was defined on the basis of 50% reduction of color development.

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    The automation of AFRNT evaluation is based on image analysis using an AID reader and ViruSpot software. Until now, imaging analysis has not been commonly used for neutralisation tests, while focus reduction assays using peroxidase staining of infected cells are common (Falsey et al., 2009; Vaidya et al., 2010; Eggers et al., 2000; Fournier et al., 2007). Only a few studies have described the use of imaging analysis for neutralisation tests.

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    However, we have previously demonstrated nearly identical serologic response by EIA using the either CAN 97-83 (group A) and CAN 98-75 (group B) strains of hMPV as antigen regardless of the infecting viral strain [8]. In addition, we have recently found that serum neutralizing antibody titers to group A and group B viruses are highly correlated (R = 0.87) [27]. Minimal differences in neutralizing activity directed against group A and group B viruses is not surprising since the conserved F protein has been found to be highly immunogenic and protective, whereas the antigenically more diverse G protein is not [28].

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