Microneutralization assay for the measurement of neutralizing antibodies to human metapneumovirus

Abstract 

Background

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

Objective

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 ∼50pfu of hMPV A or B strain for 60min 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.

Results

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).

Conclusion

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