Enterovirus seroneutralization assays
Neutralizing antibodies were measured in serum or plasma against 41 EV
serotypes listed in Table 2 as previously described23. Viruses were isolated and cultivated, and
seroneutralization assays were performed using the A549, Vero, RD (all
bought from American Type Culture Collection) and GMK (from the National
Institute for Health and Welfare, Finland) cell lines. Most of the
viruses were analyzed using a standard plaque neutralization assay24, whereas viruses that did not form clear plaques
were analyzed using a microneutralization assay (CAV5, CAV6, CAV10,
CAV16, EV71, EV78 and EV94). The neutralization assay was considered
positive when the serum dilution 1:4 reduced the number of plaques by
more than 75% (plaque assay) or inhibited the ability of the virus to
kill cells (microneutralization assay). The serum/plasma samples
analyzed were taken between 1 and 5 years of age. The sample volume was
adequate for the analysis of neutralizing antibodies against all 41
serotypes in 183 children. In 83 children the volume was adequate for
the analysis of 39-40 serotypes and in nine children for 4-21 serotypes.