A simplified and standardized neutralization enzyme immunoassay for the quantification of measles neutralizing...

A simplified and standardized neutralization enzyme immunoassay (Nt-EIA) was developed to detect measles virus growth in Vero cells and to quantify measles neutralizing antibody. Heat-inactivated sera were diluted serially 4-fold and tested in duplicate. The 50% reduction point (50%RP) of virus growth was calculated using the Reed-Muench formula and the neutralizing antibody titre of test sera was converted into mIU/ml by comparing their 50%RP with that of the international standard serum. The optimal virus input and incubation time were found to be 50-100 plaque forming unit (PFU)/well and 64-72 h, respectively. The simplified Nt-EIA had a good reproducibility with only 3.7-4.2% of duplicate tests having a ratio > 4 in an evaluation of intra assay variation and the coefficients of variance were 2-9% in an evaluation of inter assay variation. In addition, the simplified Nt-EIA had a high sensitivity(98.6%), specificity (100%) and agreement (98.8%) in qualitative comparison with plaque reduction neutralization test (PRNT). In quantitative comparison, the correlation coefficient between Nt-EIA and PRNT was 0.83 without log transformation or 0.77 after log transformation and 90% of 61 positive sera had a ratio < 4 between antibody titre tested by the two methods. The simplified Nt-EIA is thus a suitable alternative to the PRNT for the quantification of measles neutralizing antibody.