The measles plaque reduction neutralization (PRN) assay detects functional neutralizing antibodies which offer the best correlate to protection in vivo . Prior to development of the PRN assay, the hemagglutination inhibition (HAI) assay had been in widespread use, but has been replaced by the PRN assay, which has superior sensitivity and consistency than the HAI assay. The PRN assay is regarded as the gold standard for evaluation of the humoral immunity to measles.
The PRN test requires enumeration of plaques formed by the cytopathic effect induced by measles virus growth in Vero cells. Assays are generally performed in 12- or 24-well plates using serial dilutions of test serum alongside reference or standard serum samples. Results can be quantified and compared to the protective level of neutralizing antibody in mIU/ml. The serum titre used to determine measles immunity is based on the evaluation of PRN titres that appeared to provide protection from clinical disease during a measles outbreak . In that study, an outbreak of measles occurred at a Boston college that included students who had participated in a blood drive prior to exposure. The PRN titre that corresponded to the protective titre was ≥120 mIU/ml when standardized against the WHO measles antibody international standard (currently the WHO 3rd international standard; NIBSC 97/648).
The PRN titre is calculated from the dilution that reduces the number of plaques by 50%, transformed into mIU/ml using a conversion based on the concentration of the measles international standard and the end point titre of the standard in the assay run. Standardization and use of appropriate controls to ensure test validity of PRN assays is critical. PRN assays are difficult to standardize, are technically demanding, and take up to seven days to complete. In practice, there is significant inherent variation in the assay, which is addressed by testing the serum in triplicate . Therefore, the PRN assay is impractical for large-scale surveys and is used primarily for vaccine immunogenicity trials and for studies leading to vaccine licensure and can be used to validate IgG EIA data.
In many countries, there are several commercially available measles IgG EIAs. The commercial EIAs vary according to the antigen source (e.g., purified viral lysate or recombinant viral proteins) and the test format. Many assays utilize a recombinant measles nucleoprotein for the antigen as antibodies directed to this protein are the most abundant following infection or vaccination . While absence of an immune response to the nucleoprotein suggests susceptibility to infection, it is the neutralizing antibodies that are directed against the measles hemagglutinin (and to a lesser extent, the fusion protein) that confer protective immunity . The performance of different kits is variable, particularly for serum with antibody measured in the low positive range. However, positive results using well-validated EIAs show a good correlation with results obtained by the measles PRN assay [7,8,12]. The EIAs that are described as semi-quantitative include a formula for converting optical density values into titre units, but the titres are intended to evaluate whether a significant rise in titre has occurred and are not equivalent to a PRN titre.
Suboptimal sensitivity for detection of measles IgG by EIA is a particular concern in cohorts with vaccine-induced immunity. Studies have demonstrated that serum specimens that fall into the equivocal range for IgG using several of the commercial EIA kits often have PRN titres ≥120 mIU/ml [7,8,12]. One approach is to screen the serum specimens by EIA and those specimens with results in the equivocal range (and a percentage of negatives) can be evaluated by PRN [7,8].
There has been over 10 years of experience with an in-house multiplex immunoassay (MIA) based on Luminex Technology [8,20]. It is basically a bead-based ELISA. The method is easy to multiplex in one reaction and requires less serum than required for testing in an EIA. Beads can be engineered in the laboratory and the antigen is linked to color-coded beads. The assays have correlated well with other methods such as PRN and has both anti-F and anti-H IgG functionality.