There has not been an opportunity to evaluate the level of neutralizing antibodies that are protective against rubella in a similar manner as was done for measles. Instead, expert committees established a minimum level of antibodies that are considered protective against rubella disease based on studies of persons exposed to wild rubella virus [5]. At the time of those studies, the HAI assay was the gold standard technique for detection of anti-rubella antibodies. However, the HAI could be affected by nonspecific inhibitors of agglutination, producing false-positive results. Serial dilutions ≥1:8 were not affected by this and thus an HAI titre of 8 (1:8) was set as the threshold titre to define immunity to rubella. The corresponding concentration in international units was 15 IU/ml [5].
Improvements in methodology led to the replacement of the HAI by other methods including passive latex agglutination tests, immunofluorescent assays, radial haemolysis tests and virus neutralization. However, most laboratories have adopted the EIA for detection of rubella IgG antibodies. Most of the rubella IgG EIAs have been calibrated against an international rubella standard available at the time of assay development and provide quantitative results in IU/ml. Many of the first generation of rubella IgG EIAs have been replaced by automated systems and some of these utilize different detection systems including chemiluminescence [11].
A reduction of the cut-off or threshold for protection from 15 to 10 IU/ml has been accepted by most programmatic users of rubella assays [11]. However, the absence of detectable rubella-specific IgG does not necessarily correspond to susceptibility, nor does the presence of antibody measured at >15 IU/ml guarantee protection, as some rubella reinfections have occurred despite demonstration of titres >15 IU/ml [5]. Rubella IgG EIAs are calibrated using either of the two contemporary rubella standards, RUB-1-94 or RUBI-1-94.
The origin and description of the rubella standards is complex and beyond the scope of this manual and is available elsewhere [10,14]. However, the international standards for rubella were not developed according to metrological principles. This compromises the suitability of the standard for use as an analyte for calibration of rubella IgG EIAs. Investigators have recognized the need for standardization among rubella IgG assays and have raised questions regarding the accuracy and appropriateness of the 10 IU/ml cut-off in assessing immunity to rubella. Many have also voiced concerns regarding the validity of quantitative reporting of rubella IgG results [10-14].
Similar to tests that measure measles neutralizing antibody, the assays for measuring rubella neutralizing antibody titre have not been suitable for use in population studies. However, a rubella virus-specific immunocolorimeric neutralization assay, adapted to a high-throughput system for measuring neutralizing antibody titres with high sensitivity and good reproducibility has been described [21]. The neutralization titres (Nt) provided by this assay were shown to have good concordance with results of serum specimens tested by a commercial immunoblot (IB) test [11]. In a study comparing the sensitivity and specificity of eight commercial rubella IgG assays, serum specimens with Nt/IB concordant results were used for the evaluation. The results supported evidence in previous reports that showed no loss in specificity when equivocal results obtained in the rubella IgG EIAs are interpreted as positives. The protocol for the rubella neutralization assay (developed at CDC) is provided in Annex 9.1.