Mumps virus-neutralizing antibodies are believed to be the most predictable surrogate marker of protective immunity. However, assays used to detect neutralizing antibodies, such as the plaque reduction neutralization (PRN) assay, are labor-and time-intensive and consequently are often supplanted by the more rapid and inexpensive enzyme immunoassay (EIA) technique. For virus infections for which international antibody standards exist and are bridged to clinical studies of protection (e.g., measles and rubella), the EIA has been successfully used to determine immune surrogate endpoints, yet no such international reference exists for mumps serology. Since both virus-neutralizing and nonneutralizing antibodies are measured in the EIA, in the absence of a mumps serological standard, the EIA may be prone to yielding false-positive results when utilized for assessing surrogate markers of protective immunity. Moreover, since mumps virus-specific antibody titers are generally low in comparison to antibody levels induced by other viruses and EIA procedures often employ relatively high serum dilution factors, the EIA may be prone to yielding false-negative results. To examine these issues, a PRN assay and two commercially available EIA kits were used to evaluate wild-type mumps virus serological responses in human serum samples from the pre-mumps vaccine era. Our results indicate that the PRN assay is a more sensitive and specific method of measuring serological responses to wild-type mumps virus.Protective efficacy field studies have shown that mumps virus-neutralizing antibody titers as low as 1:2 provide protection against mumps (10, 30-32). Accordingly, virus neutralization assays, such as the plaque reduction neutralization (PRN) assay, have long been the "gold standard" in determining the presence of protective immunity against mumps virus infection (3, 24, 32). The PRN assay measures the serum dilution (titer) capable of preventing 50% of plaque formation by mumps virus in cell cultures. Although virus neutralization assays may be the most predictive technique for assessing protective immunity, these assays are often not standardized and are extremely skilled labor-and time-intensive, making examining large numbers of human sera by PRN assay difficult. In contrast, the enzyme immunoassay (EIA) technique is simpler to perform and provides rapid, quantitative results and, thus, is the most widely used technique in clinical serology testing. However, since the EIA does not distinguish neutralizing from nonneutralizing antibodies, this assay may be prone to yielding false-positive results in the context of assessing protective immunity. In many cases, e.g., measles and rubella serology, an international standard referenced to a protective serum titer is used within the context of the EIA to provide a reasonable immune surrogate marker of protection. No such mumps standard exists. Further, since mumps virus-neutralizing antibody titers are often low (less than or equal to 1:8) and EIA procedures require initial serum dil...