Mumps Virus-Specific Antibody Titers from Pre-Vaccine Era Sera: Comparison of the Plaque Reduction Neutralization Assay and Enzyme Immunoassays

Abstract

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...

Estimates of Mumps Seroprevalence May Be Influenced by Antibody Specificity and Serologic Method

“…Several reports have indicated that mumps-neutralizing antibody does not correlate particularly well with mumps IgG ELISA measurements (25)(26)(27)(28)(29)(30)(31)(32)(33). An explanation for these observations is that mumps antibodies predominantly measured by ELISAs which use whole-virus antigen differ from those measured by plaque neutralization tests.…”

“…Although neutralizing antibody is thought to be essential for protection (40) and PRN titration is generally considered to be the gold standard for mumps serology, previous attempts to determine a reliable threshold for protective PRN titers have been unsuccessful (20). To add further ambiguity, there have been several reports of poor correlations between mumps PRN titration and various other serologic methods, such as ELISA and immunofluorescent assays (IFA) (25)(26)(27)(28)(29)(30)(31)(32)(33)(34). These studies indicated that mumps serologic data must be interpreted with caution.…”

“…As a result, it has been widely replaced by the faster and more convenient ELISA. Interestingly, however, numerous reports (25)(26)(27)(28)(29)(30)(31)(32)(33)(34) indicate that the correlations between mumps PRN titers and IgG ELISAs, which use whole virus as the target antigen, range from poor (32) to significant but imperfect (31). Here, we report the development of mumps NP-and HN-specific ELISA methods that facilitate the analysis of sera with higher degrees of resolution than whole-virus ELISA methods.…”

Safety and immunogenicity of a live attenuated mumps vaccine

“…The cross-neutralization assay using SP-A strain of F genotype and S79 strain of A genotype was applied to determine the neutralizing antibody titer, according to previously described methods. 36 The sera were diluted in series, and the neutralization assay was performed on Vero cells using diluted 500 CCID50 SP-A and S79 viruses for incubation at 37 °C over 7 d for CPE observation. There is no known protective level of neutralizing antibody (antibody titer) for mumps, and there are no other immune parameters that correlate with protection from the mumps disease.…”

“…Some researchers report that protective efficacy field studies have shown that mumps virus-neutralizing antibody titers as low as 1:2 provide protection against mumps, 37 and mumps virus-neutralizing antibody titers are often low (less than or equal to 1:8). 36 Therefore, considering the above factors, the concentration of one-half of the lower limits of detection and quantittation (LLOQ) is used as the standardard assigned to a negative sample for the purpose of calculating GMTs. For comparison purposes, the ELISA assay was also performed using a mumps IgG ELISA reagent kit (Demeditec Corp., demum01), in which, the IgG measured by the purified mumps antigen included the neutralizing antibody against mumps virus and the antibody interacting with the virus.…”