Detection of rubella virus-specific immunoglobulin G (IgG), IgM, and IgA antibodies by immunoblot assays

Abstract

Immunoblot (IB) assays were developed for detection of rubella virus (RV)- specific immunoglobulin G (IgG), IgM, and IgA antibodies in human serum following natural infection or immunization. IB assays performed under nonreducing conditions were compared with those performed under reducing conditions and with immunoprecipitation assays. Significant loss of antigenicity (>90%) of RV E1 and E2 proteins was observed when IB assays were performed in the presence of 2-mercaptoethanol as compared with assays under nonreducing conditions. In contrast, the antigenicity of RV capsid protein was not influenced by reducing agents. Sensitivity of IB for RV-specific IgG antibodies was determined to be 0.01 IU/ml under nonreducing conditions. In the determination of RV-specific IgM and IgA antibodies by IB, pretreatment of serum with protein G to remove competing high-affinity RV-specific IgG or rheumatoid factor significantly improved assay sensitivity. IB assays were observed to be superior to immunoprecipitation assays in their ability to better define the specificities of RV-specific antibodies and to detect antibodies of all immunoglobulin classes. However, the conformational sensitivity of RV protein antigenicity should be an important consideration in the interpretation of RV-specific antibodies by IB assays.