Molecular fine-specificity analysis of antibody responses to human cytomegalovirus and design of novel synthetic-peptide-based serodiagnostic assays

To identify single immunodominant marker proteins which can replace complex virion antigen in serodiagnostic assays, we investigated in detail the mol ecular fine specificity of antibody responses in different individuals with latent or active human cytomegalovirus (HCMV) infection. An overview of th e HCMV proteins recognized by human antibodies was obtained by immunoblotti ng. For selected immunodominant proteins the epitope fine specificity of th e antibody response was determined by a peptide-scanning enzyme-linked immu nosorbent assay (ELISA). Epitope clusters were synthesized as combination p eptides and were used for further serologic analysis of immunoglobulin M (I gM) and IgG reactivities with panels of sera from different groups of patie nts in comparison to those with cytomegalovirus (CMV) virion antigen. Sever al serum samples had significantly higher reactivities with peptides than w ith the CMV virion antigen. However, individual serum samples occasionally recognized diverse peptide epitopes, stressing the importance of using comb inations of peptides in serologic assays. From these studies we were able t o define a specific combination of peptides derived from pp52 (UL44) and pp 150 (UL32) for the specific and highly sensitive early detection of HCMV Ig M, whereas a combination of peptides from pp150 (UL32), gB (UL55), and pp28 (UL99) was selected to give optimal and specific reactivity with HCMV IgG. On the basis of the results obtained with these peptide combinations, new, highly specific serodiagnostic assays were constructed. These assays had s ensitivities of 98.9 and 96.4% for IgG and IgM, respectively, in comparison with the results obtained with the "gold standard," the virion antigen-bas ed ELISA. From the results of this study we conclude that specific combinat ions of highly defined synthetic peptides can replace complex HCMV virion e xtracts used in current serodiagnostics and may add to further standardizat ion of HCMV serology.