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 molecular fine specificity of antibody responses in different individuals with latent or active human cytomegalovirus (HCMV) infection. An overview of the HCMV proteins recognized by human antibodies was obtained by immunoblotting. For selected immunodominant proteins the epitope fine specificity of the antibody response was determined by a peptide-scanning enzyme-linked immunosorbent assay (ELISA). Epitope clusters were synthesized as combination peptides and were used for further serologic analysis of immunoglobulin M (IgM) and IgG reactivities with panels of sera from different groups of patients in comparison to those with cytomegalovirus (CMV) virion antigen. Several serum samples had significantly higher reactivities with peptides than with the CMV virion antigen. However, individual serum samples occasionally recognized diverse peptide epitopes, stressing the importance of using combinations of peptides in serologic assays. From these studies we were able to define a specific combination of peptides derived from pp52 (UL44) and pp150 (UL32) for the specific and highly sensitive early detection of HCMV IgM, 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 sensitivities of 98.9 and 96.4% for IgG and IgM, respectively, in comparison with the results obtained with the "gold standard," the virion antigen-based ELISA. From the results of this study we conclude that specific combinations of highly defined synthetic peptides can replace complex HCMV virion extracts used in current serodiagnostics and may add to further standardization of HCMV serology.
Greijer
A E
AE
Organon Teknika, Boxtel, The Netherlands. a.greijer@teknika.btl.akzonobel.nl
van de Crommert
J M
JM
Stevens
S J
SJ
Middeldorp
J M
JM
D000914:Antibodies, Viral / Q000276:immunology*; D000918:Antibody Specificity; D003587:Cytomegalovirus / Q000276:immunology* / Q000302:isolation & purification; D003586:Cytomegalovirus Infections / Q000276:immunology* / Q000821:virology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D016056:Immunodominant Epitopes / Q000032:analysis / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology; D016030:Kidney Transplantation; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology*; D012680:Sensitivity and Specificity; D012698:Serologic Tests; D014184:Transplantation, Homologous
D000914:Antibodies, Viral; D016056:Immunodominant Epitopes; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D010455:Peptides