Development of a microwell adapted immunoblot system with recombinant antigens for distinguishing human herpesvirus (HHV)6A and HHV6B and detection of human cytomegalovirus

Background: The human cytomegalovirus (HCMV) and the human herpesvirus 6 (HHV6) are widely distributed in the human population. The variants A and B of HHV6 are closely related to each other and cannot be distinguished by common serological methods like enzyme-linked immunosorbent assay (ELISA) or immunofluorescence test (IFT). The aim of this study was to develop a microwell-adapted blot system for specificity detection of human cytomegalovirus and human herpesvirus 6A and 6B (HHV6A, HHV6B) that combines the advantages of ELISA (automation and multiplex detection) and immunoblotting (antigen-specific antibody detection with high specificity). Methods: Ten HCMV, five HHV6A and five HHV6B antigens were expressed as fusion proteins and tested with sera of children (n=30), of healthy young adults (n=30) and of older adults (n=30) in a newly developed microblot system. Results: Sensitivity and specificity of HCMV and HHV6 microblots were comparable to commercially available ELISA, IFT and to line assay tests. The advantage of the HHV6 microblot is the possibility of distinguishing between HHV6A-monovalent sera, HHV6B-monovalent sera and HHV6A/B-polyvalent sera. Most sera of children younger than 2 years showed only HHV6B antigen positivity, while most sera of adults and children aged over 2 years reacted with HHV6A and B proteins, although predominance for HHV6B was observed. Conclusions: The authors were able to detect HCMV positive sera and to distinguish between HHV6A-monovalent sera, HHV6B-monovalent sera and HHVA/B-polyvalent sera with the new developed microblot system. Predominance of HHV6B was observed in sera of children and adults.