title: Development of a microwell adapted immunoblot system with recombinant antigens for distinguishing human herpesvirus (HHV)6A and HHV6B and detection of human cytomegalovirus.
abstract: 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.
authors.affiliation: Institute of Virology, Medical Faculty Carl Gustav Carus, TU Dresden, Dresden, Germany. andrea.thaeder-voigt@gewebenetzwerk.de
authors.forename: Andrea
authors.initials: A
authors.lastname: Thäder-Voigt
authors.forename: Enno
authors.initials: E
authors.lastname: Jacobs
authors.forename: Werner
authors.initials: W
authors.lastname: Lehmann
authors.forename: Dirk
authors.initials: D
authors.lastname: Bandt
mesh_terms: D000328:Adult; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D057205:Automation, Laboratory; D002648:Child; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli; D005260:Female; D015654:Herpesvirus 6, Human / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D008297:Male; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D019349:Roseolovirus Infections / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D012680:Sensitivity and Specificity; D014169:Transformation, Bacterial; D055815:Young Adult
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D011994:Recombinant Proteins