title: Specifics on the refinement and application of two serological assays for the detection of antibodies to HHV-8.
abstract: BACKGROUND
  Serologic assays for the detection of antibodies to human herpesvirus type 8 (HHV-8) are important for epidemiological studies and to further investigate the proposed pathogenesis of the virus in cancer. Although a variety of assays are available, a lack of optimization and standardization makes their usefulness uncertain, and may be responsible for the controversy concerning the prevalence of infection.
  
  
  OBJECTIVE
  To refine an indirect immunofluorescent assay (IFA) for the detection of latent antibodies and a recombinant ORF 65 ELISA for the detection of lytic antibodies in order to increase their ability to differentiate individuals at higher and lower risk for HHV-8 infection.
  
  
  METHODS
  Sera from Kaposi's sarcoma (KS) patients and blood donors (BDs) were used to modify assay parameters in an attempt to better discriminate between the two populations. Modifications included methods of substrate fixation, incubation times, sample dilution, and antigen/conjugate concentrations.
  
  
  RESULTS
  Optimal modifications to the latent IFA included acetone fixation of substrate, and dilution of sera to 1:64 which enhanced detection of HHV-8 antibodies from 68 to 92% in the KS population. Similarly, successful refinement of the ORF 65 ELISA to increase the signal-to-noise ratio included the use of 88 ng of ORF 65 antigen per well and serum dilutions of 1:50. Optical density-to-cut-off ratios directly correlated with titers, thereby introducing a strategy to predict antibody concentrations. The ORF 65 ELISA and the latent IFA were both able to discriminate between the two populations but with different efficiencies.
  
  
  CONCLUSIONS
  Although neither the latent IFA nor the ORF 65 ELISA produced perfect test indices, improvement in their performances was noted following the optimization strategies. The ELISA produced better detection of antibodies to the virus than the IFA and permitted prediction of sample titers, thus improving cost and time effectiveness.
authors.affiliation: Department of Pathology, School of Medicine, University of Maryland, 725 West Lombard Street, Baltimore, MD, USA. dedelman@umaryland.edu
authors.forename: D C
authors.initials: DC
authors.lastname: Edelman
authors.forename: F
authors.initials: F
authors.lastname: Ketema
authors.forename: R D
authors.initials: RD
authors.lastname: Saville
authors.forename: J
authors.initials: J
authors.lastname: Herman
authors.forename: A M
authors.initials: AM
authors.lastname: Sill
authors.forename: P S
authors.initials: PS
authors.lastname: Gill
authors.forename: W A
authors.initials: WA
authors.lastname: Blattner
authors.forename: N T
authors.initials: NT
authors.lastname: Constantine
mesh_terms: D000914:Antibodies, Viral / Q000097:blood*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D019084:Fluorescent Antibody Technique, Indirect; D019288:Herpesvirus 8, Human / Q000276:immunology* / Q000502:physiology; D006801:Humans; D011994:Recombinant Proteins / Q000276:immunology; D012514:Sarcoma, Kaposi / Q000175:diagnosis* / Q000821:virology*; D014764:Viral Proteins / Q000276:immunology; D017735:Virus Latency
chemical_list: D000914:Antibodies, Viral; C119019:ORF65 protein, human herpesvirus 8; D011994:Recombinant Proteins; D014764:Viral Proteins