TY - JOUR
T1 - Specifics on the refinement and application of two serological assays for the detection of antibodies to HHV-8
AU - Edelman, Daniel C.
AU - Ketema, Fassil
AU - Saville, Rebecca D.
AU - Herman, Joseph
AU - Sill, Anne M.
AU - Gill, Parkash S.
AU - Blattner, William A.
AU - Constantine, Niel T.
N1 - Funding Information:
The authors wish to express their appreciation to: Thomas Schulz (Liverpool, England) for recombinant HHV-8 ORF 65 antigen and the DHFR control; David Waters and Vicki Coalter from SAIC-Frederick, NCI-FCRD (Frederick, MD) who provided the original IFA and ELISA protocols developed by Thomas Schulz and Guy Simpson; Marvin Reitz and J. Scott Foulke for HeLa and 293T cell lines along with tissue culture support and advice; Sandra Columbini-Hatch for contributing the BCBL-1 cell line obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: BCBL-1 from Michael McGrath and Don Ganem. This research was supported in part by grants from the Maryland Industrial Partnerships (MIPS) and the University of Maryland (Intramural).
PY - 2000/5
Y1 - 2000/5
N2 - 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. Objectives: 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. Study design: 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. Copyright (C) 2000 Elsevier Science B.V.
AB - 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. Objectives: 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. Study design: 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. Copyright (C) 2000 Elsevier Science B.V.
KW - Enzyme-linked immuno-sorbent assay (ELISA)
KW - Herpesvirus
KW - Human herpesvirus type 8 (HHV-8)
KW - Indirect immunofluorescent assay (IFA)
KW - Kaposi's sarcoma
KW - Optimization
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U2 - 10.1016/S1386-6532(99)00085-2
DO - 10.1016/S1386-6532(99)00085-2
M3 - Article
C2 - 10738141
AN - SCOPUS:0034017420
VL - 16
SP - 225
EP - 237
JO - Journal of Clinical Virology
JF - Journal of Clinical Virology
SN - 1386-6532
IS - 3
ER -