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. 2010 Apr 30;356(1-2):39-46.
doi: 10.1016/j.jim.2010.02.015. Epub 2010 Mar 4.

Detection of antibodies to Kaposi's sarcoma-associated herpesvirus: a new approach using K8.1 ELISA and a newly developed recombinant LANA ELISA

Georgina L Mbisa  1 , Wendell Miley, Christine J Gamache, William K Gillette, Dominic Esposito, Ralph Hopkins, Michael P Busch, George B Schreiber, Richard F Little, Robert Yarchoan, Betty A Ortiz-Conde, Nazzarena Labò, Denise Whitby
Affiliations

Detection of antibodies to Kaposi's sarcoma-associated herpesvirus: a new approach using K8.1 ELISA and a newly developed recombinant LANA ELISA

Georgina L Mbisa et al. J Immunol Methods. .

Abstract

Detection of antibodies to Kaposi's sarcoma-associated herpesvirus (KSHV or Human herpesvirus 8) is a topic of ongoing controversy. KSHV expresses multiple antigens and host responses are highly variable. We have previously described an algorithm for determining KSHV infection based on K8.1 ELISA and LANA immunofluorescence assay (IFA). Here we describe the development of a recombinant ELISA for LANA and an improved testing strategy using ELISAs for LANA and K8.1. We assessed mammalian and baculovirus expression systems for the production of full-length recombinant LANA. We evaluated the performance of LANA ELISAs using human serum samples from several sources including blood donors and clinical patients diagnosed with Kaposi's sarcoma and compared them to LANA IFA. Both LANA ELISAs exhibited comparable sensitivity and specificity to LANA IFA but showed considerably greater reliability. The LANA ELISA can thus be used in conjunction with the previously described K8.1 ELISA to enable the highly sensitive and specific detection of antibodies to KSHV. Use of this testing strategy will provide a more accurate and reliable diagnostic assessment of KSHV status.

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Figures

Figure 1
Figure 1. SDS PAGE and immunoblot analysis of Insect or Mammalian derived KSHV LANA
Five micrograms of insect derived protein (A), and 1.7 micrograms of mammalian derived product (B), fractions 1 and 2, were analyzed by SDS PAGE and immunoblot. For each of the two products, the most abundant elution fraction (F2) is showed.
Figure 2
Figure 2. Titration of a panel of clinical samples using KSHV LANA ELISA (Insect or Mammalian derived) or LANA IFA
Average performance of the insect (Sf9) and mammalian cell (293E) derived LANA ELISAs (a) and the LANA IFA (b) on a panel of titrated serum samples from the NIH-NCI clinic tested repeatedly. Error bars show titer OD variation between experiments.
Figure 3
Figure 3. Repeated testing of a diluted positive control sample using KSHV K8.1 and LANA ELISAs
Performance of the K8.1 ELISA (a) and the LANA ELISA (b) on a diluted KS patient positive control sample repeatedly tested. The cutoff for each plate is shown as a gold line. The blue line indicates the average OD value of the positive control.
Figure 4
Figure 4. Repeated titration of a positive control sample using KSHV K8.1 and KSHV LANA ELISA
Performance of the K8.1 ELISA (a) the insect cell derived LANA ELISA (b) on a positive control titrated on 15 individual plates.
Figure 5
Figure 5. ROC curve of the KSHV K8.1 and LANA ELISAs (training set)
Receiver operator characteristic of the KSHV k8.1 ELISA (a) and KSHV LANA ELISA (b) applied to the training set. The red mark indicates the optimal cutoff.
Figure 6
Figure 6. ROC curve of the KSHV K8.1 and LANA ELISAs (validation set)
Receiver operator characteristic of the KSHV k8.1 ELISA (a) and KSHV LANA ELISA (b) applied to the validation set. The red mark indicates the optimal cutoff.
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