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. 2012 Dec;62(6):516-26.

Reassessing the detection of B-virus-specific serum antibodies

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Reassessing the detection of B-virus-specific serum antibodies

David Katz et al. Comp Med. 2012 Dec.

Abstract

B virus, a natural pathogen of macaques, can cause a fatal zoonotic disease in humans. Serologic screening of macaques by titration ELISA (tELISA, screening test) and by Western blot analysis (WBA, confirmatory test) is one of the principle measures to prevent human infection. Here we slightly modified these 2 tests and reevaluated their correlation. We developed a high-throughput tELISA and used it to screen 278 sera simultaneously against the homologous BV antigen and the heterologous antigens of Papiine herpesvirus 2 and Human herpesvirus 1. More sera (35.6%) were positive by the BV-ELISA than by the HVP2-ELISA (21.6%) or HSV1-ELISA (19.8%). The superiority of the homologous tELISA over the heterologous tELISA was prominent in low-titer sera. WBA confirmed only 21% of the tELISA-positive sera with low or intermediate antibody titers. These sera might have contained antibodies to conformational epitopes that could not be detected by WBA, in which denatured antigens are used, but that could be detected by tELISA, which detects both linear and conformational epitopes. WBA confirmed 82% of the tELISA high-titer sera. However, WBA defined the remaining 18% of sera, which were negative by tELISA, as nonnegative. This finding can be attributed to the difficulties encountered with the subjective interpretation of results by WBA. Together, the current results indicate the inadequacy of WBA as a confirmatory assay for sera with low antibody titers.

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Figures

Figure 1.
Figure 1.
The gray zone of BV-indeterminate samples for a P:N range (1.8 to 3.5) around a cutoff P:N value of 2.5 that includes 5% to 95% of positive sera as determined by the automated 96-well tELISA for macaque sera tested at the dilution of 1:5.
Figure 2.
Figure 2.
The effect of reagent volume (25, 30, 40, or 50 µL) on the titration curves of a standard BV-positive macaque serum pool tested by the automated 384-well tELISA. Data are given as OD405 units (mean ± 1 SD; n = 4).
Figure 3.
Figure 3.
Percentage of positive sera in each titer group (50 EU or less, 500 EU or less, and 5000 EU or less) from among 278 sera tested by using homologous (BV) and heterologous (HVP2, HSV1) automated 384-well tELISA. Bars represent 90% confidence intervals.
Figure 4.
Figure 4.
Antibody titration of a positive-control standard macaque serum pool in automated 384-well tELISA by using BV, HVP2, and HSV1 antigens. Data are given as OD405 units (mean ± 1 SD; n = 7).
Figure 5.
Figure 5.
Percentages of sera positive (POS), negative (NEG), and indeterminate (IND) by WBA in different tELISA titer groups.

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