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Reassessing the detection of B-virus-specific serum antibodies
David Katz1, Wei Shi, Martin J Wildes
1Viral Immunology Center, Georgia State University, Atlanta, GA, USA. dkatz@gsu.edu
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.
Insights
B virus causes fatal zoonotic disease in humans. Modified titration ELISA (tELISA) and Western blot analysis (WBA) were reevaluated, revealing WBA
Area of Science:
- Veterinary Virology
- Zoonotic Disease Research
- Immunological Assays
Background:
- B virus (BV) is a macaque pathogen posing a significant zoonotic risk to humans.
- Serological tests, including titration ELISA (tELISA) and Western blot analysis (WBA), are crucial for preventing human B virus infection.
- Current diagnostic methods require reevaluation for improved accuracy and reliability.
Purpose of the Study:
- To reevaluate the correlation between modified titration ELISA (tELISA) and Western blot analysis (WBA) for B virus diagnosis.
- To assess the performance of homologous versus heterologous antigen testing in tELISA.
- To determine the suitability of WBA as a confirmatory test for low-titer sera.
Main Methods:
- Development of a high-throughput titration ELISA (tELISA) for screening.
- Screening of 278 macaque sera against homologous B virus (BV) and heterologous Papiine herpesvirus 2 (HVP2) and Human herpesvirus 1 (HSV1) antigens.
- Confirmation of tELISA results using Western blot analysis (WBA) with denatured antigens.
Main Results:
- Homologous BV-tELISA detected more positive sera (35.6%) compared to HVP2-ELISA (21.6%) and HSV1-ELISA (19.8%), especially in low-titer samples.
- WBA confirmed only 21% of low/intermediate tELISA-positive sera, potentially missing antibodies to conformational epitopes.
- WBA confirmed 82% of high-titer sera but identified 18% of tELISA-negative sera as non-negative, suggesting issues with WBA's subjective interpretation.
Conclusions:
- Homologous tELISA is superior to heterologous tELISA for detecting B virus antibodies, particularly at low titers.
- Western blot analysis (WBA) demonstrates inadequacy as a confirmatory assay for sera with low antibody titers.
- The study highlights the limitations of WBA in detecting conformational epitopes and the challenges of subjective result interpretation.
