Abstract
Different enzyme-linked immunosorbent assay (ELISA)–based techniques for the detection of antibodies to Rift Valley fever virus (RVFV) have been developed in recent years, but their diagnostic sensitivity was not directly compared. In addition, their use might still be restricted to high biocontainment facilities when sera to be tested are collected from viremic individuals. In this study, we report on direct comparison of various ELISA forms for the detection of anti-RVFV antibody in preinactivated sera using a simple thermochemical treatment. Results in naive and treated sera from experimentally infected sheep demonstrate that inactivation method used had no adverse effect on ELISA readings, but the assays analyzed differ in their ability to detect the early humoral responses to infection with RVFV. The IgM-capture ELISA was slightly more sensitive than the IgG-sandwich ELISA to detect early humoral response after infection. The indirect IgG ELISA, using Protein G Horseradish Peroxidase, was less sensitive in detecting seroconversion than the IgG-sandwich ELISA, but this problem was alleviated when using anti-sheep IgG conjugated with Horseradish Peroxidase. The high concentration of viral antigen in sheep sera collected shortly after infection might contribute to false-positive results in the inhibition ELISA, but its ability to detect seroconversion was comparable to that of IgM-capture ELISA.
Introduction
Materials and Methods
Enzyme-linked immunosorbent assays
The following ELISAs were directly compared using serial bleeds from experimentally infected sheep: IgG-sandwich ELISA based on gamma-irradiated, sucrose-acetone-extracted mouse liver RVF virus (RVFV) whole antigen (Paweska et al. 2003), IgM-capture ELISA based on the same antigen (Paweska et al. 2003), an inhibition ELISA based on gamma-irradiated tissue culture RVFV whole antigen (Paweska et al. 2005), and an indirect ELISA based on recombinant RVFV N protein (Fafetine et al. 2007).
Experimental sheep sera
Serial sera were obtained from three sheep experimentally infected with wild-type RVF virus as described previously (Le Roux et al. 2009).
Thermochemical inactivation
Sheep sera were inactivated as described by Jansen van Vuren and Paweska (2009). Briefly, an equal volume of 1% Tween20 in phosphate-buffered saline was added to each serum and incubated at 56°C for 1 h. Inactivated sera were tested for complete inactivation on 24–48-h-old Vero cell monolayers and in 2–3-day-old suckling mice. Cells were monitored for cytopathic effect until 14 days after inoculation and mice until 10 days postinfection (p.i.).
Results
The immune responses in sheep after experimental infection with wild-type RVFV were monitored using the IgG sandwich, indirect, IgM capture, and inhibition ELISAs. There was no significant difference in detection of antibodies between naive versus inactivated serum using any of the ELISAs, but these assays differed in their ability to detect the early humoral responses to infection with RVFV (Fig. 1). The IgM-capture ELISA was able to detect seroconversion on day 4 p.i. compared with day 5 p.i. with the IgG-sandwich ELISA. The inhibition ELISA yielded false-positive results on days 2 and 3 p.i. as a result of the capturing of viral antigen in highly viremic sera on days 2 and 3 p.i. (results not shown). The recombinant N protein–based IgG ELISA, using Protein G Horseradish Peroxidase, was less sensitive in detecting seroconversion (day 9 p.i.) than the IgG-sandwich ELISA (day 5 p.i.). This problem was alleviated when replacing Protein G with anti-sheep IgG Horseradish Peroxidase (Fig. 1).
Comparison of immune responses in three experimentally infected sheep as measured by testing naive (solid lines) versus thermochemically inactivated (dotted lines) sera by IgM-capture ELISA (−), inhibition ELISA (×), indirect ELISA Protein G Horseradish Peroxidase (•), indirect ELISA anti-sheep IgG Horseradish Peroxidase (○), and IgG-sandwich ELISA (▪). Note the level of sensitivity of indirect ELISA when using different HRPO conjugates, as indicated by the dotted circle. ELISA, enzyme-linked immunosorbent assay.
Conclusion
A simple thermochemical method for inactivation of RVF virus in serum specimens had no adverse effect on the level of detectable IgG and IgM antibodies in various forms of ELISA directly compared in this study; however, they differ in their ability to detect early humoral responses. This might be of practical importance for early recognition of infections and outbreaks.
Footnotes
Acknowledgments
The authors would like to thank the International Atomic Energy Agency (IAEA) (Technical contract no. 15274/R0) and the Poliomyelitis Research Foundation (PRF grant number 08/14) for supporting the study.
Disclosure Statement
No competing financial interests exist.