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Clinical and Vaccine Immunology, July 2006, p. 814-817, Vol. 13, No. 7
1071-412X/06/$08.00+0     doi:10.1128/CVI.00035-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Ross River Virus in Vertebrate Sera

Nidia M. M. Oliveira,1,{dagger} Annette K. Broom,1 John S. Mackenzie,2,{ddagger} David W. Smith,3 Michael D. A. Lindsay,1,4 Brian H. Kay,5 and Roy A. Hall2*

Arbovirus Surveillance and Research Laboratory, Department of Microbiology, The University of Western Australia, Nedlands, Western Australia,1 School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Queensland,2 Division of Health Sciences, The Western Australian Centre for Pathology and Research, Queen Elizabeth II Medical Centre, Nedlands, Western Australia,3 Mosquito-Borne Disease Control Branch, Western Australian Department of Health, Perth, Western Australia,4 Queensland Institute of Medical Research and Australian Centre for International and Tropical Health and Nutrition, Brisbane, Queensland, Australia5

Received 27 January 2006/ Returned for modification 6 March 2006/ Accepted 5 May 2006


   ABSTRACT
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We describe the development of an epitope-blocking enzyme-linked immunosorbent assay (ELISA) for the sensitive and rapid detection of antibodies to Ross River virus (RRV) in human sera and known vertebrate host species. This ELISA provides an alternative method for the serodiagnosis of RRV infections.


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Ross River virus (RRV) is a mosquito-borne alphavirus that is endemic in Australia and Papua New Guinea. The primary hosts of the virus include humans, kangaroos, wallabies, and horses (2, 11, 12, 15). RRV is the major etiological agent of epidemic polyarthritis in humans, which is characterized by severe arthritis with possible development of a rash and mild fever or chills and for which there is no specific treatment. Effective surveillance of virus activity in its natural transmission cycle, with appropriate warnings to the public, is the best measure available for minimizing this disease. Traditionally, hemagglutination inhibition (HI), neutralization, and immunofluorescence assays (2, 7, 13) have been the means for detecting RRV antibodies in both human and animal sera. Enzyme-linked immunosorbent assay (ELISA) has also been used to specifically detect RRV immunoglobulin M in human sera (6, 17). Previously, epitope-blocking assays were developed for sensitive and specific detection of seroconversions to the medically important flaviviruses Murray Valley encephalitis virus (8, 9) and West Nile virus (3, 4, 10) in avian and mammalian sera. In this study, an epitope-blocking ELISA was developed for the rapid detection of RRV antibodies in both animal and human sera to improve the efficiency of seroepidemiological studies.

This study used seven isolates of RRV, obtained over 30 years from different regions in Australia, as well as the closely related alphaviruses Chikungunya virus, Getah virus, Barmah Forest virus (BFV), Semliki Forest virus, and Sindbis virus (Table 1). RRV ELISA antigen was produced by propagation of the prototype RRV strain T48 on Vero cells in serum-free medium. Virus supernatant was clarified at 4,000 x g for 15 min at 4°C and stored in 1-ml aliquots at –80°C. Polyclonal antisera were produced in New Zealand half-lop rabbits by intravenous inoculation with 50 µg purified virus/200 µl phosphate-buffered saline and bled at day 14 postinoculation (Table 1). Hyperimmune antisera were not used due to the enhanced cross-reactions observed after multiple immunizations. Nonreactive control sera were collected from nonimmune animals. Clinical samples of human sera (PathCentre, Western Australia [WA] State Health Department, QE11 Medical Centre, Nedlands, Australia) and samples from kangaroos and horses were collected as part of an ongoing seroepidemiological study of RRV in parts of WA (13). These samples were previously tested for RRV antibodies by standard assays (1, 6, 7). Titers are presented as the reciprocal of the highest dilution of antibody to completely neutralize or inhibit RRV. In developing the epitope-blocking ELISA, the protocol described by Hall et al. (9) was adapted. U-bottomed 96-well polyvinyl chloride plates were coated with an optimal concentration of RRV ELISA antigen at 50 µl/well under appropriate biological containment and incubated overnight at 4°C in coating buffer (0.1 M carbonate/bicarbonate, pH 9.6). Antigen-coated plates were washed twice with wash buffer, and nonspecific sites were blocked with 100 µl blocking buffer (0.05 M Tris, 1 mM EDTA, 0.15 M NaCl, 0.05% [vol/vol] Tween 20, 0.2% [wt/vol] casein, pH 8.0) for 1 hour at room temperature (RT). Reference or test sera were added (50 µl/well) in duplicate at dilutions of 1/10 and 1/100 in blocking buffer and incubated for 2 hours at RT. Nonimmune chicken and rabbit sera were used as nonreactive controls. Without removal of serum, 50 µl of monoclonal antibody (MAb) (hybridoma culture supernatant diluted in blocking buffer) was added to each well, and after gentle agitation the plates were incubated at RT for 1 hour. Plates were washed four times and bound MAb detected by incubation with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Bio-Rad) diluted in blocking buffer for 1 hour at RT. Plates were washed six times and enzyme activity visualized by the addition of 100 µl substrate solution [1 mM 2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid] (ABTS) and 3 mM H2O2 in a citrate/phosphate buffer, pH 4.2). Quantitative results were determined by measuring the optical density (OD) at 405 nm, and percent inhibitions were calculated as 100 – [OD (test)/OD (negative control) x 100]. A threshold of 20% inhibition by the test serum was considered "positive" for RRV antibodies (9).


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  		TABLE 1. Neutralization titer and percent inhibition of MAb binding in the epitope-blocking ELISA produced by rabbit antisera to reference RRV strains and other alphaviruses

 
Three MAbs (3B2, G8, and B10), produced to the E2 protein of reference RRV strains as previously described (5, 16), were assessed in the assay. The most sensitive and specific reactions were obtained using MAb 3B2, which was specifically inhibited from binding to RRV antigen in the presence of antisera to all seven reference RRV strains (Table 1). Thus, unless there is a major antigenic shift in circulating RRV strains, detection of RRV antibodies directed to this immunodominant epitope should be effective. Furthermore, using this MAb, there was no cross-reactivity (<20% MAb inhibition) with rabbit antisera to the closely related alphaviruses (Table 1). The results with field samples of kangaroo and horse sera showed ELISA to be as sensitive (100%) and specific (95%) as conventional neutralization for detecting RRV antibodies in these known vertebrate hosts (Table 2).


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  		TABLE 2. Comparison of the specificity and sensitivity of the epitope-blocking ELISA with virus neutralization to detect anti-RRV antibodies in kangaroo and horse sera

 
When used to test a panel of clinical RRV-positive human samples, the blocking ELISA exhibited 97% sensitivity and 98% specificity for the detection of antibody (Tables 3 and 4). This assay's ability to differentiate between seroconversion to BFV and RRV is also important, because both viruses are causative agents of epidemic polyarthritis and they circulate in the same geographical region (13). The acute-phase samples that were negative by ELISA (5007112N and 5028481W) (Table 3) may represent individuals who fail to mount a detectable immune response to the 3B2 epitope early in infection, as the convalescent-phase paired sample to 507112N (5013418) (Table 4) was subsequently shown to be positive by ELISA. The definitive criterion for confirming a recent viral infection is a fourfold or greater increase in the neutralization or HI titer of a paired serum sample (14). We propose that a twofold or greater increase in the percent inhibition of MAb by paired serum samples in the blocking ELISA be used as the criterion for serodiagnosis of recent RRV infection (Table 4). Past seroconversions to RRV could be identified by a greater than 20% inhibition by a single serum sample (Table 3) (9). In conclusion, the RRV epitope-blocking ELISA described in this paper provides a feasible alternative for the rapid diagnosis of clinical human RRV infection and for application in preclinical serosurveillance of susceptible vertebrate hosts (12, 15).


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  		TABLE 3. Comparison of the RRV-specific epitope-blocking ELISA and standard diagnostic techniques for detection of antibodies to RRV in human seraa

 

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  		TABLE 4. Comparison of the epitope-blocking ELISA and standard diagnostic techniques for the detection of RRV antibodies in paired serum samplesa

 


   ACKNOWLEDGMENTS
 
We thank Brad Blitvich for reviewing the manuscript and Ian Marshall, Debbie Phillips, and Ian Fanning for providing virus isolates.

These studies were supported by a grant from the Western Australian Health Department, and N. M. M. Oliveira was funded by a scholarship from the Queensland Institute of Medical Research.


   FOOTNOTES
 
* Corresponding author. Mailing address: School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Qld. 4072, Australia. Phone: 61 7 33654647. Fax: 61 7 33654620. E-mail: roy.hall{at}uq.edu.au. Back

{dagger} Present address: Laboratory of Molecular Virology, National Institute of Mental Health, Room 5C-20, 49 Convent Drive, Bethesda, MD 20892. Back

{ddagger} Present address: Australian Biosecurity CRC, Curtin University of Technology, GPO Box U1987, Perth, WA 6845, Australia. Back


   REFERENCES
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Clinical and Vaccine Immunology, July 2006, p. 814-817, Vol. 13, No. 7
1071-412X/06/$08.00+0     doi:10.1128/CVI.00035-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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