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Journal of Virological Methods 177 (2011) 140– 146

Contents lists available at ScienceDirect

Journal of Virological Methods

j ourna l ho me p ag e: www.elsev ier .com/ locate / jv i romet

alidation of an IgM antibody capture ELISA based on a recombinantucleoprotein for identification of domestic ruminants infected with Rift Valley

ever virus

oy Williamsa,∗ , Charlotte Elizabeth Ellisa , Shirley Jacqueline Smithb , Christiaan Abraham Potgieterb ,avid Wallacea, Vuyokazi Epipodia Mareledwanea, Phelix Antipas Ochola Majiwaa

ARC-Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort, Gauteng, 0110, South AfricaDeltamune(Pty) Ltd., P.O. Box 14167, Lyttelton, Gauteng, 0140, South Africa

rticle history:eceived 29 March 2011eceived in revised form 14 July 2011ccepted 20 July 2011vailable online 30 July 2011

eywords:ift Valley fever virus

gMntibody capture ELISAomestic ruminants

a b s t r a c t

The presence of competent vectors in some countries currently free of Rift Valley fever (RVF) and globalchanges in climate, travel and trade have increased the risk of RVF spreading to new regions and haveemphasised the need for accurate and reliable diagnostic tools for early diagnosis during RVF outbreaks.Highly sensitive viral detection systems like PCR have a limited use during outbreaks because of theshort duration of viraemia, whereas antibodies like specific IgM which are serological indicators of acuteinfection, can be detected for up to 50 days after infection. Using the highly conserved and immunogenicrecombinant nucleoprotein of RVF virus in an IgM capture ELISA, the risk of laboratory infection associatedwith traditional serological methods is avoided. The use of pre-coated/pre-blocked ELISA plates and theconjugation of the recombinant nucleoprotein with horseradish peroxidase simplified and shortened theassay procedure. Results showed the assay to be highly reproducible with a lower detection limit equal

ecombinant nucleoproteinalidation

to that of a commercial competition ELISA. By receiver operating characteristic (ROC) curve analysis thearea under curve (AUC) index was determined as 1.0 and the diagnostic sensitivity and specificity at aPP cut-off value of 4.1 as 100% and 99.78% respectively. The results of this study demonstrated that theIgM capture ELISA is a safe, reliable and highly accurate diagnostic tool which can be used on its own orin parallel with other methods for the early diagnosis of RVF virus infection and also for monitoring ofimmune responses in vaccinated domestic ruminants.

© 2011 Elsevier B.V. All rights reserved.

. Introduction

Rift Valley fever virus (RVFV) belongs to the Phlebovirus genus,amily Bunyaviridae and was first identified in 1930 during an out-reak of sudden deaths and abortions among sheep in the greaterift Valley of Kenya (Daubney et al., 1931; Daubney and Hudson,932). The virus is primarily transmitted by mosquitoes and causes

potentially severe disease in humans and livestock in most coun-ries of the African continent and Madagascar (Gerdes, 2004). In000 the first outbreak of Rift Valley fever (RVF) outside Africaccurred in the Arabian Peninsula (Balkhy and Memish, 2003),hile it was detected for the first time recently on an island in

he Archipelago of Comores between Mozambique and Madagas-ar (Sissoko et al., 2009). The disease has re-emerged recently inenya and South Africa after long periods of absence (Pepin et al.,

∗ Corresponding author. Tel.: +27 12 529 92107; fax: +27 12 529 9285.E-mail address: WilliamsR@arc.agric.za (R. Williams).

166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2011.07.011

2010). In South Africa recent outbreaks of RVF have occurred withgreater frequency and out of season in areas where it was leastexpected.

The potential impact of climate change on the distribution ofRVFV vectors (Dufour et al., 2008; Gale et al., 2010; Martin et al.,2008) together with the intensification of international trade inlive animals, has increased the risk of RVF spreading into the Nearand Middle East and eventually into Europe. Several warnings havebeen issued recently on this potential danger for RVF-free coun-tries (Breiman et al., 2008; Davoust et al., 2008; Weaver and Reisen,2010) with urgent requests for a coordinated effort to identify andmonitor potential vectors of the RVFV in the boundary regions ofthe Mediterranean and Middle Eastern countries. There is also anurgent demand for accurate and reliable diagnostic assays to beable to detect active infection as soon as possible in order to initiate

control and avoid spread of the disease.

Numerous techniques are available in the diagnoses of RVF byvirus isolation (Anderson et al., 1989; Shope and Sather, 1979), anti-gen detection (Meegan et al., 1989; Niklasson et al., 1983), nucleic

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R. Williams et al. / Journal of Vir

cid amplification (Drosten et al., 2002; Garcia et al., 2001) andetection of specific antibodies (Paweska et al., 2005; Swanepoelt al., 1986). Although viraemia in individuals infected with RVFVeaches high levels, it is of short duration and viral detection sys-ems like PCR and antigen-detecting ELISAs have a limited use,ven though they are highly sensitive (Pepin et al., 2010). Theequirement for specialized laboratory equipment and well-trainedersonnel are additional constraints for using sophisticated diag-ostic procedures when outbreaks occur in remote regions. On thether hand, specific IgM antibodies which are serological indicatorsf acute infection, can be detected as early as 4 days after initialxposure to RVFV (Paweska et al., 2003a; Jansen van Vuuren et al.,007) and can persist for up to 50 days after infection (Niklassont al., 1984; Paweska et al., 2003b).

Conventional serological assays for RVF make use of live or inac-ivated virus antigen, requiring vaccinated laboratory personnelnd/or bio-containment facilities to limit the risk of exposure. Inddition, these assays are unable to distinguish between differentlasses of immunoglobulins and are unsuitable for automation orcreening of large numbers of sera (Wright et al., 1993). Recenttudies have shown that the nucleocapsid (N) protein, the mostbundant and immunogenic viral component in the RVF virion,ppears to be highly conserved among members of the Bunyaviri-ae family (Gauliard et al., 2006; Magurano and Nicoletti, 1999;apalahti et al., 1995). Furthermore, antigenic cross-reactivitytudies in animals (Davies, 1975; Swanepoel, 1976; Swanepoelt al., 1986) and an indirect ELISA based on a recombinant N pro-ein (Paweska et al., 2007) provided no proof of any other Africanhleboviruses which could induce an antibody response that couldbscure the serodiagnosis of RVF.

This paper describes the development and validation of an ELISAor the detection of IgM antibodies to RVFV infection in livestockased on a recombinant nucleoprotein and demonstrates the suit-bility of the assay for early diagnosis of RVF during outbreaks.

. Materials and methods

.1. Production of nucleoprotein

.1.1. Cloning of the N protein into the pStaby expression systemThe gene encoding the nucleocapsid gene of RVFV 35/74

Genbank accession number EF530207) was amplified usingrimers containing restriction enzyme sites for NdeI and XhoIo facilitate cloning into E.coli expression plasmid pStaby1.2Delphi Genetics, Gosslies, Belgium). Primers were synthe-ised by Inqaba Biotechnical Industries (Pretoria, South Africa).he primer sequences are as follows: forward primer 5′-GATCCATGGACAACTATCAAGAGCTTGCG-3′, reverse primer 5′-TCGAGGGCGTACAAGACAGCAGCC-3′. The RT-PCR and cloningas done according to standard procedures.

.1.2. Large scale expression of N proteinTo prepare large quantities of the rN protein, 50 ml LB medium

as inoculated with a stab of clone pStaby SE1-rN from the −70 ◦Crozen glycerol stocks and grown overnight at 37 ◦C with shakingt 250 rpm. The next day 1 l LB was inoculated with the overnightulture and grown at 37 ◦C with shaking (250 rpm) until the A600eached 0.6. The expression of the N protein was induced byddition of IPTG (Roche, Mannheim, Germany) to a final concen-ration of 1 mM. The flask containing the culture was then movedo an incubator at 18 ◦C and grown overnight. The cells were

arvested by centrifugation for 10 min at 6164 × g and 4 ◦C. Theupernatant was discarded and the cells were lysed for 30 minith 50 ml BugBuster (Novagen, Darmstadt, Germany), contain-

ng 1250 U benzonase nuclease (Novagen, Darmstadt, Germany),

l Methods 177 (2011) 140– 146 141

25 U rLysozyme (Novagen) and 1 tablet complete mini proteaseinhibitor, EDTA-free cocktail (Roche). The cell extract was thencentrifuged at 6164 × g for 10 min at 4 ◦C, the cleared cell lysatecollected and kept at 4–8 ◦C until purification the next day.

2.1.3. Purification of N proteinThe cell lysate was loaded on to a HIS-Select Cobalt affin-

ity column (Sigma, St. Louis, USA), pre-equilibrated with 50 mMNa2HPO4/0.3 M NaCl/20 mM imidazole, pH 8.0. The column waswashed with this buffer until the A280 was <0.1. The N proteinwas then eluted with 50 mM Na2HPO4/0.3 M NaCl/250 mM imi-dazole, pH 8.0. Fractions of 3 ml per tube were collected with afraction collector and the A280 of each fraction was determined.The protein-containing fractions were pooled and kept at 4 ◦C.

2.1.4. Conjugation of N protein to horseradish peroxidise (HRP)Conjugation of the N protein to HRP was done as described by

Nakane and Akira Kawaoi, 1974.

2.2. ELISA pre-coat an pre-block procedure

Affinity purified rabbit anti-sheep IgM (Koma Biotech, Seoul,South Korea) was used as a capture antibody to coat ELISAmicrotiter plates. The antibody was diluted with PBS to 1 �g/ml and100 �l of the diluted antibody was added to each well of PolysorpELISA microplates (Nunc, Roskilde, Denmark). Plates were incu-bated overnight at room temperature and then washed three timeswith 300 �l/well TST buffer (50 mM Tris/150 mM NaCl/0.1% Tween20, pH 8.0) the following day. The plates were then blocked with100 �l/well Stabilcoat buffer (Surmodics, Eden Prairie, USA) andincubated for 1 h at room temperature. The buffer was discardedwithout washing and plates tapped dry and allowed to dry in a flowhood for 2 h. Finally plates were vacuum-sealed in plastic bags andstored at 4–8 ◦C.

2.3. ELISA assays

2.3.1. IgM capture ELISAPositive and negative control sera and test sera were diluted

1:100 in dilution buffer (6% milk powder in TST buffer) and 100 �lwere added per well according to plate layout. Dilution bufferserved as the conjugate control. Plates were incubated for 1 h at37 ◦C and then washed three times with 300 �l TST buffer per well.The N–HRP conjugate was diluted 1:10,000 in dilution buffer and100 �l added per well. The plates were incubated for 60 min at37 ◦C and washed as before. Hundred microlitre of a TMB ready-to-use substrate (Zymed, San Francisco, USA) were added to eachwell and the reaction was allowed to proceed until the OD reached0.400–0.600 at 650 nm. The reaction was stopped with 100 �l stopsolution (2N H2SO4) per well and the final absorbance was read at450 nm.

OD readings were converted to PP values (percentage of positivecontrol serum) using the following equation:

%PP = [(Mean OD of test sample − Mean OD of negative control)

/(Mean OD of positive control − Mean OD of negative control)] × 100

2.3.2. RVF competition ELISAA commercial kit for competition ELISA (cELISA) (ID Screen

Rift Valley fever multi-species ELISA, IDVet Innovative Diagnos-tics, Montpellier, France) was used according to manufacturer’sinstructions for detecting RVF-specific antibodies. Given that it hasa validated diagnostic sensitivity and specificity of 100% (Comtet

1 ological Methods 177 (2011) 140– 146

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after had a combination of IgG and IgM antibodies. Both thesegroups of samples were important for evaluation of the IgM ELISA’sdiagnostic accuracy. The first group (only IgM) contained the ini-tial low-level IgM samples which were of major importance in

Table 1Statistical parameters of IgM ELISA PP values of negative sheep, goat and cattle sera.

42 R. Williams et al. / Journal of Vir

t al., 2010), the kit was used as the standard of reference for eval-ating the IgM capture ELISA.

.3.3. Indirect IgG ELISAThe indirect IgG ELISA was performed essentially has been

escribed by Jansen van Vuuren et al. (2007).

.4. Assay repeatability

Positive and negative RVF control sera and a conjugate controlere tested on 15 separate plates each on a different day by the

ame operator. For each plate, the mean OD value of the 4 replicatesf each control was calculated. For the positive control, coefficientsf variation were calculated within each plate (CV = standard devi-tion of 4 replicate OD values/mean of 4 replicate OD values × 100)nd between plates (CV = standard deviation of 4 replicate OD val-es/mean of the 4 replicate OD values of 30 plates × 100).

.5. Reference sera

A group of 24 sero-negative sheep were experimentally infectedith different doses of a virulent RVFV strain M35/74 in a RVF dose-

itration trial at the ARC-Onderstepoort Veterinary Institute, Southfrica. Blood samples were collected daily for the first 10 days and

hen intermittently up to day 27 after infection. No vaccine wasdministered at any stage of the trial. A total of 183 samples col-ected during the first 19 days period after infection and whichested positive with the cELISA, were selected as IgM-positive ref-rence sera.

A total of 461 serum samples that tested negative with theELISA, were used as IgM-negative reference sera. They consisted of05 sheep sera from the RVF dose-titration trial described above,48 bovine sera collected during a RVF outbreak at Irene, Southfrica and 108 goat sera selected from routine diagnostic samples

ested at ARC-Onderstepoort Veterinary Institute, South Africa.

.6. Analysis of IgM capture ELISA

.6.1. Analytic sensitivityThe assay’s analytic sensitivity (lower detection limit) was eval-

ated by testing, in parallel with the cELISA, a panel of positive serahich were serially diluted in negative serum and ranging from

ery high to very low positive. IgM ELISA results were expressed asercentage Positive (PP) with a positive–negative cut-off value of.1 as determined by receiver operating characteristic (ROC) curvenalysis.

.6.2. Diagnostic accuracyA receiver operating characteristic curve analysis was per-

ormed using MedCalc for Windows, version 11.5.1.0 (MedCalcoftware, Mariakerke, Belgium). The ability of the assay to differen-iate between positive and negative IgM samples was quantified bysing the area under curve (AUC) index (Hanley and Mcneil, 1982;teyerberg et al., 2001) where AUC values range from 0.5 (purelyandom discrimination) to 1.0 (perfect discrimination). ROC curvenalysis was used to determine diagnostic sensitivity, specificitynd predicted values for a range of cut-off values.

. Results

.1. Reproducibility

.1.1. Pre-coated/pre-blocked plate stabilityThe mean OD values (±1SD) of an IgM positive control serum

hich was tested on 10 pre-coated/pre-blocked plates (96 repli-ates per plate) over a period of 147 days, are shown in Fig. 1. The

Fig. 1. Mean OD values (±1SD) of IgM-positive control serum tested on pre-coatedplates.

mean OD was 1.087 ± 0.216 (1SD) and indicate a stable rack lifeover a period of at least five months.

3.1.2. Assay repeatabilityFour replicates of the positive and negative RVF control sera and

a conjugate control were tested on 15 separate plates each on adifferent day. The mean OD values of each of these three test con-trols showed a high degree of reproducibility. The mean OD values(±2SD) for the positive control ranged between 0.900 and 2.100while the negative and conjugate control values did not exceed0.020 and 0.018 respectively. Fig. 2 shows the mean OD values for(a) the positive control serum, (b) the negative control serum and(c) the conjugate control. The % CV for the replicate OD values ofthe positive control serum within plates and between plates did notexceed 10% in both cases. Fig. 3 shows the % CV of the OD values ofthe positive control (a) within plates and (b) between plates.

3.2. Reference sera

3.2.1. Negative seraThe group of negative reference sera consisted of samples from

sheep, goats and cattle which tested negative with the cELISA.Table 1 shows the basic statistical parameters of the IgM ELISA PPvalues for each animal species.

T-test analysis showed no significant difference in the mean PPvalues of negative samples of sheep, goats and cattle.

3.2.2. Positive seraFig. 4 shows the ability of each of the three assays to detect

the antibody response of the 24 experimentally infected sheep asa group. The IgM ELISA could detect IgM antibodies as early as4 days after infection while the IgG ELISA started detecting IgGantibodies from 9 days after infection. The results of the cELISAclosely correlated with the combined antibody response detectedby the IgM and IgG ELISA. cELISA-positive samples collected beforeday 9 therefore contained only IgM while samples collected there-

Species n Maximum Minimum Mean SD

Sheep 205 6.1 −0.4 0.13 0.731Cattle 148 3.5 −1.0 0.24 0.753Goat 108 4.1 −0.4 0.20 0.691

R. Williams et al. / Journal of Virological Methods 177 (2011) 140– 146 143

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Fig. 2. Mean OD values (±2SD) of 4 replicates from 15 individual plates of

etermining an accurate positive-negative cut-off point while theecond group (IgG and IgM) were necessary to demonstrate thebility of the assay for selective detection of IgM when IgG is alsoresent in a sample.

.3. Analytic sensitivity

The commercial cELISA was used as a comparison to evaluate thenalytical sensitivity of the IgM ELISA. A RVF-IgM positive serum

as serially diluted in a negative serum to produce a series of 11

amples varying from very strong to very weak and then testedith both assays. Fig. 5 shows that the lower detection limit of the

gM ELISA corresponds very well to that of the cELISA.

Fig. 3. Percentage CV of the OD values of the positive con

sitive control serum, (b) negative control serum and (c) conjugate control.

3.4. Diagnostic accuracy

Because the commercial cELISA kit was used as the standardof reference for validation, IgM ELISA results of the reference serawere classified as true positive (TP) or true negative (TN) if theywere in agreement with the cELISA results; alternatively they wereconsidered either false positive (FP) or false negative (FN) if theydiffered from the cELISA results. Using the IgM ELISA results of thepositive and negative reference sera, a ROC curve analysis was done(MedCalc Software, Mariakerke, Belgium) with a 95% confidence

interval. Fig. 6 shows the ROC curve for the IgM ELISA with the AUCequal to 1. The AUC is a ROC curve index indicating the probabilitythat a positive event is classified as positive given all possible valuesof the test. For a random pattern the AUC = 0.5 (dotted line in the

trol serum (a) within plates and (b) between plates.

144 R. Williams et al. / Journal of Virological Methods 177 (2011) 140– 146

Fig. 4. Average of IgM ELISA, IgG ELISA and cELISA results of 24 infected sheep for the first 27 days after infection.

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raph). A well discriminating assay should have an AUC between

.87 and 0.9 while an assay above 0.9 is considered as excellent.

In Fig. 7(a) the sensitivity and specificity of the IgM ELISA haveeen plotted against the whole range of possible PP values. The

Fig. 6. ROC curve of IgM ELISA with AUC equal to 1.

uted positive and negative control serum.

cross-over point of the two lines, one depicting sensitivity and theother specificity, indicate optimal sensitivity and specificity – inthis case at a PP value of 4.1. Fig. 7(b) shows another perspective ofthe same data, indicating that a PP value of 4.1 is the best possiblecut-off value for minimizing both false positive and false negativeresults.

The cut-off value for an ELISA can be adjusted for different targetpopulations as well as for different diagnostic purposes (Jacobson,1998). Table 2 shows the criterion values and coordinates of theROC curve with PP cut-off values of the IgM ELISA ranging between3.0 and 6.5.

4. Discussion

Rift valley fever is endemic in a number of countries of Eastand West Africa but qualifies as a re-emerging disease in severalother countries including South Africa. It constrains livestock pro-ductivity, endangers lives of people who live in close proximity tolivestock and threatens livelihoods of many who depend on live-stock or trade in livestock products. Furthermore, the presence of

insect vectors capable of transmitting the virus in some RVF-freecountries, the potential impact of climate change and increasedinternational trade and travel have increased the risk of RVF spread-ing to new regions.

R. Williams et al. / Journal of Virological Methods 177 (2011) 140– 146 145

Table 2Criterion values and coordinates of the ROC curve ranging from 3.0 to 6.5 PP.

PP cut-off Sensitivity (%) 95% CI Specificity (%) 95% CI +LR −LR

>3.0 100.00 98.0–100.0 98.26 96.6–99.2 57.62 0.000>3.1 100.00 98.0–100.0 98.48 96.9–99.4 65.86 0.000>3.2 100.00 98.0–100.0 98.70 97.2–99.5 76.83 0.000>3.4 100.00 98.0–100.0 98.92 97.5–99.6 92.20 0.000>3.5 100.00 98.0–100.0 99.57 98.4–99.9 230.50 0.000>4.1 100.00 98.0–100.0 99.78 98.8–100.0 461.00 0.000>4.6 99.45 97.0–100.0 99.78 98.8–100.0 458.48 0.006>5.0 98.91 96.1–99.9 99.78 98.8–100.0 455.96 0.011>5.8 98.91 96.1–99.9 100.00 99.2–100.0 0.011>6.3 98.36 95.3–99.7 100.00 99.2–100.0 0.016

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I – confidence interval; +LR – positive likelihood ratio; −LR – negative likelihood r

Control of this disease and its management require continu-us surveillance and accurate diagnosis. For both of these, robustiagnostic tests are required. By using a recombinant RVFV nucle-protein in an IgM antibody capture ELISA, the shortcomingsf conventional serological assays were addressed, including theealth risk to laboratory staff, the need for bio-containment facili-ies outside endemic areas and the risk of the live virus falling intohe wrong hands. In addition, the soluble and highly purified nucle-protein can be produced in large quantities that will allow for lessxpensive, fully automated mass-screening of sera.

The evaluation of an IgM ELISA presented two challenges:

general scarcity of IgM-positive serum samples and the facthat the virus neutralisation test, which normally serves as thegolden standard’ for RVF assays, detects both IgG and IgM. A RVFV

ig. 7. ROC curve analysis of IgM ELISA results showing that a PP value of 4.1 (arrow)s the optimum cut-off value where (a) sensitivity and specificity are optimal andb) where false positives and false negatives are minimal.

0 99.2–100.0 0.022

dose-titration trial, performed as part of the development of arecombinant RVFV vaccine, provided a valuable collection of sam-ples which demonstrated the dynamics and individual variationsof the IgM and IgG immune responses elicited by different doses ofthe virulent virus (Fig. 4). It could arguably be considered as a com-parative model for naïve populations during a natural outbreak ofRVF where infected vectors transmit varying doses of the virus tohosts. The sera obtained in this trial made it possible to evaluate theability of the assay to detect low levels of IgM in the early stagesof infection and its ability to detect IgM when IgG antibodies arealso present in the later stages of infection. Although the lack ofIgM-positive goat and cattle sera prevented objective assessmentof the performance of the assay in these two species, it has beenshown that rabbit antiserum against sheep IgM reacts equally wellwith the IgM of goats and cattle (Fafetine et al., 2007; Paweska et al.,2003a). The results of this study did however show that there wasno significant statistical difference in the PP values of the negativesera of sheep, goats and cattle.

The use of pre-coated/pre-blocked ELISA plates with a stablerack life of at least five months and the conjugation of the recom-binant nucleoprotein with horseradish peroxidase, simplified andshortened the assay procedure. Validation results showed the assayto be highly reproducible with its analytic sensitivity comparingfavorably to that of the cELISA.

ROC curve analysis provides a simple graphical means of evalu-ating sensitivity and specificity and allows for selection of specificcut-off values to obtain desired levels of accuracy, determined byfactors such as disease prevalence and the cost of incorrect classi-fication of test results (Paweska et al., 2003b). In this study, ROCcurve analysis determined a PP cut-off value of 4.1 for the assayto attain the highest combined sensitivity and specificity of 100%and 99.78% respectively while the calculated AUC of 1.0 indicatesan excellent ability to discriminate between positive and negativeanimals.

5. Conclusion

The data presented show that the IgM capture ELISA is a safe,reliable and highly accurate diagnostic tool which will be par-ticularly useful for detecting IgM specific antibodies in animalswhich have an active RVFV infection, enabling immediate controlmeasures to be implemented. The assay will also be useful for mon-itoring of immune responses in vaccinated animals.

Acknowledgements

The authors would like to thank the Department of Agriculture,Forestry and Fisheries (DAFF) and GALVmed for partial funding ofthis work and also Mr Henry Harris for his technical assistance.

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