A monoclonal antibody-based antigen-capture enzyme-linked immunosorbent assay ( ELISA ) for the detection of bluetongue virus

A monoclonal antibody-based antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of bluetongue virus (BTV) in cell culture lysates and blood samples from sheep. The monoclonal antibody 3E2 and 1C11 specific to BTV VP7 were used as capture antibody and detection antibody, respectively. The assay has detected BTV 1-22 specifically, and had no crossreactivity with the closely related epizootic hemorrhagic disease virus (EHDV) serotypes 5. The limit of sensitivity of the assay was 9 ng/ml for purified recombinant BTV VP7 and 10 0.5 TCID50/ml for BTV-5. The coefficient of variation (CV) of intra-assay and inter-assay range from 3.45 to 6.10%. The developed antigen-capture ELISA showed good coincident rate (100%) with INGEZIM BTV DAS in 5 serotypes BTV and 8 blood samples from sheep. Therefore, the antigen-capture ELISA may be useful for testing large number of samples in a convenient and short time.


INTRODUCTION
Bluetongue (BT) is an insect-borne viral disease of ruminants.Among domestic animals, clinical disease occurs most often in sheep, and can result in significant morbidity.The economic consequences of the outbreak were dramatic.For instance, in the Netherlands, the estimated total net costs of the 2006 and 2007 outbreak were 200 million Euros (Velthuis et al., 2010).It has been included in the World Organization for Animal Health (OIE) list of notifiable diseases (formerly List A) (OIE, 2011).The distribution of BT is determined by the geographic distribution of the arthropod vector and extends globally between latitudes 35°S and 53°N (Martin et al., 2008;Orru et al., 2004).Outbreaks have occurred in many countries in northern and western Europe since 2006 (Saegerman et al., 2008;Carpentera et al., 2009;Kampen and Ortega et al, 2010).
Bluetongue virus (BTV) is the prototype member of the genus Orbivirus within the family Reoviridae.Thus far 26 serotypes are recognized.BTV is non-enveloped with a double shelled structure and a double-stranded (ds) 10 segment RNA genome.The virus contains 7 structural proteins and its genome encode also for 5 non structural proteins: NS1, NS2, NS3, NS3a and NS4.The outer capsid proteins, VP2 and VP5, are the serotype determinants and are responsible for generation of serotype-specific neutralizing antibody.The antibodies against VP7, the major core protein, will specifically detect the whole BTV serogroup.And the genomic segment 5, encoding NS1, is the most highly conserved of the 10 segments (Roy, 1989).
Traditionally, laboratory confirmation of BTV is done by intravenous egg inoculation followed by passages in mammalian cells (World Organization for Animal Health, 2008).Virus isolation is tedious and may take up to 5 weeks for completion.Consequently, alternative methods for virus detection have been sought, which include enzyme-linked immunosorbent assay (ELISA), immunoelectron microscopy, reverse transcription polymerase chain reaction (RT-PCR) (Yin et al., 2008), real-time RT-PCR (Yin et al., 2010), bio-barcode assay (Yin et al., 2011) and so on.ELISA techniques have a number of advantages including being economical, specific and rapid.Additionally, large number of clinical or laboratory samples could be screened by the assay in a very short time during sero-epidemiological studies.In this study, a monoclonal antibody-based antigen-capture ELISA was developed for detecting of VP7 protein.
BTV-5 propagated in confluent monolayers of baby hamster kidney (BHK)-21 cells was purified through gradient centrifugation.Balb/c mice were immunized with the purified BTV-5 antigens.Splenocytes from the immunized mice were fused with SP2/0 myeloma cells, and positive hybridoma clones were screened through the expressed recombinant VP7.Monoclonal antibodies (MAb) 3E2 and 1C11 specific to BTV VP7 were prepared and determined to IgG2b (к) (Yang et al., 2008).

The antigen-capture ELISA
In the antigen-capture ELISA, flat bottom, 96-well plates were incubated with 3E2 MAb and incubated overnight at 4°C.After three washes with PBST, 1% bovine serum albumin (BSA) blocking solution was added to the wells and incubated at 37°C for 60 min and washed three times with PBST.VP7 or BTV samples were added to the wells and incubated at 37°C for 60 min.After three washings, horseradish peroxidase-conjugated 1C11 MAb was added to the wells and incubated at 37°C for 30 min.The solutions were developed by adding 3,3',5,5'-tetramethylbenzidine (TMB) at room temperature.Reactions were stopped after 15 min by the addition of 2 M H2SO4.The absorbance in each well was read at 450 nm wavelength on an ELISA reader.A value twice (or more) the mean OD value of the negative antigen control was considered as the positive/negative cut-off value (positive to negative (P/N) ratio≥2).
The working dilutions of the capture and detection antibodies were selected by chequer board titration, and the best and most satisfactory result was obtained at a 1:2000 dilution for the capture antibody and a 1:1000 dilution for the detection antibody, while the dilution of the antigen (recombinant VP7) and negative control was a 1:10 dilution.At this dilution, the protein content of the controls was between 10 and 15 μg per well, and the P/N ratio was 4.0.These dilutions of reagents were followed throughout the study.

Evaluation of the antigen-capture ELISA
The specificity of the antigen-capture ELISA was confirmed by performing it to detect BTV serotypes 1-22 and the closely related orbivirus epizootic hemorrhagic disease virus (EHDV), using reference strains of serotype 5 (China Animal Health and Epidemiology Center, China).
The sensitivity of the assay was evaluated by conducting the antigen-capture ELISA to detect the recombinant VP7 protein at different concentrations and viral 10-fold serial dilutions of a BTV-5 strain cultured in BHK-21 cell, respectively.
The antigen-capture ELISA was carried out to detect strongpositive sample (0.25 μg/mL VP7), weak-positive sample (0.010 μg/ml VP7) and negative sample (distilled water) to determine the reproducibility of the assay.The samples were tested on 5 separate occasions, with 3 identical samples each time.The coefficients of variation (CV) were analyzed according to sample to negative (S/N) ratio (OD450 value).
Five serotypes of BTV cultured in BHK-21 cell were 10-fold diluted respectively, and these BTV samples and eight sheep blood samples (a gift from China Animal Health and Epidemiology Center) were processed using the antigen-capture ELISA and INGEZIM BTV DAS (INGENASA, Madrid, Spain) to investigate the coincidence rate.

The specificity
Different serotypes BTV and EHDV-5 were detected using the antigen-capture ELISA, and the results showed that BTV serotypes 1-22 in the infected cell culture supernatant were all detected as positive, whereas EHDV 5 tested as negative (Table 1).

The sensitivity
VP7 of BTV at different concentrations was detected using the antigen-capture ELISA, and the results indicated that the assay could detect VP7 at concentrations as low as 9 ng/ml protein.Then, 10-fold serial diluted BTV-5 was tested using the assay.Results showed that titers as low as 100.5 TCID50/ml BTV-5 were detected positively (Table 2).

The reproducibility
The reproducibility of the antigen-capture ELISA was evaluated by detecting the VP7 protein.And the results showed that the CVs (%) of the strong-positive sample, weakpositive sample and negative sample were 3.79, 4.55 and 3.45 intra-test respectively, and 5.38, 6.10 and 4.88 intertest, respectively (Table 3).

Coincidence analysis
Five serotypes of 10-fold diluted BTV and eight sheep blood samples were detected using the antigen-capture ELISA and INGEZIM BTV DAS.And the results indicated   that the coincidence rate of the antigen-capture ELISA and INGEZIM BTV DAS was 100% in detecting BTV-5, 14, 16, 21, 22 (10 1.57 , 10 1.81 , 10 2.67 , 10 2.93 , 10 3.50 TCID 50 /ml) and these blood samples (Table 4).Serological assays provide evidence of earlier animal exposure to BTV.VP7 has a highly conserved sequence, displays antigenicity across all serotypes, and is the major group-specific antigen (Mertens et al., 2005).Not surprisingly, VP7 is frequently used in immunoassays designed to detect BTV (Nagesha et al., 2001;Reddington et al., 1991).Many countries use ELISAs that use Abs raised against BTV to detect the virus, even though ELISAs that utilize MAbs that specifically recognize VP7 have greater specificity (Afshar et al., 1992;Reddington et al., 1991).

DISCUSSION
A recent study demonstrated that the sensitivity of the polyclonal antibody-based sandwich ELISA was estimated to be between 10 2.4 and 10 2.6 TCID 50 /ml with different serotypes of BTV (Chank et al., 2009).In the present study, the analytical detection limit of the antigen-capture ELISA for VP7 protein and BTV-5 was 9 ng/ml VP7 and 10 0.5 TCID 50 /ml BTV-5, respectively.The sensitivity of the assay was compared with the real-time RT-PCR

Table 1 .
Detection of BTV and EHDV strains cultured in BHK-21 cell with the antigen-capture ELISA.

Table 2 .
Detection of BTV VP7 and BTV at different concentrations with the antigen-capture ELISA.
a P: positive;b N: negative.

Table 3 .
Reproducibility of the antigen-capture ELISA for positive and negative samples.
bS/N: sample to negative ratio.