Monoclonal Antibody-based Competitive ELISA Research Articles

Development of a novel monoclonal antibody-based competitive ELISA for antibody detection against bovine leukemia virus

Infection with bovine leukemia virus (BLV) leads to enzootic bovine leukosis, the most prevalent neoplastic disease in cattle. Due to the lack of commercially available vaccines, reliable eradication of the disease can be achieved through the testing and elimination of BLV antibody-positive animals. In this study, we developed a novel competitive ELISA (cELISA) to detect antibodies against BLV capsid protein p24. Recombinant p24 protein expressed by Escherichia coli, in combination with the monoclonal antibody 2G11 exhibiting exceptional performance, was used for the establishment of the cELISA. Receiver-operating characteristic curve analysis showed that the sensitivity and specificity of the assay were 98.85 % and 98.13 %, respectively. Furthermore, the established cELISA was specific for detecting BLV-specific antibodies, without cross-reactivity to antisera for six other bovine viruses. Significantly, experimental infection of cattle and sheep with BLV revealed that the cELISA accurately monitors seroconversion. In a performance evaluation, the established cELISA displayed a high agreement with Western blotting and the commercial BLV gp51 cELISA kit in the detection of 242 clinical samples, respectively. In conclusion, the novel p24 cELISA exhibited the potential to be a reliable and efficient diagnostic tool for BLV serological detection with a broad application prospect.

Post-Vaccination Sero-Monitoring of Peste des Petits Ruminants in Sheep and Goats in Karnataka: Progress towards PPR Eradication in India.

Peste des petits ruminants (PPR) presents economic challenges in enzootic countries impacting small ruminant productivity. The state of Karnataka, India, implemented a mass vaccination campaign in alignment with the PPR-Global Eradication Programme (GEP) and the National Strategic Plan for PPR eradication. This study was conducted from January to March 2023 to assess seroconversion in post-vaccinated goats and sheep at the epidemiological unit (epi-unit) level, aligning with the World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) guidelines in the PPR Global Control and Eradication Strategy (GCES). Before vaccination, 3466 random serum samples were collected from small ruminants of three age groups (6-12 months, 1-2 years, and >2 years) across 116 epi-units, spanning 82 taluks in 28 districts. Post-vaccination sero-monitoring included 1102 serum samples collected from small ruminants of the 6-12-month age group only, across 111 epi-units covering 64 taluks in 23 districts. The PPRV antibody status was determined using an indigenous hemagglutinin (H) protein monoclonal antibody-based competitive ELISA kit. Pre-vaccination, the PPR seropositivity rates were 55%, 62%, and 66% in the age groups of 6-12 months, 1-2 years, and >2 years, respectively, with a 61% PPRV antibody prevalence across all the age groups. Notably, 41% of the epi-units exhibited antibody prevalence rates of ≥70%, indicating a substantial population immunity, possibly attributed to the previous vaccination program in the state since 2011. In contrast, only 17% of the epi-units had below 30% seroprevalence rates, emphasizing the need for intensified vaccination. Statistical analysis of the data revealed significant correlations (p < 0.05) between the presence of PPRV antibodies and host factors such as species, breed, and sex. Post-vaccination seroprevalence in the 6-12 months age group was found to be 73.4%, indicating the use of an efficacious vaccine. On the evaluation of vaccination immunity in the 6-12 months age group, it was revealed that over 69% of the epi-units achieved a response surpassing ≥70%, indicating a significant improvement from 42% of the epi-units in pre-vaccination. For active PPR eradication, a mass vaccination campaign (>95% coverage) targeting small ruminant populations aged >4 months is advocated, aiming to achieve the desired herd immunity of >80%. This study offers crucial insights into PPR baseline seroprevalence/immunity status and vaccine efficacy, guiding national strategies towards a PPR-free India and further supporting the global eradication initiative.

Estimating Vaccine Potency Using Antibody-Based Competition Assays.

The issues of vaccine potency and stability constitute formidable challenges associated with the development and readiness of vaccines for biodefense. In most instances, the vaccines will be stockpiled (at considerable cost) for years and used only in the rare event of a public health emergency. It is therefore imperative that there be means to readily monitor overall stability of the stockpiled vaccines, preferably using reliable in vitro assays, without the need for expensive and labor-intensive animal studies. In this chapter, we describe an in vitro monoclonalantibody-based competition ELISA known as RiCoE for assessing the potency of a ricin toxin subunit vaccine. RiCoE can be applied to drug substance and drug products adsorbed to aluminum salts adjuvant. While RiCoE is specific for ricin toxin, the general methodologies and protocols described herein are amenable to virtually any subunit or even virus-like particle-based vaccine. Ultimately, RiCoE-like assays may replace or at least reduce the need for animal studies in vaccine potency determinations.

Proteome Modulation in Peripheral Blood Mononuclear Cells of Peste des Petits Ruminants Vaccinated Goats and Sheep.

In the present study, healthy goats and sheep (n = 5) that were confirmed negative for peste des petits ruminants virus (PPRV) antibodies by monoclonal antibody-based competitive ELISA and by serum neutralization test and for PPRV antigen by s-ELISA were vaccinated with Sungri/96. A quantitative study was carried out to compare the proteome of peripheral blood mononuclear cells (PBMCs) of vaccinated goat and sheep [5 days post-vaccination (dpv) and 14 dpv] vs. unvaccinated (0 day) to divulge the alteration in protein expression following vaccination. A total of 232 and 915 proteins were differentially expressed at 5 and 14 dpv, respectively, in goats. Similarly, 167 and 207 proteins were differentially expressed at 5 and 14 dpv, respectively, in sheep. Network generated by Ingenuity Pathway Analysis was “infectious diseases, antimicrobial response, and inflammatory response,” which includes the highest number of focus molecules. The bio functions, cell-mediated immune response, and humoral immune response were highly enriched in goats at 5 dpv and at 14 dpv. At the molecular level, the immune response produced by the PPRV vaccine virus in goats is effectively coordinated and stronger than that in sheep, though the vaccine provides protection from virulent virus challenge in both. The altered expression of certain PBMC proteins especially ISG15 and IRF7 induces marked changes in cellular signaling pathways to coordinate host immune responses.

Development and evaluation of a monoclonal antibody-based competitive ELISA for the detection of antibodies against H7 avian influenza virus

BackgroundH7 subtype avian influenza has caused great concern in the global poultry industry and public health. The conventional serological subtype-specific diagnostics is implemented by hemagglutination inhibition (HI) assay despite lengthy operation time. In this study, an efficient, rapid and high-throughput competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of antibodies against H7 avian influenza virus (AIV) based on a novel monoclonal antibody specific to the hemagglutinin (HA) protein of H7 AIV.ResultsThe reaction parameters including antigen coating concentration, monoclonal antibody concentration and serum dilution ratio were optimized for H7 antibody detection. The specificity of the cELISA was tested using antisera against H1 ~ H9, H11 ~ H14 AIVs and other avian viruses. The selected cut-off values of inhibition rates for chicken, duck and peacock sera were 30.11, 26.85 and 45.66% by receiver-operating characteristic (ROC) curve analysis, respectively. With HI test as the reference method, the minimum detection limits for chicken, duck and peacock positive serum reached 20, 21 and 2− 1 HI titer, respectively. Compared to HI test, the diagnostic accuracy reached 100, 98.6, and 99.3% for chicken, duck and peacock by testing a total of 400 clinical serum samples, respectively.ConclusionsIn summary, the cELISA assay developed in this study provided a reliable, specific, sensitive and species-independent serological technique for rapid detection of H7 antibody, which was applicable for large-scale serological surveillance and vaccination efficacy evaluation programs.

A neutralizing monoclonal antibody-based competitive ELISA for classical swine fever C-strain post\u2013vaccination monitoring

BackgroundVirus neutralization test (VNT) is widely used for serological survey of classical swine fever (CSF) and efficacy evaluation of CSF vaccines. However, VNT is a time consuming procedure that requires cell culture and live virus manipulation. C-strain CSF vaccine is the most frequently used vaccine for CSF control and prevention. In this study, we presented a neutralizing monoclonal antibody (mAb) based competitive enzyme-linked immunosorbent assay (cELISA) with the emphasis on the replacement of VNT for C-strain post–vaccination monitoring.ResultsOne monoclonal antibody (6B211) which has potent neutralizing activity against C-strain was generated. A novel cELISA was established and optimized based on the strategy that 6B211 can compete with C-strain induced neutralizing antibodies in pig serum to bind capture antigen C-strain E2. By testing C-strain VNT negative pig sera (n = 445) and C-strain VNT positive pig sera (n = 70), the 6B211 based cELISA showed 100% sensitivity (95% confidence interval: 94.87 to 100%) and 100% specificity (95% confidence interval: 100 to 100%). The C-strain antibody can be tested in pigs as early as 7 days post vaccination with the cELISA. By testing pig sera (n = 139) in parallel, the cELISA showed excellent agreement (Kappa = 0.957) with VNT. The inhibition rate of serum samples in the cELISA is highly correlated with their titers in VNT (r2 = 0.903, p < 0.001). In addition, intra- and inter-assays of the cELISA exhibited acceptable repeatability with low coefficient of variations (CVs).ConclusionsThis novel cELISA demonstrated excellent agreement and high level correlation with VNT. It is a reliable tool for sero-monitoring of C-strain vaccination campaign because it is a rapid, simple, safe and cost effective assay that can be used to monitor vaccination-induced immune response at the population level.

Development and evaluation of recombinant antigen and monoclonal antibody based competition ELISA for the sero- surveillance of surra in animals

Trypanosoma evansi, a haemoflagellated protozoan parasite, is responsible for chronic as well as the acute debilitating disease called surra in a wide range of herbivores and carnivores including domestic and wild animals. Since the parasite is having wide host range, there is a need for diagnostic test which can detect the T. evansi specific antibody in different species of animals for generating sero-surveillance data. In the present study we developed and evaluated competitive enzyme immunoassay using monoclonal antibodies (MAbs) raised against recombinant variable surface glycoprotein (rVSG) of T. evansi. The immunoreactivity of the developed MAbs (IgG3-subtype) was evaluated by immunoblot as well as ELISA and subsequently used in the development and standardization of competitive ELISA (C-ELISA). Further, the serological data generated from the C-ELISA using reference samples constituting true positive or surely infected (35), true negative (45), sero-positive (225) and sero-negative (215) samples and was analyzed statistically. The true positivity/negativity was determined by thin blood smear examination and diagnostic PCR assay, While, seropositivity/seronegativity of the reference samples was determined through standard reference tests. The data showed the diagnostic sensitivity of 92.6% and specificity of 96.4% with Cohen's kappa value of 0.88. In order to determine the utility of C-ELISA in detecting T. evansi antibodies in different species of animals, the assay was further evaluated with 1361 field sera sample comprising bovine, horse, donkey and camel. Since the C-ELISA described herein has showed high sensitivity and specificity, this single test can be explored in the sero-surveillance of T. evansi in a wide range of animals.

Prevalence of Peste-des-petits-ruminant virus antibodies in cattle, buffaloes, sheep and goats in India

The present study describes the prevalence of Peste-des-petits-ruminant virus (PPRV) antibodies in cattle, buffaloes, sheep and goats carried out during the period 2011 using the serum samples randomly collected from different villages of five states of India. A total of 1,498 serum samples [n=605 (cattle); n=432 (buffaloes); n=173 (sheep); n=288 (goats)] were collected from 52 districts in five states (Andhra Pradesh, Gujarat, Jammu and Kashmir, Maharashtra and Rajasthan) of India and were screened for PPRV-specific antibodies by using PPR monoclonal antibody-based competitive ELISA kit. Analysis of 1,498 samples indicates that an overall seroprevalence of 21.83% with 11.07% in cattle, 16.20% in buffaloes, 45.66% in sheep and 38.54% in goats. This report presents the results of PPRV-specific antibodies in situations where the subclinical, inapparent or nonlethal or recovery of infection was suspected in cattle, buffaloes, sheep and goats. The presence of PPRV antibodies demonstrate that bovines are exposed to PPRV infection and it implies the importance of cattle and buffaloes as subclinical hosts for the virus besides widespread presence of the disease in sheep and goats. Further, the study showed that the prevalence of PPRV antibodies in apparently healthy livestock under natural situation, 21.83% of the animals were protected from PPRV re-infection. This inturn help in the implementation of disease control strategies such as vaccination in that particular geographical area.

Preparation and validation of monoclonal antibody-based indirect competitive ELISA for detecting testosterone levels

This paper presents the generation of monoclonal antibodies (mAbs) against testosterone (TES) through cell fusion technology, and the development of a mAb-based indirect competitive enzyme-linked immunosorbent assay (icELISA) method to detect TES residue in bovine edible tissues. Under optimal conditions, this assay exhibited a working range of 0.04–19 ng/mL, with half-maximum inhibition (IC50) and limit of detection values of 0.27 and 0.02 ng/mL, respectively. After three sample pretreatment procedures were checked, a simple dilution method was adopted for further use. The stabilisation studies demonstrated that the icELISA kits can be stored for at least 180 to 240 days, at 4°C and −20°C, respectively. When applied to bovine samples, the data from icELISA and gas chromatography coupled to mass spectrometry showed excellent correlation (r2=0.9923 in muscle, 0.9884 in liver and 0.9957 in kidney). Therefore, this assay has the potential to be incorporated into a quantitative monitoring programme for the rapid screening of TES residue in foods.

Seroprevalence of Peste des petits ruminants in cattle and buffaloes from Southern Peninsular India

This study describes seroprevalence of Peste des petits ruminants (PPR) in cattle and buffaloes carried out during the period 2009-2010 using the randomly collected serum samples from different parts of Southern peninsular India. The report presents the results of PPR virus (PPRV)-specific antibodies in situations where either the subclinical or inapparent or non-lethal infection was there in cattle and buffaloes. A total of 2,548 serum samples [cattle = 1,158, buffaloes = 1,001, sheep = 303 and goat = 86] were collected and screened for PPRV antibodies by using a PPR monoclonal antibody-based competitive ELISA kit. Analysis of 2,159 serum samples indicates an overall 4.58% prevalence of PPRV antibody in cattle and buffaloes. The presence of PPRV-specific antibodies demonstrates that cattle and buffaloes are exposed to PPR infection naturally, and the transmission mode may be direct or indirect. Further, it implies the importance of bovines as subclinical hosts for the virus besides widespread presence of the disease in sheep and goats in the country.

Competitive ELISA for the Detection of Antibodies to Rift Valley Fever Virus in Goats and Cattle

Rift Valley fever virus (RVFV) is one of the important emerging viral diseases of serious impact in public health and animal hygiene both in human and animal industries. In this study, we developed a monoclonal antibody-based competitive ELISA for the detection of antibodies to RVFV in goats and cattle. The recombinant N protein of RVFV was expressed in E. coli with a six-histidine tag, and the purified N protein was used for detecting antigen with a competitive monoclonal antibody against RVFV antibodies. The competitive ELISA (C-ELISA) could detect antibodies at 9-11 days after inoculation in goats and cattle with a sensitivity of 94.7% (virus neutralization titer >32) and specificity of 99.7%, respectively. In addition, the C-ELISA did not show any cross-reactivity with positive sera against arboviruses such as Akabane, Aino, Chuzan, Ibaraki and bovine ephemeral fever virus, which are prevalent viral agents in ruminant animals throughout Southeast Asia. The results of the present study indicate that the C-ELISA is a simple, rapid and convenient serodiagnostic method for RVFV in goats and cattle.

The detection of antibody against peste des petits ruminants virus in Sheep, Goats, Cattle and Buffaloes

Monoclonal antibody-based competitive ELISA (C-ELISA) has been used for the specific measurement of antibodies to peste des petits ruminants (PPR) viruses in sheep, goats, cattle and buffalo. Serum samples from sheep (n = 232), goats (n = 428), cattle (n = 43), buffalo (n = 89) were tested. The animals had not been vaccinated against rinderpest or PPR. Findings suggested that the sero-positive cases were significantly higher in sheep (51.29%) than in goats (39.02%) (P = 0.002). The overall sero-prevalence of PPRV in small ruminants was 43.33%. The PPR antibodies seroprevalence was 67.42% in buffalo and 41.86% in cattle which was significantly higher in buffalo (P = 0.005). The overall sero-prevalence of PPRV in large ruminants was 59.09%. Cattle and buffalo sera showed a high prevalence of antibody against PPR virus which may explain the difficulty experienced in achieving high post-vaccination immunity levels against rinderpest. Because antibodies against PPR virus are both cross-neutralizing and cross-protective against rinderpest virus, further vaccination in the presence of antibodies against PPR virus may be a waste of national resources. It was also suggested that antibodies to PPR virus could prevent an immune response to the rinderpest vaccine. This paper presents serological evidence for the transmission of PPR virus from sheep and goats to cattle and buffalo and highlights the need to include PPR serology in the sero-monitoring programme to give a better indication of national herd immunity of sheep and goats against PPR.

Seroprevalence of Neospora caninum Antibodies in Dogs in India

Neospora caninum is one of most important causes of abortion in cattle worldwide, and dogs are an important risk factor for N. caninum infection in cattle. Antibodies to N. caninum were determined in 184 (126 rural, 58 urban) dogs from the Punjab State, India, using commercial monoclonal antibody-based competitive ELISA and found in 16.8% of the animals. The prevalence of N. caninum antibodies was significantly higher in rural dogs (21.4%, 27 of 126) than city dogs (6.9%, 4 of 58). To our knowledge this is the first report of N. caninum infection in canines from India.

Short Communication: Comparison of Diagnostic Efficacy of a Monoclonal Antibody-based Competitive ELISA Test with a Similar Commercial Test for the Detection of Antibodies to Peste des Petits Ruminants (PPR) Virus

Short Communication: Comparison of Diagnostic Efficacy of a Monoclonal Antibody-based Competitive ELISA Test with a Similar Commercial Test for the Detection of Antibodies to Peste des Petits Ruminants (PPR) Virus

Monoclonal antibody-based competitive ELISA for simultaneous detection of rinderpest virus and peste des petits ruminants virus antibodies

An experimental competitive enzyme-linked immunosorbent assay (morbillivirus cELISA) using a recombinant N antigen (rRPV N) expressed in a baculovirus and a ruminant morbillivirus (RPV and PPRV)-specific monoclonal antibody (P-13A9) was developed for simultaneous detection of rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) antibodies and its diagnostic performance was evaluated. A set of known reference antisera against RPV and PPRV belonging to different lineages, experimental sera from cattle vaccinated for a RPV of Asian lineage, and field sera from cattle and sheep/goat populations known to be positive (West Africa) and negative (Korea) for RPV and PPRV were used for the evaluation. Morbillivirus cELISA results on the panel of experimental RPV and PPRV antisera showed high correlation ( r=0.97) between the whole virus and the rRPV N antigens, suggesting that the rRPV N contains a ruminant morbillivirus-specific antigenic determinant recognized by the P-13A9 and it may be suitable as an ELISA antigen in place of the whole virus. Morbillivirus cELISA detected anti-RPV and anti-PPRV antibodies in all reference RPV and PPRV antisera containing VN titers ≥1:8, suggesting that the assay can simultaneously detect antibodies against RPV and PPRV. Anti-RPV antibody was detected by morbillivirus cELISA in vaccinated cattle as early as the VNT and continued to be detectable by both the cELISA and the VNT until termination of the study. When applied to field samples from Africa, morbillivirus cELISA showed good agreement with a RP cELISA kit (kappa value of 0.86) in bovine sera and with a peste des petits ruminant cELISA kit (kappa value of 0.81) in caprine/ovine sera. Usefulness of morbillivirus cELISA using the rRPV N protein was discussed.

Antigenic sites of foot-and-mouth disease virus (FMDV): an analysis of the specificities of anti-FMDV antibodies after vaccination of naturally susceptible host species.

Of the known neutralizing antigenic sites of foot-and-mouth disease virus (FMDV), site 1 or A, formed in part by the G-H loop of VP1, has historically been considered immunodominant because of evidence implicating its importance in the induction of a protective immune response. However, no systematic study has been done to determine the relative importance of the various specificities of antibodies against the known neutralizing antigenic sites of FMDV in the polyclonal immune response of a natural host after vaccination. In this report, we have adopted a monoclonal antibody-based competition ELISA and used antibodies specific to sites 1, 2 and 3 to provide some insight into this issue. Following vaccination of the three main target species, cattle, pigs and sheep, with an O1 serotype strain, results indicate that none of these three antigenic sites can be considered immunodominant in a polyclonal serum. Interestingly, pigs did not respond to epitopes on the carboxy terminus end of VP1 as efficiently as the ruminant species. In addition to the known sites, other as yet undefined sites might also be important in the induction of a protective immune response. Possible implications for the design of new vaccine strategies for foot-and-mouth disease are discussed.

Investigation of serum cartilage oligomer protein (COMP) levels in rheumatoid arthritis (RA)

RA is a disease characterised by an inflammatory process in the synovium and a degradation process of the joint cartilage. When articular cartilage matrix is degraded by a disease process, protein fragments are produced and some of them subsequently appear in the blood circulation and can be used to monitor cartilage degradation. COMP was first described by D.Heinegard as a noncollagenous protein primarily found in articular cartilage. COMP levels of serum and synovial fluids have been shown to have potential as prognostic markers of osteoarthritis and RA progression as well. For the detection of COMP mainly monoclonal antibody based competition ELISA systems have been described. Recently on the basis of the research group of D.Heinegard a two-site sandwich ELISA test from AnaMar Medical AB has been available in wich two monoclonal antibodies directed against separate antigenic determinant of COMP molecule are applied. Using the COMP ELISA test of 'AnaMar' we measured the serum COMP levels of 47 patients who fulfilled the ACR criteria of RA. Duration of the disease varied between 3–6 years. CRP and RF levels were also measured from the same sample. Patients were grouped by clinical activity, radiological progression and also by RF and CRP positivity. Our results showed that there was no difference between the serum COMP levels of seropositive and seronegative patients (11.4 vs. 10.71 ng/ml). Serum COMP levels of clinically active patients (11.7 ng/ml) were higher than of patients with inactive disease (10.5 ng/ml) (P < 0.08). The average COMP levels of the radiologically progressive group was higher (12.5 ng/ml) than that of the non-progressive ones (10.3) (P < 0.06). Statistically the highest difference was found between patients with elevated CRP (12.3 ng/ml) and those with normal CRP levels (8.8 ng/ml) (P < 0.03). Significant correlation was also found between serum CRP and COMP levels R:0.54 (P < 0.003). These results confirm previous findings that the serum COMP level reflects the actual degree of cartilage destruction ongoing during the inflammatory process of arthritis, thus can be a useful marker in predicting radiological progression and in monitoring the effectiveness of the treatment of RA.

Development of a monoclonal antibody-based cELISA for the analysis of sulfadimethoxine. 2. Evaluation of rapid extraction methods and implications for the analysis of incurred residues in chicken liver tissue.

Several rapid extraction methods were evaluated for use with a monoclonal antibody-based competitive inhibition ELISA (cELISA) to detect sulfadimethoxine (SDM) in chicken liver tissue. These methods included extraction of the samples with (1) aqueous buffer with or without ultrafiltration, (2) acetonitrile/water, (3) methanol/water, or (4) acetone. The organic extraction methods were evaluated with or without solvent evaporation prior to dilution into assay buffer for the cELISA. The aqueous-based extraction methods were compatible with the cELISA. However, of the organic extraction methods, only the acetone liver extract with solvent evaporation prior to analysis was compatible with the cELISA. The cELISA method coupled to aqueous- or acetone-based sample extraction as well as an HPLC method was evaluated for the analysis of chicken liver tissues fortified with SDM at levels from 0.2 to 0.025 ppm. Mean SDM recoveries for the HPLC method and for the cELISA method using samples prepared by aqueous extraction, aqueous extraction and ultrafiltration, or acetone extraction, evaporation, and reconstitution were 68.9, 95.7, 60.1, and 52.5%, respectively. For the analysis of samples obtained from an SDM incurred residue study, HPLC and cELISA analysis of the same organic extract gave results that were highly correlated (R(2) = 0.976; p < 0.0001). However, results obtained from the analysis of aqueous extracts by cELISA did not correlate well with those obtained by HPLC (R(2) = 0.61, p > 0. 0006). This was attributed to the coextraction of cross-reactive SDM-related residues that were not quantified by the HPLC method. The presence of these residues should be considered during data interpretation when ELISA methods coupled with rapid aqueous extraction of samples are used in SDM residue monitoring programs.

Validation of a monoclonal antibody-based ELISA to detect antibodies directed against swine vesicular disease virus

A simple, rapid and sensitive competitive monoclonal antibody-based ELISA for the detection of antibodies directed against swine vesicular disease virus (SVDV) was developed. The ELISA was validated using field sera originating from SVDV-infected and non-infected Dutch pig herds, reference sera obtained from the Community Reference Laboratory for Swine Vesicular Disease at the Institute for Animal Health, Pirbright Laboratory, UK, and sera from animals infected experimentally. When testing 4277 sera originating from non-infected Dutch pig herds and collected as part of the national screening program, this ELISA had only 0.6% false positive results, whereas ≈2% of false positive results were obtained with a conventional blocking ELISA used until recently. A sensitivity relative to the virus neutralisation test of >97% was achieved when testing sera collected from Dutch pig farms where an outbreak of SVDV had occurred. All international reference sera scored consistently correct. Sera collected sequentially from pigs experimentally infected with SVDV isolates representing all currently recognized antigenic groups, were scored positive slightly earlier by the ELISA compared to the virus neutralisation test. This monoclonal antibody-based competitive ELISA for SVDV antibodies designated the Ceditest ELISA for SVDV-Ab, is as sensitive but more specific than the ELISA used until recently. Because sera are tested at a single dilution (1:5), incubations are carried out at room temperature and test results are available within 3 h, this ELISA is simple, easy to automate and therefore very suitable for screening large numbers of serum samples.

Reduction of singleton reactors against swine vesicular disease virus by a combination of virus neutralisation test, monoclonal antibody-based competitive ELISA and isotype specific ELISA

Pigs which are serologically positive for swine vesicular disease virus (SVDV) but which show no clinical signs and for which there is neither a relevant history of the disease on the holding nor contact with a known outbreak are considered as singleton reactors. False positive serological results for an epizootic disease, like SVD, in a non-vaccinated population or in imported animals are of great concern to international trade. For the virus neutralisation test, the gold standard for SVD, singleton reactors are found at a level of 1–3/1000. Singleton reactors also occur when other serological testing methods are used. The number of animals finally considered as singleton reactors can be reduced considerably by performing three different serological tests (virus neutralisation test, monoclonal antibody-based competitive ELISA and isotype specific ELISA) on the same serum. A serological profile of the animal can be derived by analysing the results in greater detail. This procedure can reduce considerably the number of pig holdings on which a prohibition of movement and trade needs to be imposed without requiring analysis of supplementary samples.