R.P. Achterberg

Bead-based suspension array for simultaneous detection of antibodies against the Rift Valley fever virus nucleocapsid and Gn glycoprotein

A multiplex bead-based suspension array was developed that can be used for the simultaneous detection of antibodies against the surface glycoprotein Gn and the nucleocapsid protein N of Rift Valley fever virus (RVFV) in various animal species. The N protein and the purified ectodomain of the Gn protein were covalently linked to paramagnetic Luminex beads. The performance of the resulting multiplex immunoassay was evaluated by testing a comprehensive and well-characterized panel of sera from sheep, cattle and humans. The suitability of this multiplex immunoassay to differentiate infected from vaccinated animals (DIVA) was investigated by testing sera from lambs vaccinated with a paramyxovirus vaccine vector expressing the RVFV surface glycoproteins Gn and Gc. The results suggest that the bead-based suspension array can be used as a DIVA assay to accompany several recently developed experimental vaccines that are based on RVFV glycoproteins, and are devoid of the N protein.

A bead-based suspension array for the serological detection of Trichinella in pigs

The feasibility of using bead-based suspension arrays to detect serological evidence of Trichinella in pigs was assessed. Trichinella spiralis excretory-secretory antigen was covalently coupled to paramagnetic beads and used to bind serum antibodies, which were subsequently detected using anti-swine antibody. The assay was evaluated by testing pig sera from farms where trichinellosis was endemic and comparing the results with those obtained using two commercially available ELISAs. With cut-offs established by receiver operating characteristic (ROC) analysis, digestion-negative sera from a Trichinella-free population of pigs were deemed seronegative. When anti-swine antibody was replaced with protein A/G, higher test sensitivity (94% vs. 88%) at similar test specificity (95%), was achieved. The potential use of this assay in species other than swine was also demonstrated by testing human sera.

Towards a peptide-based suspension array for the detection of pestivirus antibodies in swine

Classical swine fever (CSF) is a highly contagious and lethal disease in swine. Serological tests for the diagnosis of CSF need not only to detect antibodies against CSFV, but also need to differentiate these from antibodies against other pestiviruses. To investigate the possibilities of specific peptide-based serology, various synthetic peptides that represent a well-described linear epitope of the CSFV E2 protein (TAVSPTTLR) were used to test the viability of a peptide-based suspension array for the detection of antibodies against pestiviruses in swine. The results show that N-terminally biotinylated peptides can bind to avidin conjugated beads, and function in detection of the corresponding monoclonal antibody WH303. There are indications that the length of the spacer between epitope and biotin affect the efficiency of the peptide-antibody interaction. A protocol was established that enables probing for antibodies in porcine sera, where neutravidin-blocking of serum and the use of empty control beads for normalization was crucial. With a set of porcine sera with antibodies against various pestiviruses, the proof of concept of a peptide-based suspension array for specific detection of antibodies against pestiviruses in porcine sera was demonstrated.

Exploring target-specific primer extension in combination with a bead-based suspension array for multiplexed detection and typing using Streptococcus suis as a model pathogen:

We investigated the feasibility of an assay based on target-specific primer extension, combined with a suspension array, for the multiplexed detection and typing of a veterinary pathogen in animal samples, using Streptococcus suis as a model pathogen. A procedure was established for simultaneous detection of 6 S. suis targets in pig tonsil samples (i.e., 4 genes associated with serotype 1, 2, 7, or 9, the generic S. suis glutamate dehydrogenase gene [gdh], and the gene encoding the extracellular protein factor [epf]). The procedure was set up as a combination of protocols: DNA isolation from porcine tonsils, a multiplex PCR, a multiplex target-specific primer extension, and finally a suspension array as the readout. The resulting assay was compared with a panel of conventional PCR assays. The proposed multiplex assay can correctly identify the serotype of isolates and is capable of simultaneous detection of multiple targets in porcine tonsillar samples. The assay is not as sensitive as the current conventional PCR assays, but with the correct sampling strategy, the assay can be useful for screening pig herds to establish which S. suis serotypes are circulating in a pig population.

A bead-based suspension array for the detection of Salmonella antibodies in pig sera

BackgroundSlaughter pigs are monitored for the presence of the zoonotic pathogen Salmonella, using both serology and bacteriology. ELISAs used to investigate pig herds are based on the detection of antibodies against components of the Salmonella cell envelope. Nearly all Salmonella isolates in food-producing animals are serovars of Salmonella enterica subspecies enterica, distributed over various serogroups as determined by the composition of their lipopolysaccharide (LPS). ELISAs for Salmonella serology are usually based on serogroup B and C1 LPS, often combined with serogroup D or E LPS. Although C2 LPS may improve serology, use of C2 LPS in a broad ELISA was never achieved.ResultsTo enable detection of serum antibodies against Salmonella in pigs, a bead-based suspension array was developed with five LPS variants (B, 2× C1, C2, D1), each conjugated to a different bead set using triazine chemistry. Reactivity of the beads was confirmed with rabbit agglutination sera and with experimental pig sera. With a mixture of bead sets, 175 sera from slaughter pigs were investigated for the presence of antibodies against Salmonella. With a combination of ROC analysis (B and D LPS) and a prevalence estimation based on historic data (C LPS), individual cut-offs were defined for each LPS-conjugated bead set, and assay performance was evaluated.Results of the suspension array (BC1C1C2D) suggest that more pigs are seroconverted than indicated by a commercial BC1D1-ELISA, and that most of these extra seropositive samples give a signal on one of the beads with C LPS. These results show that expansion of a standard panel with more C LPS variants improves antibody detection.ConclusionsA suspension array for Salmonella serology in pigs was developed, that detects more seropositive sera than ELISA, which is achieved by expanding the panel of Salmonella LPS variants, including C2 LPS. The results demonstrate that bead-based suspension arrays allow for testing of pig sera, with the advantage of being able to set cut-offs per antigen. Ultimately, this type of assay can be applied in routine veterinary serology to test for antibodies against multiple Salmonella serovars (or other pathogens) in one single serum sample, using up-to-date antigen panels.