Bead-Based Multiplex Assay for the Simultaneous Detection of Antibodies to African Swine Fever Virus and Classical Swine Fever Virus

Abstract

African swine fever (ASF) and Classical swine fever (CSF) are both highly contagious diseases of domestic pigs and wild boar. In the last years, several cases of both diseases have been reported in the Caucasus, Russian Federation and Eastern Europe. Thus, the probability of encountering these two viruses in the same area is increasing. Since differentiation by clinical or post-mortem examination is not possible, laboratory tools for differential diagnosis are required. In the present work, we have developed a triplex bead-based assay using some of the most immunogenic antigens of each virus, for the simultaneous detection of antibodies; i.e. the VP72 and VP30 of ASF virus (ASFV) and the E2 protein of CSF virus (CSFV). The assay was firstly set up and optimized using well characterized reference serum samples specific for each pathogen. Then, a panel of 352 sera from experimentally infected animals with either ASFV or CSFV were analyzed in the multiplex assay. A collection of 253 field negative sera was also included in the study. The results of the multiplex analysis were compared to those obtained by two commercially available ELISAs for detection of antibodies against ASFV or CSFV, and considered in this study as the reference techniques. The data obtained showed values of 97.3% sensitivity and 98.3% specificity for detection of antibodies to ASFV and 95.7% of sensitivity and 99.8% specificity for detection of antibodies to CSFV. This multiplex assay allows the simultaneous and differential detection of antibodies against ASFV and CSFV, providing a valuable tool for surveillance studies. Moreover, this method is rather versatile, offering the possibility of increasing the panel of antigens from other swine diseases that could be of interest for a differential diagnosis along with ASF and CSF.

Development of a multiplex assay for antibody detection in serum against pathogens affecting ruminants

“…The coupling of antigens to beads was described recently by Aira et al (2019). Briefly, the five target proteins were covalently coupled to five different regions (regions #12, #18, #21, #25 and #30) of carboxylated magnetic microspheres (Luminex) using the xMAP® Cookbook by Angeloni et al (2016).…”

Rapid Multianalyte Microfluidic Homogeneous Immunoassay on Electrokinetically Driven Beads

“…Previous approaches aiming to simultaneously carry out multiple biomarkers’ detection mainly relied on the use of fluorophores embedded in the beads [ 39 , 40 ]. This technology, developed by Luminex, associates a barcode defined as a ratio of fluorophore dye in the bead to each analyte of interest [ 41 ], thus allowing for efficient detection of distinct markers in a flow cytometry setup [ 42 ]. Despite its performance, the main limitation of this solution is the need to integrate at least two fluorescent filters within the readout platform to ensure barcode reading.…”

Understanding and combatting African Swine Fever

“…Sero-surveillance can be used to demonstrate the disease-free status in a country, provided that it is combined with active or passive surveillance based on virus detection. Recently, a multiplex test for antibody detection, using the Luminex platform and including different target antigens of both ASFV and CSF virus, showed the increased sensitivity of the assay in comparison with the individual ELISAs used as reference in the study (Aira et al, 2019). This could especially be of interest when analysing field samples, where animals can react differently to viral exposure and present diverse levels of antibodies to each of the virus proteins.…”