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.

Keywords: African swine fever; antibody; classical swine fever; diagnosis; multiplex.

Figure 1

Establishment of optimal conditions for the development of a multiplex bead-based assay. X Axis shows the dilution value of the sera employed and Y Axis shows the Median Fluorescence Intensity (MFI). Response to different antigens is shown: (°) signal of bead #12 coupled to VP72, (x) signal of bead #15 coupled to VP30, and () signal of bead #25 coupled to E2, using a reference serum for ASFV (A), a reference serum for CSFV (B) and a negative serum for both diseases (C).

Figure 2

Validation of the bead-bead assay. The left panels represent a dot diagram where each dot represents an individual sample: results obtained for VP72 coupled to bead #12 (A) VP30 coupled to bead #15 (B), and E2 antigen coupled to bead #25 (C), The horizontal solid line corresponds to the cutoff values in each assay, according to the Medcalc software. X Axis shows the positive (1) or negative (0) classification of samples according to the ELISA used as reference technique in this study and Y Axis shows Median Fluorescence Intensity (MFI) obtained in the developed assay The right panels show a ROC curve analysis based on the data obtained in the bead-bead assay.

Figure 3

Stratified analysis of positive samples to ASFV according to days post-infection. X axis shows the percentage of positive samples within each group. Y axis shows different days post infection clustered as follows: 0–7 dpi, 8–15 dpi, 16–28 dpi, and > 1 month pi. Error bars show the 95% confidence interval for each bin of data. Statistical significance has been calculated according to a McNemar test, *p < 0.05.

Figure 4

Analysis of weak positive samples in pooled conditions. X axis shows the dilution of the whole pool in assay buffer and Y axis the Median Fluorescence Intensity (MFI). Signals of weak positive sample for ASFV (A) or CSFV (B), spiked in a 4 (—) or 9 (- - -) negative sera matrix are represented for (°) bead #12 coupled to VP72, (x) bead #15 coupled to VP30 and () bead #25 coupled to E2. Cut off values for different antigens were established at 3500 (VP72), 3700 (VP30) and 5000 (E2) MFI.