ELISA Methods Based on Monoclonal Antibodies for the Serological Diagnosis of Lumpy Skin Disease

Abstract

Lumpy skin disease (LSD) is a notifiable, transboundary cattle disease that spreads rapidly and has a relevant economic impact. The etiological agent is the LSD virus (LSDV), genus Capripoxvirus, and family Poxviridae. To date, LSDV is widely present in Africa, Asia, and in transcontinental regions like Russia, Turkey, and the Middle East, thus representing a continuous threat to free neighbours. Appropriate serosurveillance programs can complement disease control, inform about its spread, and enable the assessment of vaccination campaigns. Since reliable and practical diagnostic tools could improve serological surveillance, this study aimed to produce and characterize monoclonal antibodies (MAbs) that allowed us to develop ELISA tests for the serological detection of LSD. Four MAbs recognizing a 35 kDa viral protein were selected and used to develop and optimize competitive and indirect ELISAs. Both assays detected seroconversion within 14 days postinfection (dpi) in 18 cattle experimentally infected with LSDV and sequentially sampled for up to 4 weeks. The two novel ELISAs detected also antibodies raised by other capripoxviruses: as observed in cattle, both assays revealed seroconversion within 14 dpi in all nine sheep experimentally infected with sheeppox virus (SPPV), while in eight goats infected with goatpox virus (GTPV) competitive ELISA identified seropositivity earlier and in more animals compared to indirect ELISA. Overall, the sensitivity performance of both developed ELISAs resulted comparatively superior to those of virus neutralization test and the commercial Id.Vet ELISA. Testing of about 200 negative sera from each species recorded a single false-positive cattle in the indirect ELISA, which gave a specificity of 99.5%, whereas for the competitive ELISA, the diagnostic specificity was 100% irrespective of the species tested. The results enable concluding that both new tests correctly detect anti-LSDV antibodies in cattle and can also be reliable tools to recognize antibodies to SPPV and GTPV.

Figure 1

Sandwich ELISA performed with all combinations of MAbs used as capture and detector antibody (peroxidase-conjugated MAb). LSD crude viral antigen was diluted and tested (shown in the x-axis); on the y-axis, the recorded value of optical density (OD) is indicated.

Figure 2

Values distribution obtained examining a population of 192 LSD-negative cattle sera. Results are expressed as percentage of inhibition in the competitive ELISA (a) and as optical density (OD) in the indirect ELISA (b), and the values are reported on the x-axis. On the y-axis, the number of analyzed samples is shown.

Figure 3

Seroconversion detected in cattle sera collected at different time points after experimental infection with three LSDV strains. Six cattle were infected with the vaccine strain Neethling, eight with a 2016 isolate from North Macedonia, and four with a 2019 isolate from Nigeria. Sera were tested in competitive ELISA (a) and indirect ELISA (b). On the x-axis, dpi are shown, while on the y-axis the percentage of inhibition (a) and the optical density (OD) value (b) are reported. The dashed line indicates the cutoff value for both analyses.

Figure 4

Frequency values distribution obtained examining a population of 187 goatpox-negative goat sera. Results are expressed as percentage of inhibition in the competitive ELISA tests (a) and as optical density (OD) in the indirect ELISA test (b). On the x-axis, the percentages of inhibition (a) and the OD value (b) are reported, while on the y-axis the number of the analyzed samples is shown.

Figure 5

Frequency values distribution obtained examining a population of 241 sheepox-negative sheep sera. Results are expressed as percentage of inhibition in the competitive ELISA test (a) and as optical density (OD) in the indirect ELISA test (b). On the x-axis, the percentages of inhibition (a) and the OD value (b) are reported, while on the y-axis, the number of the analyzed samples is shown.