Development and validation of an indirect enzyme immunoassay for detection of ovine antibody to Brucella ovis

Abstract

An indirect enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Brucella ovis infection was developed. The assay uses a mouse monoclonal antibody to bovine IgG 1 horseradish peroxidase (HRPO) conjugate that cross-reacts with immunoglobulin from sheep and a purified antigen from Brucella ovis. The ELISA data were read and analyzed according to a targeting procedure. The ELISA results were compared with a cold complement fixation test (CFT). Sera from 675 rams from three uninfected flocks were used to determine the ELISA cut-off value (O.D. 405 nm: 0.095) and the diagnostic specificity of the ELISA (100%) and the CFT (99.69% ± 0.42). The ELISA cut-off value was corroborated by receiver operating characteristic (ROC) analysis. Six hundred and forty semen and serum samples from 419 rams from two naturally infected flocks were collected before and after mating-time during two consecutive years. All semen samples were cultured and Brucella ovis was isolated from 28 samples. Sera from the 28 rams with positive semen were used to determine the diagnostic sensitivity of the ELISA (96.43% ± 6.8) and of the CFT (including suspected positive samples with titers of 1:5; 88.89% ± 11.85). Considering the CFT suspicious and the anti-complementary reactions as positive resulted in a diagnostic sensitivity value of 89.28% ± 11.46. Six hundred and ten serum samples from the 640 sera were used to determine relative sensitivity (excluding sera with 1:5) at: ELISA/CFT 97.26% ± 3.74 and CFT/ELISA was 71.72% ± 8.87. The percent agreement, beyond chance measured by the Kappa index was 79.7. Relative sensitivity ELISA/CFT (including 1:5 titers in the CFT as positive) was 94.9% ± 4.83 and CFT/ELISA was 72.84% ± 8.59. The Kappa index was 79.4.