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<p>Many serological tests are available for brucellosis diagnosis in ruminants. Some are rapid, objective and cost-effective compared to other tests, but others are highly complex, time consuming, and present subjectivity in results interpretation. Here, we optimized and validated an in-house rapid competitive ELISA (cELISA) using a monoclonal antibody against <i>Brucella abortus</i> oligo-polysaccharide (MAb BC68) for the serodiagnosis of caprine brucellosis and evaluated its putative utility in bovine serum and caprine milk samples. All assays were performed using samples from female animals that tested either consistently negative or consistently positive in both the buffered plate antigen (BPA) and complement fixation (CF) tests. Optimization of cELISA conditions and HRP-conjugation of BC68 allowed reducing incubation time from 4&#xa0;h in 4 steps to 1.5&#xa0;h in 2 steps. Using 499 goat serum samples, a cut-off value of 20.28% of inhibition (%INH) was determined by receiver operator characteristic (ROC) analysis. Then, the BC68-HRP cELISA was successfully validated in an independent goat serum sample with a good relative sensitivity (rSe = 90.28%, 95% CI = 86.31–93.19%) and a high relative specificity (rSp = 98.34%, 95% CI = 96.17–99.29%), showing a great agreement with both BPA/CF concordant results (<i>k</i> = 0.892, <i>n</i> = 593) and a commercial iELISA kit (<i>k</i> = 0.936, <i>n</i> = 217), by Cohen’s kappa analysis. On the other hand, an even better optimization of BC68-HRP cELISA parameters was achieved for bovine serum samples (<i>n</i> = 216), with a cut-off value of 25.50%INH and an associated rSe of 96.15% (95% CI = 90.53% − 98.49%) and rSp of 100% (95% CI = 96.68–100%). Finally, a proof-of-concept study suggested a potential utility of BC68-HRP cELISA for brucellosis diagnosis in caprine milk, with a cut-off value of 38.73%INH and an associated Se of 71.43% (95% CI = 50.04–86.19%) and Sp of 89.80% (95% CI = 78.24–95.56%) (<i>n</i> = 70). In conclusion, this work suggests that BC68-HRP cELISA would be useful as a time-saving and easy test for serodiagnosis of ruminant brucellosis. Furthermore, exploratory results encourage a further, larger optimization study in milk samples.</p>

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Evaluation of a competitive ELISA for detecting the anti-Brucella antibodies in serum and milk of goats and cattle

  • 期刊论文

  • 发表日期：

    2025年12月26日

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  • CN Foster,
  • UA Rossi,
  • MP Saracino,
  • MC Ferrero,
  • PC Baldi,
  • CA Rossetti

摘要

Many serological tests are available for brucellosis diagnosis in ruminants. Some are rapid, objective and cost-effective compared to other tests, but others are highly complex, time consuming, and present subjectivity in results interpretation. Here, we optimized and validated an in-house rapid competitive ELISA (cELISA) using a monoclonal antibody against Brucella abortus oligo-polysaccharide (MAb BC68) for the serodiagnosis of caprine brucellosis and evaluated its putative utility in bovine serum and caprine milk samples. All assays were performed using samples from female animals that tested either consistently negative or consistently positive in both the buffered plate antigen (BPA) and complement fixation (CF) tests. Optimization of cELISA conditions and HRP-conjugation of BC68 allowed reducing incubation time from 4 h in 4 steps to 1.5 h in 2 steps. Using 499 goat serum samples, a cut-off value of 20.28% of inhibition (%INH) was determined by receiver operator characteristic (ROC) analysis. Then, the BC68-HRP cELISA was successfully validated in an independent goat serum sample with a good relative sensitivity (rSe = 90.28%, 95% CI = 86.31–93.19%) and a high relative specificity (rSp = 98.34%, 95% CI = 96.17–99.29%), showing a great agreement with both BPA/CF concordant results (k = 0.892, n = 593) and a commercial iELISA kit (k = 0.936, n = 217), by Cohen’s kappa analysis. On the other hand, an even better optimization of BC68-HRP cELISA parameters was achieved for bovine serum samples (n = 216), with a cut-off value of 25.50%INH and an associated rSe of 96.15% (95% CI = 90.53% − 98.49%) and rSp of 100% (95% CI = 96.68–100%). Finally, a proof-of-concept study suggested a potential utility of BC68-HRP cELISA for brucellosis diagnosis in caprine milk, with a cut-off value of 38.73%INH and an associated Se of 71.43% (95% CI = 50.04–86.19%) and Sp of 89.80% (95% CI = 78.24–95.56%) (n = 70). In conclusion, this work suggests that BC68-HRP cELISA would be useful as a time-saving and easy test for serodiagnosis of ruminant brucellosis. Furthermore, exploratory results encourage a further, larger optimization study in milk samples.