Development and validation of a gold nanoparticle-enhanced ELISA for serodiagnosis of Ehrlichia canis in dogs

This study aimed to develop a gold nanoparticle-enhanced enzyme-linked immunosorbent assay (ELISA; Nano-ELISA) for serodiagnosis of Ehrlichia canis in dogs, compare it to a conventional indirect ELISA (i-ELISA) using dsb TaqMan quantitative polymerase chain reaction (qPCR) as the reference standard, and characterize the gold nanoparticle (AuNP) conjugates. Blood from 200 dogs clinically suspected of canine monocytic ehrlichiosis (CME) was tested by qPCR using a prespecified quantification cycle (Cq) threshold of ≤ 38; 115 (57.5%) were positive, and 85 (42.5%) were negative. An additional 30 clinically healthy dogs were recruited separately and used only for cut-off determination and cross-reactivity testing. Both ELISA formats used horseradish peroxidase (HRP)-conjugated Protein A/G with formalin-inactivated E. canis antigen; the Nano-ELISA incorporated AuNP-Protein A/G-HRP conjugates to amplify the signal. AuNPs and conjugates were characterized by transmission electron microscopy, dynamic light scattering, zeta potential, and ultraviolet-visible (UV-Vis) spectroscopy. The Nano-ELISA achieved a sensitivity of 98.3% (95% confidence interval [CI]: 93.9-99.5%), specificity of 97.6% (95% CI: 91.8-99.4%), accuracy of 98.0% (95% CI: 95.0-99.2%), area under the receiver operating characteristic curve (AUC) of 0.980 (95% CI: 0.953-1.000), and Cohen's κ of 0.96. The i-ELISA achieved a sensitivity of 96.5% (95% CI: 91.4-98.6%), specificity of 94.1% (95% CI: 87.0-97.5%), accuracy of 95.5% (95% CI: 91.7-97.6%), AUC of 0.953, and Cohen's κ of 0.91. McNemar and DeLong comparisons showed no statistically significant difference between formats (p = 0.50; p > 0.05). One of 10 Anaplasma platys-positive sera cross-reacted in the i-ELISA (3.3% across the 30-serum heterologous panel); no cross-reactivity was observed for the Nano-ELISA, although a result of 0/10 per pathogen does not exclude cross-reactivity (upper 95% CI 30.8%). The Nano-ELISA serologically detects infections with or exposure to E. canis and shows high agreement with dsb TaqMan qPCR in this single-centre cohort of clinically suspected dogs.