The ELISA system consists of a N. caninum tachyzoite antigen treated with Triton X-100, chicken immunoglobulin and a milk-derived protein block, and peroxidase (POD)-labeled Protein AG conjugate. Non-specific reactions in indirect fluorescent antibody test (IFAT)-negative sera with the use of POD-labeled anti-bovine IgG significantly decreased with the use of POD-labeled Protein AG. There was no overlap of ELISA values for negative and positive gold standard sera in the gold standard panel. Anti-sera against Sarcocystis cruzi, Hammondia hammondi, Toxoplasma gondii, and Besnoitia wallacei failed to show a positive reaction in Protein AG ELISA (PAG-ELISA). ELISA values highly correlated with the log-transformed IFA titer (correlation coefficient =0.866, index of linearity =0.979). In the field samples, IFAT-negative sera showed ELISA values of 0.156±0.017 (mean±95% confidence interval), and IFAT-positive sera showed ELISA values of 0.697±0034. The negative and positive sera were significantly different from each other (p<0.0001). A cut-off ELISA value, resulting in the lowest total number of diagnostic errors and the response operation characteristic (ROC) curve, was 0.360. This cut-off value yielded 0.885 of kappa values, a 92.4% of sensitivity and a 96.5% of specificity. Thus, PAG-ELISA is simple, stable, specific, sensitive, and objective. PAG-ELISA is well suited to epidemiological surveys, other large tests of N. caninum infection, or the detection of latent infection by N. caninum. Furthermore, Protein AG reacts to almost all mammalian immunoglobulin, eliminating the need for specific anti-immunoglobulin antibodies. (Immunopathology Section, Department of Immunology TEL +81-298-38-7833)
