The yield average of NcSRS2 recombinant obtained was 1 mg/L of E. coli culture. The recombinant NcSRS2 solubilized with the two protocols (urea 8 M and N-lauroyl sarcosine) at the same concentration (0.5 μg/mL) were tested in the ELISA-NcSRS2. However, when we performed ELISA using rNcSRS2 solubilized in urea, all OD mean values obtained using the sera were negative (data not shown). The ELISA-NcSRS2 results

obtained in the present selleck chemicals study were achieved using NcSRS2 solubilized in N-lauroyl sarcosine. In order to access the specificity of NcSRS2 protein with the sera from cattle infected with N. caninum we performed a WB analysis using an unrelated protein and positive and negative pooled bovine sera ( Fig. 1). The reaction of the rNcSRS2 with the sera was observed only in the positive sera. The 100 positive and 258 negative sera from MS state, and 94 positive and 45 negative sera from RS state, as determined by IFAT were evaluated using ELISA-NcSRS2. The standardization of ELISA-NcSRS2 www.selleckchem.com/screening/ion-channel-ligand-library.html was achieved using the 497 sera. Correlation between those two diagnostic tools was assessed using ROC analysis. Considering a cut-off value of 0.095 for the 100 IFAT-positive sera, 10 sera were found to be ELISA-negative. Of the 352 IFAT-negative sera, 18 were above the cut-off point when assessed by ELISA-NcSRS2. To access the repeatability of the ELISA-NcSRS2, experiments

intra plate using the same sera was performed. The results obtained shown that the repeatability was maintained between the plates. In order to confirm theses results we selected

the 18 IFAT-negative sera with absorbance above cut-off (≤0.095) for Western blotting analysis. All sera reacted with the truncated recombinant NcSRS2 protein (data not shown), showing the newly developed Fossariinae ELISA to be more sensitive than IFAT, and provide an excellent tool for detection of N. caninum infection. Fig. 2A shows the frequency distribution of the IFAT-positive and negative samples. Based on ROC analysis (Fig. 2B), a mean ELISA OD value of 0.095 was chosen as the threshold to distinguish between positive and negative samples, yielding a specificity of 96% and a sensitivity of 95%. Using this cut-off point (OD ≤ 0.095), the test’s negative predictive value ranged from 99.4% (for 10% prevalence) to 68.8% (for 90% prevalence) and the positive predictive value ranged from 74.5% to 99.5%, depending on the prevalence of the disease in a particular area (Fig. 3). In order to evaluate the accuracy in the absence of a gold standard the TAGS analysis was performed with the results of the ELISA and IFAT with the sera from the two populations of cattle (from MS and RS). The analysis shows a good sensibility and specificity of the tests, the ELISA-NcSRS2 had 100% and 96% of sensibility and specificity, respectively. The IFAT had 100% and 94% of sensibility and specificity, respectively.