Journal of Proteome Research ARTICLE Figure 3. Statistical presentation of principal component analysis and multiple antigens AUC. (A) Unsupervised principal component analysis of the signal intensity for samples from Peruvian naives and culture positive groups revealed that these two groups could be segregated on the basis of 33 selected serodianosis protein antigens and 2 Bm LPS (at 0.1 mg/mL or 0.01 mg/mL). The serodiagnostic antigens were ranked by single antigen AUC. (B) Cross-validation AUC boxplot with variance and mean value and (C) cross-validation ROC graphs show classiﬁers with increasing number of human serodiagnostic antigens. With the top 3 antigens, this classiﬁer yielded a highest sensitivity and speciﬁcity rate of 98.6% and 98.6%, respectively. All 33 antigens plus Bm LPS also produced sensitivity and speciﬁcity over 93%. Figure 4. Immunostrips probing. (A) Eineteen serodiagnostic antigens and Bm LPS at 0.01 mg/mL were printed onto nitrocellulose membrane in adjacent stripes using a BioDot jet dispenser as described in Materials and Methods. Strips were probed with culture positive or Peruvian naive sera diluted 1/200 followed by alkaline phosphatase conjugated secondary antibody and enzyme substrate. Weak reactivity in the na€ive healthy controls can be distinguished from the strong reactivity in the infected group. (B) Cross-validation ROC curve was generated and sensitivity and speciﬁcity of immunostrips test is 96% and 96%, respectively. samples, whether from na€ive individuals or individuals diagnosed to be infected. Classifier of Serodiagnostic Antigens We performed unsupervised principal component analysis and showed that the 33 serodiagnostic antigens clearly separated the Peruvian naive from culture positive groups (Figure 3A). To further determine the accuracy of distinguishing brucellosis cases from controls, cross-validation receiver operating characteristic (ROC) curves and area under the curve (AUC) boxplots were generated (Figure 3). The serodiagnostic antigens were ranked by decreasing single antigen AUC. The top four ORFs, BMEI0536 (bp26), BMEI0805 (hypothetical protein), BMEI1330 (protease Do), and BMEI 1890 (transporter), as wells as Bm LPS, all have an individual antigen AUC greater than 0.90 (Table S1, Support- ing Information). Three of the antigens were identiﬁed pre- viously on microarrays.5 Antigen bp26 (BMEI0536; AUC 0.991; BH p-value < 1 10 16) gave the best single antigen discrimination with 95% sensitivity and speciﬁcity rate. We used kernel methods and support vector machines43,49 to build linear and nonlinear classiﬁers. Variability and mean value of accuracy from this model has been shown in the AUC boxplot. The top 5 antigens all produced sensitivity and speciﬁcity over 96% (Figure 3C). With the top 3 antigens, this classiﬁer yielded a highest sensitivity and speciﬁcity rate of 98.6 and 98.6%, respectively. Altogether 33 protein antigens plus LPS produced sensitivity and speciﬁcity over 93%. Validation of Serodiagnostic Accuracy with Immunostrips Eighteen serodiagnostic proteins and Bm LPS were printed onto nitrocellulose membranes using a BioDot jet dispenser. The membrane was then cut into 3 mm strips. The individual strips were probed with 31 diﬀerent randomly selected culture positive sera and 31 Peruvian naive sera. Brucellosis patients reacted strongly against the serodiagnostic antigens with variable signal intensities among the patients. Na€ive samples showed much lower reactivity against these serodiagnostic antigens (Figure 4A). The ROC curve (Figure 4B) shows that this immunostrip test 4817 dx.doi.org/10.1021/pr200619r |J. Proteome Res. 2011, 10, 4813–4824