Parasitol Res (2012) 110:2187–2191 Healthcare). A positive sample was recorded if a band could be seen at 27 kDA in the Western blot Tsol-p27 and at 36 kDa in the Western blot TsolHSP36. The membranes were blocked for 2 h with 5% skim milk in phosphate- buffered saline (PBS, pH 7.5) and then rinsed in washing buffer containing PBS and 0.05% Tween 20. Thereafter, the membranes were incubated first for 3 h with human sera diluted 1:500 in 5% skim milk–PBS, and then for 1 h with rabbit anti-human IgG conjugated with peroxidase (Sigma) diluted 1:3000 in 5% skim milk–PBS. The subsequent washing steps and detection procedures were performed according to the ECL Plus manual (GE Healthcare). Immunodot blot analysis of Tsol-p27 (in house) Immunodot blot analysis was carried out using a nitrocellulose membrane (GE Healthcare), which was pre-wetted with dis- tilled water and then placed into a bio-dot blot apparatus (Bio- Rad, Hercules, CA, USA). This was followed by fixation of 10 μg of recombinant Tsol-p 27 protein that had been purified using glutathione agarose and his-selected nickel beads. The apparatus was left at room temperature for 1 h to allow antigen immobilization. The dots were subsequently blocked for 1 h with 5% skim milk in PBS containing 0.05% Tween 20 and then washed three times with PBS containing 0.05% Tween 20. Thereafter, the dots were incubated for 4 h with human sera diluted 1:1000 in 5% skim milk–PBS and washed three times with PBS containing 0.05% Tween 20, and this was followed by addition of 100 μl of rabbit anti-human IgG conjugated with peroxidase (Sigma) diluted 1:4000 in 5% skim milk–PBS. The subsequent washing steps and detection procedures were per- formed according to the ECL Plus manual (GE Healthcare). All analyses of the serum samples were performed in duplicate. Results To increase the efficacy and reduce the background of the in house immunodot blot method, the Tsol-p27 protein was purified using his-selected nickel magnetic agarose and glutathione-agarose beads. Figure 1 shows the Tsol-p27 antigen in a 10% SDS-PAGE, visualized by staining with Coomassie Brilliant Blue. The sensitivity and specificity values were determined based on the positive and negative results for Tsol-p27 antigen obtained by commercial EITB analysis, the gold standard method. Of the 100 investigated serum samples from epileptic patients, 13 were positive and 87 negative by EITB. The same number of serum samples was analyzed using immunodot blot Tsol-p27, and an example of the results for sera from epileptic patients and patients with positive and negative serology to Chagas diseases is pre- sented in Fig. 2. Corresponding results obtained by the 2189 Fig. 1 SDS-PAGE analysis of Tsol-p27 antigen. Lane 1, cells from induced E. coli; lane 2, purified recombinant GST/Tsol-p27; lane 3, Tsol-p27 digested with precision protease; lane 4, Tsol-p27 after puri- fication using glutathione-agarose and his-selected nickel magnetic beads commercial ELISA and in house Western blot Tsol-p27/ TsolHSP36 are shown (Tables S1 and S2 in the Electronic supplementary material). Table 1 summarizes the perfor- mance of the different immunodiagnostic tests compared to the gold standard method EITB for detection of anti- bodies in serum from epileptic human subjects. The immunodot blot Tsol-p27 was found to offer 86.7% sensitivity and 97.8% specificity, with a positive predictive value (PPV) of 86.7% and a negative predictive value (NPV) of 97.8%. Corresponding values for the other tests were as follows: ELISA, 86.7% sensitivity and 94.6% spec- ificity (PPV 72.2%, NPV 97.8%); Western blot Tsol-p27, 76.4% sensitivity and 95.6% specificity (PPV 76.5%, NPV 98.9%); Western blot TsolHSP36, 61.9% sensitivity and 86.1% specificity (PPV 48.1%, NPV 91.5%, Table 2). Discussion Immunodiagnosis of human cysticercosis has been accom- plished using a variety of methods, including indirect hem- agglutination tests, ELISA, indirect fluorescent antibody tests, radioimmunoassay, complement fixation assays, im- munoelectrophoresis, dot blot, and EITB (Deckers and Fig. 2 Immunodot blot using the Tsol-p27 antigen. a Probing done with two positive serum controls (A1–A2) followed by sera from seven positive epileptic cases (A3–A9) and two negative serum controls (A10–A11). b, c Probing done with negative serum samples from epileptic cases (b), and with sera from patients with positive (1–6) or negative (7–11) serology for Chagas disease (c)