The membrane was vacuum washed with a solution of Tween and TBS. Blocking solution, fetal calf serum, and 1% TBS were added (200 μl per well) and left to gravity filter. This was followed by the fixation of 100 μl (5 μg/ml) of flagellar antigen to each well ( 14 Bio Dot instruction manual, Bio-Rad). The membrane was rehydrated by using 100 μl of phosphate-buffered saline per well, which was later vacuum extracted. The paper was first soaked with Tris- buffered saline (TBS), then the membrane was placed into a Bio Dot microfiltration apparatus (Bio-Rad Laboratories). Dot blot analysis was carried out by using nitrocellulose paper ( 1, 5 Bio Dot microfiltration apparatus instruction manual, Bio-Rad Laboratories, Richmond, Calif.). Antigen purity was checked in a 10% polyacrylamide gel, and the purified flagellar protein mass was determined to be 45 to 66 kDa ( 10). Antigen protein concentration was 0.7 mg/ml as determined by the Lowry method ( 14). Polymerized flagellar protein was then recovered from the pellet after 15 min of centrifugation at 15,000 × g at 4☌, was dissolved in 5 ml of distilled water, and was dialyzed with a 50,000-pm-pore-size filter into distilled water for 2 h at room temperature and then for 18 h at 4☌ into 500 ml of distilled water containing 20 g of activated carbon. Two volumes of ammonium sulfate (2.67 M) were added to the recovered supernatant (pH 7.2), and the solution was stored at 4☌ overnight. The suspension was centrifuged at 3,000 × g for 30 min, the supernatant was recovered, and the insoluble material was isolated by centrifugation at 40,000 × g for 1 h at 4☌. Bacteria were centrifuged at 500 × g for 30 min, the pellet was resuspended in 150 ml of saline, and pH was adjusted to 2. Briefly, a colony obtained from a 37☌ overnight culture in nutritive agar was subcultured into 400 ml of brain heart infusion broth (Becton Dickinson BBL) at 37☌ for 16 h. Antigen isolation and purification was carried out following procedures previously described ( 10). Serovar Typhi 1991-ICMT (isolated from the blood of a patient at the ICMT) was used to obtain the flagellar antigen. Once diagnosis of typhoid fever was confirmed by bacteriological isolation, patients were included in the study and were dot blot tested on days 1, 7, and 15. When typhoid fever was suspected, blood samples were collected for triplicate cultures ( 7, 8, 11, 17), and one serum sample was collected for dot blot analysis. They were treated at the Instituto Colombiano de Medicina Tropical (ICMT) (Medellín and Apartadó, Colombia) or Hospital Cayetano Heredia (Lima, Perú) in 19. One hundred and two volunteers older than 15 years with typhoid fever confirmed by bacteriological isolation of Salmonella enterica serovar Typhi from blood culture were included in this study.

Dot blot results