Figure. Western blot reactivity to and Silver stain analysis of Mycobacterium ulcerans culture filtrates (MUCF). A) Representative antibody responses to MUCF. M; molecular weight markers with annotations corresponding to molecular weight on the left; lanes 1-6, representative BU patient sera with reactivity to MUCF; lanes 7-10, representative antibody reactivity in healthy persons from the disease-endemic area; lanes 11-12, serologic reactivities to MUCF of two representative tuberculosis (TB) patients. TB patient sera that were serologically reactive to MUCF were included as a control for cross-reactivity. Regions of antibody reactivity corresponding to M. ulcerans antigens of 70, 38/36, and 5 kDa are noted (arrows). B) Silver-stained SDS-PAGE gel of MUCF. The identification of putative proteins corresponding to the serologically reactive 70, 38/36, and 5 kDa MUCF antigens are noted (stars). For Western blot analyses, aliquots (50 µg protein) of MUCF were resolved by discontinuous SDS-PAGE (10) with preparative 10% to 20% gradient 1-well gels (Novex, San Diego, CA) and then transferred to nitrocellulose (14). Nitrocellulose sheets were cut into 2-mm strips and stored in 5% skim milk in Tris-buffered saline, pH 7.6, until used. Antibody to MUCF was detected by probing nitrocellulose strips with sera at a 1:50 (vol:vol dilution). Bound serum antibodies were detected with alkaline phosphatase-conjugated anti-human antibody (Sigma Chemical Co., St. Louis, MO), and the substrate 4-Bromo-3-Chloro-2-Indoyl-1-Phosphate/Nitro blue tetrazoleum (Sigma Chemical Co., St. Louis, MO). All sera were tested in duplicate for confirmation of the antibody responses.