Serologic Response to Culture Filtrate Antigens of Mycobacterium ulcerans during Buruli Ulcer Disease
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.