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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.
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