Solubilization of epicuticular antigen from Dirofilaria immitis third-stage larvae.
The solubilization of epicuticle from third-stage (L3) Dirofilaria immitis larval cuticles was investigated. Cuticles collected after L3 had molted were incubated in 1.5% sodium dodecyl sulfate (SDS) at 37 degrees C with vigorous shaking. Solubilization of epicuticular layers was accomplished as demonstrated by electron microscopy. Diminished binding of an epicuticular specific monoclonal antibody (DIM-229) was seen when SDS-treated cuticles were compared to untreated cuticles in an indirect fluorescence antibody assay. Cuticles which were extracted further by boiling in 1.5% dithiothreitol (DTT) produced less protein than cuticles solubilized in SDS. Both extracts reacted with DIM-229 in an indirect enzyme-linked immunosorbent assay, indicating retention of antigenic reactivity of the solubilized epitope. SDS-polyacrylamide gel electrophoresis of SDS-derived antigens revealed, after silver staining, proteins from 12 to 77 kDa and only 1 band at 15 kDa for SDS-treated cuticles boiled in DTT. Western blot analyses of the extracts with DIM-229 were inconclusive.
Mok
M
M
School of Veterinary Medicine, University of Wisconsin, Madison.
Grieve
R B
RB
Abraham
D
D
Rudin
W
W
D000818:Animals; D000911:Antibodies, Monoclonal; D000947:Antigens, Helminth / Q000378:metabolism*; D000954:Antigens, Surface / Q000378:metabolism; D001345:Autoradiography; D015153:Blotting, Western; D004183:Dirofilaria immitis / Q000276:immunology* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005370:Filarioidea / Q000276:immunology*; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D007814:Larva / Q000276:immunology / Q000648:ultrastructure; D008854:Microscopy, Electron; D012967:Sodium Dodecyl Sulfate; D012995:Solubility
D000911:Antibodies, Monoclonal; D000947:Antigens, Helminth; D000954:Antigens, Surface; D012967:Sodium Dodecyl Sulfate