PCR mediated cDNA synthesis from minute amounts of filarial L3 RNA.
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Biomedical subjects
Publications and source records attributed to F Seeber.
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The full length cDNA of the immunodominant Ov33 protein of Onchocerca volvulus was expressed in E. coli using various vector constructs. Expression was best with the vectors pGEX2T and pCG808fx, yielding fusion protein Ov33-GST and Ov33-MBP, respectively. Purified fusion protein Ov33-GST and O. volvulus antigen extracts (OvAg) were used to compare antibody responses (IgM and IgG-subclasses) of patients infected with O. volvulus, Brugia malayi, Wuchereria bancrofti, Mansonella perstans/Loa loa and of Sudanese control sera. Sera of all groups contained IgM reacting with Ov33-GST and with OvAg. There was no IgG1 response to Ov33-GST. IgG1 responses to OvAg were only detected in filariasis sera. IgG2 and IgG3 responses were not detectable or marginal in all groups. The IgG4 reaction of onchocerciasis patients to Ov33-GST and to OvAg was high, whereas few other filariasis sera contained IgG4 antibodies to Ov33-GST and to OvAg. A serodiagnostic test for onchocerciasis based on detection of IgG4 to Ov33-GST had a sensitivity of 93.3% and a specificity of 96%. An epitope common to Ov33 and to the homologous proteins of other filarial species was demonstrated with a monoclonal antibody. Purified Ov33-MBP fusion protein was used to follow the development of the antibody response of four chimpanzees experimentally infected with O. volvulus. The data indicates that antibodies to Ov33 are induced by developing worms and later parasite stages.
We have found that the previously described fast and sensitive copper staining of proteins resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis does not interfere with the subsequent electrotransfer of these proteins to a solid support and their detection by specific antibodies. After the gel is copper stained and photographed it is simply destained and then equilibrated in transfer buffer prior to immunoblotting. We find that this treatment has no significant effect on transfer efficiency or band sharpness and is compatible with all common detection methods for the blotted proteins. It thus permits the separation of proteins to be checked in a simple way before immunoblotting is performed.
Adult Onchocerca voluvlus and infective larvae, but not microfilariae contain an immunodominant antigen (33,000 and 21,000 Mr in females, 39,000, 33,000, and 21,000 Mr in males, 133,000 Mr in infective larvae) which is recognized by an Onchocerca-specific mAb. The component is part of the reproductive organs and muscles. 96.2% of onchocerciasis sera contained antibodies detectable by immunoblotting against it. Antigen purified by immunoaffinity chromatography was specifically recognized in immunoblots by onchocerciasis sera, but not by sera from other filarial infections. The high immunogenicity, the specificity, and the occurrence in infective larvae of this antigen indicate an immunodiagnostic potential and a possible role in the immunobiology of the parasite.
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