Characterization Of Anti-Schistoma Haematobium Monoclonal Antibodies And Investigations Into Their Reactivity In The Western Immunoblot Assay

SUMMARY

Schistosoma haematobium antigens are the least studied amongst the three major human schistosomes (S. haematobium, S. mansoni and S. japonicum), mainly because of difficulty in maintaining the life cycle of this parasite in the laboratory. As a result, work on identification of potentially diagnostic and protective S. haematobium antigens lags behind. The work reported in this thesis was aimed at characterizing monoclonal antibodies recently produced against S. haematobium antigens so as to determine whether any of them are useful for specific diagnosis of urinary schistosomiasis or for protection against the disease. Nineteen monoclonal antibodies (MoAbs) recently generated against S. haematobium soluble egg and infected human urinary antigens were used in this study. Earlier attempts to characterize six of the MoAbs had revealed that one of them (Sh3/15.28) was S. haematobium species-specific, but it could not detect the antigen in infected human urine, and was therefore not suitable for developing highly desirable non-invasive, field applicable assays. Moreover, four out of the six MoAbs failed to bind the antigens that they detect in the western immunoblot assay, thereby making it difficult to further characterize them by this technique. From the studies reported in this thesis, fifteen of the MoAbs were found to be of the IgM class, three were of IgGl and one was of IgG3 subclass. Crossreactivity studies using crude antigens of S. haematobium, S. mansoni, S. japonicum12 and Necator americanus (hookworm) eggs or worms showed that two MoAbs (Sh2/15.F and Sh3/15.28) were S. haematobium species-specific, and another one (Sh3/44.3) was specific to the Egyptian strain of S. haematobium. Sh3/44.3 detected antigens in soluble egg antigens of an Egyptian strain of S. haematobium (SEAEgy) obtained from the World Health Organization (WHO) but failed to detect antigens in both egg and adult worm antigen extracts of Ghanaian strain(s) that were tested. Six MoAbs namely, Shl/71.7, Sh3/34.10, Sh3/38.2, Sh4/14.3, Sh5/32.30 and Sh5/34.10 were Schistosoma genus-specific, while the remaining ten bound cross-reactive antigens in S. haematobium and S. japonicum. Biochemical analysis o f the antigens showed that seven out of eight MoAbs namely, Sh2/15.F, Sh3/15.13, Sh3/34.10, Sh4/14.3, Sh4/16.45, Sh5/32.30 and Sh5/34.10 bound protein epitopes which were also present in ammonium sulphate precipitated proteins (P2J) from the urine of individuals infected with Ghanaian strain(s) o f S. haematobium. The eighth antibody (Sh3/44.3) bound a S. haematobium Egyptian strain-specific antigen and, therefore, could possibly not detect any antigens in P2J. All the remaining eleven MoAbs bound glycoprotein antigens. Characterization of the antigens detected, by the indirect immunoflourescent test (IFAT), showed that four MoAbs bound antigens located on the surface membranes of Ghanaian strain(s) of S. haematobium miracidia while, three others bound internally located (possibly cytoplasmic) antigens. The remaining twelve MoAbs bound both surface and internal antigens. Further characterization using the western immunoblot assay showed that reactivity of the MoAbs was influenced by the biochemical nature o f the antigens detected, the13 antibody concentration used as probes for antigen detection and the amount of current used in electrophoresis. Out of the nineteen MoAbs investigated, seventeen reacted under varying conditions of complete denaturing whilst the remaining two (Sh3/38.2 and Sh3/44.5) failed to bind antigens under both denaturing and nondenaturing conditions. All the MoAbs that detected glycoproteins epitopes (except those with generally low reactivity) reacted when diluted in Tris-buffered saline, pH 8.0 containing 2% skimmed milk as blocking agent. On the other hand, most o f the MoAbs which detected relatively low molecular weight polypeptides (