Production and Characterization of Monoclonal Antibodies against Schistosoma Haematobium Soluble Egg and Urine-Based Antigens

Abstract

Diagnosis of schistosome infections is principally based on the demonstration of parasite eggs in excreta, or by haematuria and /or proteinuria. These methods are, however limited by inadequate sensitivity or specificity. Furthermore, even though microscopy is very specific, it is also tedious and time consuming. The work reported in this thesis was conducted with the aim of producing monoclonal antibodies (MoAbs) against Schistosoma haematobium antigens, and to characterize them so as to determine if any of them would be useful in diagnosis, especially for detection of parasite antigens in the urine of infected persons. The availability of such MoAbs would pave the way for the development of more sensitive, specific and field-applicable immunological assays for diagnosis of urinary schistosomiasis caused by S. haematobium. The strategy employed in this study was to produce MoAbs, using S. haematobium soluble egg antigens (ShSEA) and infected human urine-based parasite antigens in protein extracts (UP2-IP) as immunogens. Both antigens induced substantial serum antibody responses in immunized mice, with titres as high as 1:5,000 for ShSEA and 1:50,000 for UP2-IP. The high immunogenicity of UP2-IP was attributed to human immunoglobulins associated with parasite antigens in immune complexes. All MoAbs produced, using this antigen, were found to react with UP2IP but not with ShSEA. Also, the MoAbs generated using ShSEA as immunogen did not react with UP2-IP. However, one MoAb Sh5/32.30 reacted with both ShSEA and UP2-IP. This MoAb was produced using both ShSEA and UP2-IP as immunogen, but the final booster, just before cell fusion was made with ShSEA. In all, six MoAbs were produced. These included two S. haematobium species-specific MoAbs (Sh2/15.F and Sh3/38.2), and four pan-schistosome MoAbs (Shl/71.7, Sh3/15.28, Sh4/14.3 and Sh5/32.30) that cross-reacted with antigens in S. haematobium, S. mansoni and S. japonicum egg or adult worms. Three of the MoAbs were also found to detect S. haematobium antigens in urine of infected humans. Characterization of the antigens detected, using the indirect immunofluorescent test (IFAT), Western immunoblot analysis, proteinase-K digestion and periodate oxidation, showed that the two S. haematobium species-specific MoAbs bound different antigens (one protein and one glycoprotein), and at least three different antigens. The glycoprotein species-specific antigens had molecular weights (MW) of approximately 37kDa and 46kDa. It was also found that all the glycoprotein antigenic determinants detected were located on the surface membrane, as well as on intracytoplasmic organelles in S. haematobium miracidia, whilst the protein epitopes were located only on the surface membrane. Of the six MoAbs that were produced and characterized, three of them bound protein epitopes, all of which were different from each other. One of the protein epitopes was detected by S. haematobium species-specific MoAb (Sh2/15.F), whilst the other two were detected by pan-schistosome MoAbs (Sh4/14.3 and Sh5/32.30). Interestingly, only the three protein antigens could be detected in the urine of S. haematobium infected persons. One of these protein epitopes, bound by Sh4/14.3, was shown to occur on different peptides of MW, 73kDa and 78kDa. Cross-reactivity studies with soluble egg antigens of an Egyptian strain of S. haematobium revealed that only Sh2/15.F could not react with the North African parasite stain. This ability of most of the MoAbs to detect both Ghanaian and Egyptian strains of S. haematobium suggested that the MoAbs generated in this study might not only be useful for diagnosis of urinary schistosomiasis in Ghana, but also in other parts of Africa.