Production and characterisation of polyclonal antiserum and monoclonal antibodies against Strongyloides ratti antigens and their utility for diagnosis of strongyloidiasis by coproantigen detection

Human strongyloidiasis is commonly referred to as threadworm infection. Most cases of strongyloidiasis manifest as chronic mild, or asymptomatic intestinal infection that last many years. However, in certain circumstances, parasites can rapidly multiply in a patient, disseminate more widely to other organs, and cause life threatening infection. Diagnosis of strongyloidiasis can generally be undertaken by parasitological diagnosis, usually microscopy, coproculture (Harada Mori, or Baermann), or by detection of parasitespecific antibodies by ELISA. Each of these approaches have drawbacks. Similar problems have been encountered with the diagnosis of many other intestinal parasites. One way of overcoming the shortfalls of diagnostic tests of other intestine infections has been the use of coproantigen assays. Coproantigen assays are commercially available for diagnosis of a range of human and animal helminth, protozoan, bacterial and viral infections, where their sensitivity and specificity is satisfactory. For the detection of Strongyloides spp in faeces, only one coproantigen detection assay has been published for the detection of S. ratti. In this assay, whole parasitic worm antigen was used to raise polyclonal antiserum. However, the assay was relatively insensitive, and its utility to detect human Strongyloides was not investigated. In addition, compared to research undertaken in other well-studied parasites such as Hookworms, Taenia spp. and Echinococcus spp, little is known about the identity and function of E/S proteins or indeed other proteins of Strongyloides spp. In this study, a panel of antibodies was raised against S. ratti to explore the utility of Strongyloides specific antibodies for the development of a diagnostic assay and to study the biology of the parasite. The first results chapter reports the development and optimisation of a technique to isolate S. ratti excretory/secretory (E/S) antigens, and the generation of high titre antiserum in both rabbits (α-E/S Ab) and mice (StrE/S MAb). α-E/S Ab contained antibodies that cross-reacted with rat intestinal proteins and the dominant contaminant at ~75 kDa was determined to be rat serum albumin. Many techniques were attempted to reduce the level of cross-reactive antibodies within the antibody preparation and the successful technique utilised adsorption against a combination of uninfected rat faecal supernatant, sham E/S and uninfected rat serum. This final serum was used in a faecal supernatant immunoprecipitation experiment to purify Strongyloides coproantigen. This immunoprecipitation/coproantigen complex was utilised for the generation of monoclonal antibodies. One specific monoclonal antibody (StrE/S MAb) was generated and displayed high levels of immunoreactivity with S. ratti and S. stercoralis larval and parasitic adult worm antigens, while not cross-reacting with any rat proteins. In immunolocalisation experiments, StrE/S MAb was observed to bind to specific cells in the ovaries, including the ova. In the second results chapter the utility of a polyclonal α-E/S antibody raised against S. ratti E/S to detect coproantigen is reported. High levels of cross-reactivity was observed when faecal supernatants were extracted in a common extraction buffer PBS-T or 70% Ethanol. In contrast with many published coproantigen ELISAs, the addition of foetal calf serum or rat serum in the assay did not improve the signal:noise ratio. However, when faecal supernatants were extracted in formalin levels cross-reactivity with faecal components was eliminated, while preserving the positive signal in infected samples. Coproantigen stability was explored with a panel of faecal samples preserved at 4°C, extracted and unextracted in formalin. Interestingly, formalin-extracted faecal supernatants stored at 4°C tested negative, whereas faecal samples preserved, unextracted in formalin remained coproELISA positive. Finally, three consecutive stool samples were collected from a patient infected with S. stercoralis and extracted in formalin. The stool samples collected tested negative for larvae by sodium nitrate flotation and Baermann concentration, suggesting a sub-patent infection. All three faecal supernatants tested coproELISA positive at the standard assay dilution namely 1/4 and remained positive when diluted further to 1/8. The final results chapter describes the characterisation of a monoclonal antibody raised against S. ratti parasitic adult worm extract (MAb 8C3) by molecular, histochemical and proteomic techniques. Specificity for the parasitic adult worm lifecycle stage alone was demonstrated by immunoblotting, immunoprecipitation and immunohistochemistry. Using immunohistochemistry the target organ of MAb 8C3 was identified as nerve cords in the parasitic adult worm hypodermis. Proteomic analysis identified MAb 8C3s target antigen as the 10 kDa chaperone protein, CPN10. The validity of standard proteomic analysis for the characterization of nematode proteins and the characterization of S. ratti CPN10 is discussed