Phase separation and immuno-characterization of integral membrane proteins of the tape worm echinococcus granulosus
Mutisya, Maxine Yalo
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Immune responses in human patients with suspected cystic echinococcosis (CE) have been widely used to diagnose the infection or the disease. Almost all available serological tests have been used for the diagnosis of human CE. The components of the metacestode from a variety of hosts have been used as sources of antigen in diagnostic tests. There are considerable differences between the various tests both in their sensitivity and specificity. as the sensitivity of a test increase so does the demand for improved antigens in order that sufficient specificity can be achieved to take advantage of the greater sensitivity. Difficulties with widescale application of serodiagnosis in CE occur due to limitations in supply of native parasite antigens for tests, extensive cross reactivity of cyst fluid antignes with human antibody to a wide variety of helminths, heterogeneity in response by patients and in quality control of antigen preparations derived from different batches of hydatid cysts. In this study, integral membrane proteins from the metacestode have been isolated and their efficacy as antigens in the serodiagnosis of human CE partially assessed. Nonionic detergent Triton X-114 was used in extracting antigens from the various compartments of the metacestode and submitting the detergent solubilized materials to phase separation. Ultra-centrifugation of whole extracts of the different tissues was also carried out to compared the enrichment of important antigenic components in the cellular fractions. Analyses of aliquots of the separated phases and ultra-centrifuge fractions was accomplished by sodium dodecyl sulphate-polyacrylamide gel lectrophoresis (SDS-PAGE) and immunoblotting. Antigenic and exclusively integral membrane proteins were isolated from E. granulosus protoscoleces. In particular, bands at 21 kDa, 31 kDa, and 47 kDa were highly antigenic when probed with pooled positive Echinococcus granulosis human sera. Six clones tentatively expressing fragments of immunogenic protoscoleces integral membrane antigens were isolated by employing rabbit antisera to immunoscreen an E. granulosus protoscoleces mRNA derived gt11 expression library. Furthermore, four of the six clones, identified were validated through PCR to contain inserts. The six clones identified showed high species specificity in plaque immunoassay employing individual patient sera. One clone, Eg,-1A, showed potential as a candidate in Echinococcus granulosus diagnosis.
- MST-Zoological Sciences