Serodiagnosis Of Strongyloidiasis: Identification Of Cdna Clones, Production Of Recombinant Antigens And Immunoassay Development
| dc.contributor.author | Arifin, Norsyahida | |
| dc.date.accessioned | 2017-01-26T01:50:11Z | |
| dc.date.available | 2017-01-26T01:50:11Z | |
| dc.date.issued | 2016-03 | |
| dc.description.abstract | Strongyloidiasis is a human parasitic disease caused by the nematode Strongyloides stercoralis. Infection by this parasite can cause long-term infection in humans or can disseminate to other organs, especially in individuals with immunosuppression, which commonly results in fatal outcomes. The majority of patients are asymptomatic or present with non-specific gastrointestinal complaints, and there is no gold standard method to rule out the infection. Definitive diagnosis is usually made by a combination of clinical signs and symptoms, microscopic identification, and serology test. To date, the available commercial tests are based on native parasite antigen extract, but such tests have problems of cross-reactivity with other helminthic infections. A recombinant antigen-based test is a good alternative for improved diagnostic specificity and standardized test quality, thus, the present study was conducted to achieve this goal. The initial stage of the study involved testing serum samples with the in-house IgG-, IgG4- and IgE-ELISAs in addition to a commercial IgG-ELISA kit. The results showed that the IgG-ELISA assay demonstrated the highest sensitivity using either in-house or commercial assays (84.6%, n=26), whereas the IgG4-ELISA displayed the highest specificity of 92.7% (n=55). The serum samples were categorized into different groups based on the IgG and IgG4 results for use in the subsequent immunoscreening experiments. Primary immunoscreening of the S. stercoralis phage cDNA library resulted in the selection of 20 cDNA clones from each IgG- and IgG4-phage immunoblot. These clones were subjected to secondary and tertiary immunoscreenings using individual pre-adsorbed serum samples. Finally, two cDNA clones namely Ss3a and SsIa were selected as having high potential diagnostic value. The Ss3a DNA insert was highly similar to a S. stercoralis hypothetical protein, while the SsIa DNA insert was identical to an immunoglobulin-binding protein of S. ratti. The Ss3a gene was recombinantly expressed as a GST-fusion protein in the pET42a vector and validated by MALDI TOF/TOF analysis. The diagnostic value of rSs3a protein was determined by western blot analysis using individual serum samples. Subsequently, a one-hour lateral flow dipstick test was produced with diagnostic sensitivity and specificity of 100%, respectively. Meanwhile, the SsIa gene was fused to a dual protein tag in the pET28b vector and expressed as a His-fusion protein and verified by western blot analysis and MALDI-TOF TOF. The rSs1a was then developed into a rapid (15 min) lateral flow dipstick test, which gave a sensitivity rate of 90% (n=30) and a specificity rate of 98% (n=46). This study has successfully discovered two infection markers and produced two new recombinant proteins of potential diagnostic value. Proof-ofconcept of their usefulness in a lateral flow dipstick test for serodiagnosis of strongyloidiasis was also demonstrated, and in combination may serve as a good rapid test for of human strongyloidiasis. | en_US |
| dc.identifier.uri | http://hdl.handle.net/123456789/3590 | |
| dc.language.iso | en | en_US |
| dc.publisher | Universiti Sains Malaysia | en_US |
| dc.subject | Strongyloides stercoralis | en_US |
| dc.title | Serodiagnosis Of Strongyloidiasis: Identification Of Cdna Clones, Production Of Recombinant Antigens And Immunoassay Development | en_US |
| dc.type | Thesis | en_US |
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