Expression and purification of recombinant LipL32 protein and evaluation of its use in elisa for the diagnosis of leptospirosis
Leptospirosis is a zoonotic disease caused by the spirochete, Leptospira interrogans (L. interrogans). Microscopic Agglutination Test (MAT) is a reference method for the diagnosis of Leptospirosis. However, this method is time consuming and laborious. ELISA using recombinant antigens has proven to b...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2016
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| Subjects: | |
| Online Access: | https://eprints.ums.edu.my/id/eprint/39282/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/39282/2/FULLTEXT.pdf |
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| Summary: | Leptospirosis is a zoonotic disease caused by the spirochete, Leptospira interrogans (L. interrogans). Microscopic Agglutination Test (MAT) is a reference method for the diagnosis of Leptospirosis. However, this method is time consuming and laborious. ELISA using recombinant antigens has proven to be a good diagnostic test for Leptospirosis. Leptospira outer membrane protein (OMP), LipL32 is conserved in pathogenic species, and play an important role in immunopathogenicity and serodiagnosis. The objectives of this study were, to express and purify the synthetic lipl32 gene, to determine the presence of Leptospira-specific antibodies in human serum samples using synthetic rLipL32 antigen in ELISA and to measure the level of regulatory cytokine, IL-10 for the determination of disease severity. In this study, lipl32 gene was synthetically designed and expressed in Escherichia coli (E. coli) expression vector BL21 (DE3) and the protein was purified by immobilized metal affinity chromatography (IMAC). The purified rLipL32 was used as an antigen to detect Leptospira-specific IgM and IgG by ELISA. A total of 53 human serum samples (20 MAT positive samples, 20 MAT negative samples, 11 unknown serum samples and 2 RCPA serum samples) were tested. ELISA results showed that all MAT positive serum samples were positive for IgG and only 65 % (n=13) of these samples were positive for IgM. For the MAT negative serum samples, 25 % (n=5) and 45 % (n=9) of the samples were positive for IgM and IgG respectively by ELISA. The synthetic rLipL32 based ELISA scored 71 % in the RCPA programme out of the total participants in the survey. The MAT positive serum samples were tested for the presence of the regulatory cytokine, IL-10 to determine the disease severity. The concentration of IL-10 in all human serum samples ranged from 10.8 ± 0.04 pg/mL to 31.9 ± 1.38 pg/mL. When compared to the previous study, this level of IL-10 concentrations shows the mild and non-fatal outcome of Leptospirosis disease. These results demonstrates that ELISA based on synthetic rLipL32 antigen was able to detect Leptospira-specific IgM (sensitivity 65 % and specificity 75 %) and IgG (sensitivity 100 % and specificity 55 %) in the human serum samples. Synthetic rLipL32 based ELISA is able to detect Leptospira-specific antibodies in human serum samples and has the potential to serve as a rapid diagnostic test for Leptospirosis and for determination of seroprevalence in the community. |
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