Browsing by Author "Munasinghe, M.M.E."

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Designing of immunogenic peptides from Dengue Virus NS1 region for production of monoclonal antibodies as diagnostic intermediates
(Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Munasinghe, M.M.E.; Chandrasekharan, N.V.; Korbakis, D.; Soosaipillai, A.; Diamandis, E.P.; Athapaththu, A.M.M.H.; Gunathilaka, P.A.D.H.N.; Abeyewickreme, W.
BACKGROUND: Small peptide antigens have become an essential tool for antibody production in the recent life science research applications. The immunogenicity of peptide antigens is a critical factor to induce the immune response in order to produce desired antibodies. METHODS: In the current study, we have previously determined four Dengue (DEN) serotype specific peptides, containing 28 Amino Acid (AA) residues were re-designed. The peptides were re-designed considering many factors, for instance, sequence of the Sri Lankan isolates, abundance of Cysteine residues, solubility and the length of the peptide, carrier protein to be used and several other factors such as the N-terminal and C- terminal AAs and multiple AA residues. The peptide sequences were analysed using Antigen Profiler Peptide Tool (Thermo-scientific), Peptide Property Calculator (Genscript) and Swiss-Model (Biozentrum). RESULTS: The protein sequence of the peptides were changed according to the Sri Lankan isolates (AEB98757.1, ACS32038.1, AHG23239.1 and AHN50410.1). Oxidation of Cysteine residues results in significant conformational changes. Replacement of Cysteine with Serine prevents such oxidation reactions and it often retains full biological activity. Generally, peptides with a high number of hydrophobic AA (>50%) may result insoluble peptides. Similarly, to obtain a soluble peptide, it is important to contain at least one charged AA in every five AAs. Hence, the number of hydrophobic residues in the peptides were maintained below 50% and ensured that one out of every five amino acids is charged. The length of the peptide is an important factor as long peptide increases immunogenicity, but also increases the chance for cross-reactivity while a short peptide improves the specificity, but may not be immunogenic. In order to obtain both highly conserved and variable regions among four serotypes, the peptide length was determined as 29 residues. A terminal Cysteine was added to allow peptide conjugation with carrier protein. Keyhole Limpet Hemocyanin was selected as the carrier protein due to its higher immunogenicity. N-terminal Glutamine or Aspargine and C-terminal Proline or Glycine in the sequences were avoided. Finally, the peptides sequences were determined as: DEN1; CPESSDDQRA WNIWEVEDYGFGIFTTNIW,DEN2; CAESPN TNRA WNSLEVEDYGFGVFTTNIW, DEN3;CPESPSASRAWNVWEVEDYGFGVFTTNIW and .DEN4;CSESPNERRAWNSLEVEDYGFGMFTTNIW. CONCLUSION: These peptides have a high potential to be used as peptide antigens for Monoclonal Antibody production.
Development of a quantum dot-based rapid diagnostic assay for the detection of dengue NS1 antigen.
(University of Kelaniya, 2022) Munasinghe, M.M.E.
Dengue is a mosquito bom viral disease which is considered as one of the most important and most prevalent infectious diseases in tropical and sub-tropical world. This is caused by a virus from the genus Flavivirus that has four closely related serotypes. The differentiation of the clinical symptoms of dengue fever (DF) is challenging fbr the clinicians due to its similarity with other febrile illnesses. Diagnosis of the disease as early as possible would improve the patient management, vector controlling and lower the fatality rate. The main objective in this study was to develop a cost effective rapid Lateral Flow Immunoassay (LF1A) using nanotechnology for detection of dengue infection. The specific objectives were to develop monoclonal antibodies against dengue NS1 protein as the capturing agent and to synthesize L-Cysteine capped CdTe quantum dots as the detecting agent. In this study, four serotype specific synthetic peptides (Pl, P2, P3 and P4) were designed from the NS 1 region of the serotype 1, 2, 3 and 4 respectively, considering various factors. These peptides were used to immunize four, female Balb/c mice and fusions were carried out to produce hybridoma clones. Enzyme Linked Immunosorbent Assays (ELISA) were developed to screen the antibodies reacting with peptides as well as the native protein present in clinical samples. The selected antibody was used to anchor on L-Cysteine capped CdTe quantum dots. The quantum dot conjugated antibody was used in the development of LFIA. A total number of 28 IgG secreting hybridoma clones out of 1830 growing clones produced dengue specific monoclonal antibodies. A monoclonal antibody (Pl.18) resulted from the fusion of Pl immunized mice, showed significantly high antibody response fbr all four dengue serotypes. This antibody was used as the detector antibody in LFIA. During the LFIA, a fluorescent band was visible under ultra violet light (UV) fbr the detection of dengue NS1 protein in infected urine samples implying the successful development of a cost effective LFIA fbr detection dengue. Detection of infected blood was not feasible due to fluorescent quenching resulted by high lysozyme concentration present in blood and has to be further optimized. These findings can be further used to develop a user friendly low cost diagnostic test kit fbr detection of dengue infection from urine.