Development of a quantum dot-based rapid diagnostic assay for the detection of dengue NS1 antigen.

Abstract

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.