Preparation And Characterization Of Monoclonal Antibody Against M Protein And NS1 Protein Of Dengue Virus Serotype 2

Objectives:The global prevalence of dengue virus has grown dramatically in recent decades, predominantly in Southeast Asia areas.Dengue fever (DF) and its more serious forms, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are becoming important public health problems around the world.At present, no dengue vaccine has been licensed, and there is no specific treatment for DF and DHF, from which rates of mortality are high. Therefore, there is a great demand for the rapid and specific detection of dengue infection.The diagnostic kits for dengue virus infection are mainly used full virus or recombinant E protein as detected antigens presently, and further studies are needed to evaluate their sensitivity and specificity. Our aim is to prepare monoclonal antibody (McAb) against M protein and NS1 protein of dengue virus type 2, which could be applied to the development of gold colloidal rapid diagnostic kit for the specific detection of dengue virus infection.Methods: Recombinant expression vectors pET-32a(+)/M and pQE-30/NS1 containing prm and ns1 gene were induced by optimal concentration of IPTG. Recombinant protein expressed in the supernatant was purified by Ni-NTA-chelated agarose affinity chromatography method. Purified M protein and NS1 protein were confirmed by SDS-PAGE analysis.Immunoactivity of purified recombinant proteins were confirmed by rabbit seraBalb/c mice were immunized with recombinant proteins. Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line SP2/0 cells. The hybridoma cells that secreted specific McAbs were cloned with soft agarose culture method. The immunoglobulin (Ig) subtype, the antibodies titers, and the affinity of the obtained McAbs were determined by indirect ELISA. The specificity of McAbs were tested by ELISA and Western blot analysis.Results: Recombinant M and NS1 proteins were mainly expressed in the form of supernatant in E.coli system, whereas the antigenicity of the recombinant proteins were detected by rabbit antiserum. Recombinant proteins were successfully purified from induced supernatant.Three hybridomas were screened out, namedâ…¢1A6,â…¢3D2 andâ…¢4C3. The Ig subtype ofâ…¢4 C3 andâ…¢1A6 McAb are both IgG₁, and the Ig subtype ofâ…¢3D2 is IgG_2a. The titers ofâ…¢1A6 andâ…¢3D2 McAb were 1: 10⁶ and 1:10⁵, respectively. The relative affinity constants of McAbs were determined as 10⁷. Western blot identification suggested that the McAbs reacted specifically with the recombinant proteins.Conclusions: Three high titer, specific McAbs against M protein and NS1 protein of dengue virus type 2 have been successfully prepared and primarily identified, which may be useful in the development of a rapid and convenient diagnostic kit for detection of dengue virus infetcion.