| Due to increasing global incidence of dengue disease and lack of vaccine development,early, rapid and economical detection is only way to overcome this disease. The detection ofdengue infection remains an open question. The dengue virus type2(DENV2) NS1, is the firstnon-structural glycosylated protein to be translated in the dengue virus genome. It has beenfound that NS1can be detected in the blood of infected patients as soluble serum antigen mostlyfrom days1–9and significant amount at days3-5in primary and secondary infections. So, NS1represents an interesting target antigen for the development of rapid diagnostic tools. In thisstudy, NS1was expressed in Escherichia coli and protein refolding protocol was successful. TherNS1was obtained as soluble protein with preserved structural features, which were confirmedby fluorescence spectroscopy and circular dichroism methods. Simultaneously, it was found thatrNS1protein conserved important antigenic determinants of the native virus protein andrepresents a useful reagent for the improvement of diagnostic tools.Antibodies are widely used for disease diagnostics and therapy. Serotype-specificmonoclonal antibodies against rDENV2-NS1were successfully generated by using theconventional hybridoma technology. Totally18hybridoma cell lines were obtained after theirpositive reactivity with rNS1protein. MAb3B3was selected for comparison with VHH antibodyon the basis of its no cross reaction with other His-tag proteins and strong reactivity after indirectELISA. But this conventional Mab production by hybridoma technology is laborious, costly andtime consuming especially in the cloning and selection process.The discovery of camelid heavy chain antibodies establishes a new concept inimmunology and biotechnology. Rapid, reliable, convenient selection and screening of heavychain antibody (VHH) against dengue type2rNS1protein, from a large non-immune llama VHHlibrary were derived in the present study. VHH antibodies against recombinant dengue virus type2NS1were biopanned from non-immune llama library by phage display method and purifiedfrom E.coli. Sequence analysis of VHH antibodies revealed novel CDR3regions indicating highantigenic specificity. The dissociation rate constant, KD, as an indicator of affinity is used in thepresent study. The binding affinity value of VHH (P2) was2.79×10-8which is within the range ofKDvalues of most VHHs for their target antigens while monoclonal antibody (3B3) showed5.74×10-6affinity for rNS1.Epitope mapping was performed to determine whether different or similar epitopes of VHH antibody and monoclonal antibody were involved in the diagnosis responses. Antigenicepitopes were screened by using phage display peptide library and purified VHH and monoclonalantibodies were used as target. Interestingly, same region of NS1comprising of amino acids224HWPKPHTLW232was highly conserved for both kinds of antibodies and can be consideredbinding epitope region. This homologous region in rNS1sequence (224-232) was assessed aspotential epitope which indicates that probably histidine-trptophan-typtophan or tryptophan-proline-tryptophan are key components and form the antigen epitope. So, this information isuseful to compare the binding affinities of both monoclonal and VHH antibodies, having sameantigenic epitopes of rNS1antigen, in serological diagnostic tests.Keeping this concept in mind, VHH antibody was compared with monoclonal antibodyby developing rapid diagnostic kit based on immunochromatographic assay. VHH antibodyshowed better sensitivity and specificity as compared to monoclonal antibody which might bedue to the long CDR3regions of the VHH antibodies and their ability to bind to the pocket andcleft of the targeted antigen. This demonstrates that VHH antibodies purified from E.coli aremore cost-effective tools than monoclonal antibodies and likely to be an option for developingpoint-of-care tests against DENV infection. |
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