Polyclonal antibodies against properly folded Dengue virus NS1 protein expressed in E. coli enable sensitive and early dengue diagnosis

Abstract

The non-structural 1 (NS1) protein plays an important role in dengue diagnosis because it has been detected as a soluble serum antigen in both primary and secondary infections. The NS1 protein was expressed in Escherichia coli cells, and the efficiency of four different refolding protocols was tested. All of the protocols generated dimeric NS1 in a conformation similar to that of the protein expressed by eukaryotic cells. A polyclonal antibody produced from the properly folded E. coli recombinant NS1 (rNS1) protein proved to be a useful tool for the diagnosis of Dengue virus because it detected 100% of the Dengue virus 2 (DENV2) in infected patients' sera and 60% of the DENV IgM-positive sera not detected by commercial NS1-based diagnostic kits. These data suggest a high-efficiency method for correctly folding rNS1 that maintains its structural and immunogenic properties. In addition, a detection method using the polyclonal antibody against correctly folded rNS1 seemed to be more sensitive and efficient for NS1 detection in serum, highlighting its usefulness for developing a high-sensitivity diagnostic kit.

Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity

“…After five washes with PBS, nonspecific binding sites were blocked with 200 l of 1% BSA in PBS containing 0.05% Tween 20 (PBST) for 1 h at 37°C, followed by five washes with PBST. Specific concentrations of the recombinant NS1 (rNS1) protein in dimeric form, the protocol (protocol IV) for the purification of which was published elsewhere (26), were added to each well, and the plates were incubated for 2 h at 37°C. The plates were then washed five times with PBST, followed by a 1-h incubation with an anti-NS1 polyclonal antibody (produced in a mouse in-house) diluted in PBS.…”

“…A pulldown assay was carried out using approximately 80 g/column of purified homemade anti-NS1 polyclonal antibody (26) attached to an AminoLink Plus coupling resin (co-IP kit; Pierce, USA). The resin was incubated overnight with 200 l of a mixture containing 2.5 M rNS1 protein and 2.5 M GAPDH (Sigma, USA), 2.5 M rNS1 protein and 2.5 M hemopexin (HPX; Millipore, USA), or 2.5 M rNS1 protein and 1 mg BHK-21 cell extract in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl.…”

“…The cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min. The slides were incubated in blocking solution containing 1.5% BSA in PBS and then with purified anti-NS1 mouse polyclonal (26), anti-GAPDH rabbit monoclonal, anti-GAPDH mouse monoclonal or anticalreticulin (calreticulin is an endoplasmic reticulum [ER] marker) rabbit monoclonal antibody (Abcam) that had been diluted in blocking solution for 1 h. The cells were washed and incubated for 45 min with an Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen) or an Alexa Fluor 546-conjugated goat anti-rabbit IgG antibody (Invitrogen) diluted in blocking solution. The cells were subsequently incubated with 5 mM 4=,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 5 min at room temperature.…”

“…The NS1 protein, in turn, is known to interact with a myriad of intracellular and extracellular proteins (37). To determine whether the interaction between iNS1 and GAPDH was a false-positive result or if a direct interaction occurs, a direct binding ELISA was performed with both purified recombinant NS1 (rNS1) (26) and GAPDH proteins. In this experiment, either GAPDH or BSA (control) was adsorbed onto a plate and incubated with increasing concentrations of the rNS1 protein.…”

Determination of Viremia and Concentration of Circulating Nonstructural Protein 1 in Patients Infected with Dengue Virus in Mexico

“…The recombinant protein was obtained by using a refolding protocol that enabled generation of dimeric NS1 in a conformation similar to that of the protein expressed in eukaryotic cells. 26 Serum samples obtained from DENV-1 patients were diluted 1:1,000 and those obtained from DENV-2 patients were diluted 1:200 to generate optical readings within the linear range of the curve, which ranged from 0.073 ng/mL to 0.60 ng/mL (Supplemental Figure 1). The dilution factor required for analysis of serum samples corresponding to DENV-1 and DENV-2 patients was determined empirically by testing samples at multiple dilutions.…”