Abstract
BackgroundDetection of dengue virus (DENV) soluble/excreted (s/e) form of the nonstructural-1 (NS1) glycoprotein in patient acute-phase sera is ideal for diagnosis. The commercially-available detection assays are, however, too expensive for routine use and have low specificity, particularly for the s/e NS1 glycoprotein of DENV-2 and DENV-4, which are important causes of lethal human disease worldwide.MethodsMouse monoclonal antibodies (MAbs) were generated and screened against s/e NS1 glycoprotein purified from each DENV serotype to obtain those that reacted equally with each serotype, but not with yellow fever virus (YFV) s/e NS1 glycoprotein or human serum proteins. One MAb, MAb 2C4.6, was further tested against these DENV glycoproteins in human sera using simple, peroxidase-labelled secondary antibody/substrate-developed dot-blot assays.ResultsOptimal quenching of endogenous human serum peroxidases was attained using 3% H2O2 in H20 for 5 min. MAb 2C4.6 showed an acceptable detection sensitivity of < 32 ng/ml for the s/e NS1 glycoprotein of each DENV serotype but did not cross-react with the YFV s/e NS1 glycoprotein or human serum proteins. By contrast, the LX1 epitope-specific MAb, 3D1.4, showed similar detection sensitivity against only the DENV-1 NS1 glycoprotein, consistent with results from commercial DENV s/e NS1 glycoprotein detection assays.DENV s/e NS1 glycoproteins were stable in human sera after drying on the nitrocellulose membranes and storage for one month at ambient temperature (28°C) before being processed. The total assay time was reduced to 3 h without any loss of detection sensitivity. This dot-blot format was ideal for the circulating immune complex disruption step, which is required for increased DENV s/e NS1 glycoprotein detection.ConclusionsThis is the first study to determine the detection sensitivity of MAbs against known concentrations of s/e NS1 glycoprotein from each DENV serotype. The preparation of patient serum samples for dot-blot assays can be performed by staff with a basic level of training and storage at low temperatures (e.g., -80°C) is not necessary. These simple, inexpensive (US$ 0.05/sample), robust, sensitive and relatively rapid assays, using improved MAbs such as MAb 2C4.6, should be ideal for the diagnosis of all DENV serotypes in DENV endemic regions.
Highlights
Detection of dengue virus (DENV) soluble/excreted (s/e) form of the nonstructural-1 (NS1) glycoprotein in patient acute-phase sera is ideal for diagnosis
This assay only detected the s/e NS1 glycoprotein in less than 50% (DF: 40% and dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS): 45%) of acute-phase sera from patients with secondary DENV-2 infections, at concentrations from 70 ng/ml up to 15,000 ng/ml [6]. Since these serum samples had undergone several freezethaw cycles, the results demonstrated the lability of the DENV s/e NS1 glycoprotein, probably through disruption of the relevant LD2 epitope, [4] or loss of the LD2 epitope through cleavage of the NS1 glycoprotein at the conserved dibasic amino-terminal protease cleavage site [7]
We previously showed that the s/e NS1 glycoprotein from all four DENV serotypes could be detected in Indonesian patient sera using a simple dot-blot format, when acid treatment and neutralisation steps were used [17]
Summary
Detection of dengue virus (DENV) soluble/excreted (s/e) form of the nonstructural-1 (NS1) glycoprotein in patient acute-phase sera is ideal for diagnosis. The ELISA using MAb 1H7.4 failed to detect the s/e NS1 glycoprotein in acute-phase sera from patients with primary DENV-2 infections collected in Thailand [6]. This assay only detected the s/e NS1 glycoprotein in less than 50% (DF: 40% and DHF/DSS: 45%) of acute-phase sera from patients with secondary DENV-2 infections, at concentrations from 70 ng/ml up to 15,000 ng/ml [6]. A capture ELISA using mouse and rabbit polyclonal antibodies (PAbs) detected the s/e NS1 glycoprotein in a moderate proportion of acute-phase sera from patients with either primary (77% (10/13): 40 to 2,000 ng/ml) or secondary (88% (14/16): 10 to 2,000 ng/ml) DENV-1 infections in French Guiana [8], identifying the s/e NS1 glycoprotein as a suitable target for DENV diagnostics
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