Four DENV serotypes (DENV-1 to DENV-4) have been identified, each of which may cause SDD (DHF/DSS).ĭuring DENV infections, high concentrations of the native homo-hexameric form of the nonstructural-1 (NS1) glycoprotein are secreted from infected mammalian cells along with infectious DENV virions. Up to 12 500 people (2.5% of all DF cases) die from SDD (DHF/DSS) annually. Approximately 500 000 cases result in the more severe, life-threatening forms, due to plasma leakage, severe haemorrhage, shock and organ failure called either severe dengue disease (SDD) or dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS). 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.ĭengue viruses (DENVs) are the most important vector-borne human viruses in the world, causing an estimated 50–100 million infections in 100 countries and resulting in a self-limiting febrile illness called dengue fever (DF), which is sometimes associated with haemorrhage. 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☌) is not necessary. This is the first study to determine the detection sensitivity of MAbs against known concentrations of s/e NS1 glycoprotein from each DENV serotype. This dot-blot format was ideal for the circulating immune complex disruption step, which is required for increased DENV s/e NS1 glycoprotein detection. The total assay time was reduced to 3 h without any loss of detection sensitivity. 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.ĭENV s/e NS1 glycoproteins were stable in human sera after drying on the nitrocellulose membranes and storage for one month at ambient temperature (28☌) before being processed. 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. Optimal quenching of endogenous human serum peroxidases was attained using 3% H 2O 2 in H 20 for 5 min. 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. Mouse 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. 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. Detection of RNA is termed northern blot and was developed by George Stark at Stanford.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. Neal Burnette and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. The name western blot was given to the technique by W. The method originated in the laboratory of Harry Towbin at the Friedrich Miescher Institute. Other related techniques include dot blot analysis, immunohistochemistry and immunocytochemistry where antibodies are used to detect proteins in tissues and cells by immunostaining, and enzyme-linked immunosorbent assay (ELISA). The gel electrophoresis step is included in Western blot analysis to resolve the issue of the cross-reactivity of antibodies. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. The Western blot (sometimes called the protein immunoblot) is a widely used analytical technique used to detect specific proteins in a sample of tissue homogenate or extract.
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