title: A simple, inexpensive, robust and sensitive dot-blot assay for equal detection of the nonstructural-1 glycoprotein of all dengue virus serotypes.
abstract: BACKGROUND
  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 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.
  
  
  METHODS
  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. 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.
  
  
  RESULTS
  Optimal quenching of endogenous human serum peroxidases was attained using 3% H(2)O(2) in H(2)0 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.
  
  
  CONCLUSIONS
  This 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.
authors.affiliation: Laboratorio de Enfermedades Tropicales, Departmento de Medicina, Universidad del Norte, Km 5 Antigua via Puerto Colombia, Barranquilla, Colombia. afalconar@uninorte.edu.co
authors.forename: Andrew K I
authors.initials: AK
authors.lastname: Falconar
authors.forename: Claudia M E
authors.initials: CM
authors.lastname: Romero-Vivas
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000914:Antibodies, Viral / Q000302:isolation & purification; D000956:Antigens, Viral / Q000032:analysis*; D019411:Clinical Laboratory Techniques / Q000191:economics / Q000379:methods*; D003715:Dengue / Q000175:diagnosis* / Q000821:virology; D003716:Dengue Virus / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D015151:Immunoblotting / Q000191:economics / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D012680:Sensitivity and Specificity; D044967:Serum / Q000821:virology; D013997:Time Factors; D017361:Viral Nonstructural Proteins / Q000032:analysis*; D014773:Virology / Q000191:economics / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D017361:Viral Nonstructural Proteins