A simple, inexpensive, robust and sensitive dot-blot assay for equal detection of the nonstructural-1 glycoprotein of all dengue virus serotypes

Virology Journal

Claudia ME Romero-Vivas¹  Andrew KI Falconar¹ 

[1] Laboratorio de Enfermedades Tropicales, Departmento de Medicina, Universidad del Norte, Km 5 Antigua via Puerto Colombia, Barranquilla, Colombia

关键词: Low-cost;    Sensitivity;    Dot-blot;    Monoclonal antibody;    Diagnostic;    Nonstructural-1 glycoprotein;    Dengue virus;   

Others  :  1150624
DOI  :  10.1186/1743-422X-10-126

 received in 2012-11-09, accepted in 2013-04-17,  发布年份 2013

【 摘 要 】

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% 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.

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.

【 授权许可】

   
2013 Falconar and Romero-Vivas; licensee BioMed Central Ltd.

【 图 表 】

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