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Volume 50, Number 2, April 2017

Improving dengue viral antigens detection in dengue patient serum specimens using a low pH glycine buffer treatment
 


Wen-Fan Shen, Jedhan Ucat Galula, Gwong-Jen J. Chang, Han-Chung Wu, Chwan-Chuen King, Day-Yu Chao


 

Corresponding author:

Day-Yu Chao, Corresponding author. Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung-Hsing University, Taichung, 401, Taiwan. 



 

Background and purpose: 

Early diagnosis of dengue virus (DENV) infection to monitor the potential progression to hemorrhagic fever can influence the timely management of dengue-associated severe illness. Nonstructural protein 1 (NS1) antigen detection in acute serum specimens has been widely accepted as an early diagnostic assay for dengue infection; however, lower sensitivity of the NS1 antigen-capture enzyme-linked immunosorbent assay (Ag-ELISA) in secondary dengue viral infection has been reported.

 



 

Methods:

In this study, we developed two forms of Ag-ELISA capable of detecting E-Ag containing virion and virus-like particles, and secreted NS1 (sNS1) antigens, respectively. The temporal kinetics of viral RNA, sNS1, and E-Ag were evaluated based on the in vitro infection experiment. Meanwhile, a panel of 62 DENV-2 infected patients' sera was tested. 



 

Results:

The sensitivity was 3.042 ng/mL and 3.840 ng/mL for sNS1 and E, respectively. The temporal kinetics of the appearance of viral RNA, E, NS1, and infectious virus in virus-infected tissue culture media suggested that viral RNAs and NS1 antigens could be detected earlier than E-Ag and infectious virus. Furthermore, a panel of 62 sera from patients infected by DENV Serotype 2 was tested. Treating clinical specimens with the dissociation buffer increased the detectable level of E from 13% to 92% and NS1 antigens from 40% to 85%. 



 

Conclusion:

Inclusion of a low-pH glycine buffer treatment step in the commercially available Ag-ELISA is crucial for clinical diagnosis and E-containing viral particles could be a valuable target for acute DENV diagnosis, similar to NS1 detection. 



 

Key words:

Antigen-capture ELISA, Dengue, NS1 antigen, Viral particle