Rapid and Simultaneous Detection of Dengue and Chikungunya Viruses by a Multiplex Lateral Flow Assay Using Ficolin-1, One of Human Innate Immune Defense Proteins

Abstract

license/by-nc/3.0/). The infection of dengue virus (DENV) and chikungunya virus (CHIKV) can cause global public health problems, many of which are undifferentiated. Thus, their differential diagnosis is critical to proper patient management. In this study, we present a novel multiplex diagnostic system based on the lateral flow assay (LFA) using ficolin-1 and two-color latex beads labeled with antibodies to detect the dengue virus (DENV) and chikungunya virus (CHIKV) in a single strip. We investigated the binding of ficolin-1, a human innate immune system defense protein, to the viral envelope protein (EP) and developed the LFA system that contains the capturing agent, ficolin-1, which was immobilized on the test line. Our diagnostic system could differentially detect DENV-2 and CHIKV based on the color of the detecting agent in the test line of the strip. The limit of detection (LOD) by naked-eye observation in the multiplex LFA for viral EPs was 25 nM and that for the whole virus was 1 ´ 10 6 TCID 50 /mL per strip. Thus, multiplex LFA using ficolin-1 provides a rapid method for simultaneously detecting DENV and CHIKV and can be employed to monitor the status of circulating mosquitoes in a region at risk for DENV and/or CHIKV (FBG) domain and activates the complement lectin pathway (12, 13). Recently, several reports have demonstrated that ficolin-1 binds to the viral EP and bacterial membrane and activates the complement system to initiate an antimicrobial response (11, 14, 15). In this study, we developed a novel multiplex diagnostic system based on the lateral flow assay (LFA) using ficolin-1 and two-color latex beads labeled with antibodies to detect DENV and CHIKV on a single strip. Ficolin-1 was used as the capturing agent at the test line; red latex beads conjugated with anti-DENV monoclonal antibody (mAb) and blue latex beads conjugated with anti-CHIKV mAb were used as the detecting agents in the strip test. Accumulation of the virus– colored detecting agent complex at the test line was based on the virus capture by ficolin-1. The differential diagnosis of DENV-2 and CHIKV can be achieved by observing the color of the test line. The limit of detection (LOD) by naked-eye observation in our multiplex LFA for the viral EP was 25 nM and that for the whole virus was 1 ´ 10 6 TCID 50 /mL per strip. To the best of our knowledge, this is the first study to simultaneously detect and differentiate DENV and CHIKV in a single test line with ficolin-1 on a single strip. This LFA will be a powerful tool as a multiplex platform for the rapid differential diagnosis of DENV and CHIKV and can have clinical and DENV-4 EPΔTM. The EPs with TM-domain were used enhance the secretion of EPs. The anti-CHIKV monoclonal antibody laboratory and the (USA). The anti-DENV mAb reactive with DENV serotype-1, -2, -3, and -4. The nitrocellulose