Generation and characterization of a monoclonal antibody against duck Tembusu virus envelope protein

Abstract

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. Envelope (E) protein of DTMUV is an important structural protein, which is able to induce protective immune response in target animals and can be used as specific serological diagnosis tool. In this study, a novel monoclonal antibody, designated mAb 3E9, was generated against DTMUV E protein. It is positive in indirect ELISA against both His-E protein and the purified whole viral antigen. Also, this mAb showed positive reaction with DTMUV in Western blot and indirect immunofluorescence assay, and the isotype was IgG1. End-point neutralizing assay performed in BHK-21 cells revealed that the neutralization titer of 3E9 against DTMUV JS804 strain reached 1:50. Furthermore, functional studies revealed that 3E9 blocks infection of DTMUV at a step on viral attachment. The anti-E mAbs produced in the present work may be valuable in developing an antigen-capture ELISA test for antigen detection or a competitive ELISA test for antibody detection or therapeutic medicine for DTMUV in poultry.

A Novel Diagnostic Method to Detect Duck Tembusu Virus: A Colloidal Gold-Based Immunochromatographic Assay

“…In this study, the developed ICS was based on antibodies of E protein of this virus. E protein, as a major envelop protein of flavivirus, has multi-antigenic determinants and plays an important role in mediating the host-related immune response and producing neutralizing antibodies ( Allison et al, 2001 ; Chen et al, 2005 , 2014 ; Mukhopadhyay et al, 2005 ; Pierson et al, 2008 ; Chavez et al, 2010 ; Han et al, 2016 ). Therefore, we utilized the purified monoclonal antibody A12D3 against the envelop E protein as a gold conjugate antibody, while the preparation of purified polyclonal antibody C12D1 from BALB/c mice against the envelope E protein served as the capture antibody.…”

Identification of determinants that mediate binding between Tembusu virus and the cellular receptor heat shock protein A9

“…Plaque formation was continuously observed daily for four days and TCID 50 was calculated according to the method of Reed-Muench. TMUV-monoclonal antibody was generated and maintained by our laboratory as previously described [ 8 ]. Alkaline phosphatase and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG were purchased from the Beyotime Institute of Biotechnology (China).…”

“…The HSPA9-binding peptides were detected by indirect ELISA as previously described [ 8 ]. The 96-well ELISA plate was coated with purified recombinant HSPA9 (4 µg per well) at 4℃ overnight and blocked with 1% BSA in PBST at 37℃ for 2 h. The plate was incubated with different overlapping peptides in triplicate for 1 h at 37℃.…”

Identification of a Neutralizing Monoclonal Antibody That Recognizes a Unique Epitope on Domain III of the Envelope Protein of Tembusu Virus

“…Firstly, the TMUV E protein was shown to induce protective immune response by different approaches, including immunization with E protein delivered by recombinant duck enteritis virus [5,9], and expressed in a prokaryotic system [7,10]. Furthermore, several mouse monoclonal antibodies (MAbs) (1F3, 1A5, 3B6, 3E9, 3H11, and 1G2) against E protein have been described [23][24][25][26][27]. Importantly, MAbs 3E9 and 1G2 exhibit neutralizing activity [24,27].…”

“…Furthermore, several mouse monoclonal antibodies (MAbs) (1F3, 1A5, 3B6, 3E9, 3H11, and 1G2) against E protein have been described [23][24][25][26][27]. Importantly, MAbs 3E9 and 1G2 exhibit neutralizing activity [24,27]. Finally, three epitopes, recognized by non-neutralizing MAbs 1F3, 1A5, and 3B6, have been identified in EDII (positions 87-91 and 221-225) and EDIII (positions 374-380) [25,26].…”