Development and application of a monoclonal antibody-based blocking ELISA for detection of antibodies to Tembusu virus in multiple poultry species

Lijiao Zhang,Jingliang Su,Xueying Hu,Huan Jin,Zhanhong Li

doi:10.1186/s12917-018-1537-6

Abstract

BackgroundTembusu virus (TMUV) is a member of the genus Flavivirus. Outbreak of this virus infection in duck flocks was first observed in China in April 2010, causing severe egg drop and neurological signs in laying ducks. Recently reported duck infections in southeastern Asia highlighted the need for well-validated diagnostic methods of TMUV surveillance to understand its epidemiological characteristics and maintenance in nature. Several enzyme-linked immunosorbent assays (ELISAs) for the detection of TMUV infection have been reported, but none have been applied to high-throughput diagnostics.ResultsIn this study, a monoclonal antibody (MAb) against TMUV was generated and characterized. MAb 9E4 was shown to bind specifically to a disulfide bond-dependent epitope on the domain I/II of TMUV E protein, and a blocking ELISA was established based on this MAb. The cut-off percentage inhibition value for negative sera was set at 30%. By comparison with the virus neutralization test, the specificity and sensitivity of the blocking ELISA were 96.37% and 100%, respectively, and the kappa value was 0.966, based on 416 serum samples collected from both experimentally and clinically infected ducks, geese and chickens. A good correlation (r2 = 07998, P < 0.001) was observed between the blocking ELISA and plaque reduction neutralization test (PRNT) titers. Using archived duck serum samples collected between 2009 and 2015, the seroprevalence in duck flocks raised in Northern China was estimated by blocking ELISA.ConclusionsOur MAb-based blocking ELISA provides a reliable and rapid diagnostic tool for serological monitoring of TMUV infection and evaluation of immune status following TMUV vaccination in multiple poultry species.

Highlights

Tembusu virus (TMUV) is a member of the genus Flavivirus
We have recently described the uses of a Flavivirus group-specific monoclonal antibody-based blocking Enzyme-linked immunosorbent assays (ELISA) for detection of antibody responses in ducks immunized with an inactivated TMUV vaccine under laboratory conditions [14]
The monoclonal antibody (MAb) 9E4 was selected for development of the blocking ELISA (bELISA) as it displayed high affinity to the coating virus antigen in the preliminary indirect ELISA test

Summary

Introduction

Tembusu virus (TMUV) is a member of the genus Flavivirus. Outbreak of this virus infection in duck flocks was first observed in China in April 2010, causing severe egg drop and neurological signs in laying ducks. The virus was originally isolated from mosquitoes in Malaysia in 1953 and the first animal infection case was reported in a 4-week-old broiler chicken flock in Perak State, characterized by encephalitis, growth retardation and increased blood glucose levels [1]. 2010, a severe outbreak of duck TMUV infection causing egg drop and signs of central nerve system involvement was reported in China [2]. The recent emergence of TMUV infections in duck flocks in Malaysia and Thailand provided warnings of the increasing impact on animal health [6, 7]. In addition to domestic birds, TMUV has been occasionally isolated from mosquitos, pigeons and house sparrows in the vicinity of duck farms in China [8,9,10], suggesting that wild birds may serve as a natural reservoir, carrying and

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