Abstract
BackgroundThe σB protein of Muscovy duck reovirus (DRV), one of the major structural proteins, is able to induce neutralizing antibody in ducks, but the monoclonal antibody (MAb) against σB protein has never been characterized.ResultsFour hybridoma cell lines secreting anti-DRV σB MAbs were obtained, designated 1E5, 2F7, 4E3 and 5D8. Immunoglobulin subclass tests differentiated them as IgG2b (1E5 and 4E3) and IgM (2F7 and 5D8). Dot blot and western blotting assays showed that MAbs reacted with His-σB protein in a conformation-independent manner. Competitive binding assay indicated that the MAbs delineated two epitopes, A and B of σB. Immunofluorescence assay indicated that the four MAbs could specifically bind to Vero cells infected with DRV and σB was distributed diffusely in the cytoplasma of infected cells. MAbs had universal reactivity to all DRVs tested in an antigen-capture enzyme-linked immunosorbent assay.ConclusionResults of this research provide important information about the four monoclonal antibodies and therefore the MAbs may be useful candidate for the development of a MAb capture ELISA for rapid detection of DRVs. In addition, it showed that the σB protein was located in the cytoplasma of infected cells by immunofluorescence assay with MAbs. Virus isolation and RT-PCR are reliable way for detection of DRV infection, but these procedures are laborious, time consuming, and requiring instruments. These obvious diagnosis problems highlight the ongoing demand of rapid, reproducible, and automatic methods for the sensitive detection of DRV.
Highlights
The σB protein of Muscovy duck reovirus (DRV), one of the major structural proteins, is able to induce neutralizing antibody in ducks, but the monoclonal antibody (MAb) against σB protein has never been characterized
The results showed that the antigen preparations containing the expressed His-σB protein of DRV could induce the production of MAbs
The results reveal that the MAbs capture enzyme-linked immunosorbent assay (ELISA) clearly differentiates the samples between the DRV- and mockinfected as demonstrated by absorbance values, suggesting that non-specific reactions could be markedly reduced in the MAb capture ELISA
Summary
The σB protein of Muscovy duck reovirus (DRV), one of the major structural proteins, is able to induce neutralizing antibody in ducks, but the monoclonal antibody (MAb) against σB protein has never been characterized. The Muscovy duck reovirus (DRV) consists 10 segments of double-stranded RNA (dsRNA) packaged into a nonenveloped icosahedral double-capsid shell [1,2]. All avian reovirus (ARV) encoded proteins including at least 10 structural proteins (λA, λB, λC, μA, μB, μBC, μ1C σC, σA, and σB) and 4 nonstructural proteins (μNS, P10, P17, and σNS). The σB protein of DRV encoded by S3 gene segment is structurally related to the σ3 protein of mammalian or σB of ARV [9,10,11,12] and may be functional related. The σB protein is a major constituent of the outer capsid and, like σC, is exposed to the surface of the virion [2]. σB protein induce group-specific neutralizing antibody, while protein σC induces type-specific neutralizing antibodies [4]
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