Characterization of monoclonal antibodies against Muscovy duck reovirus σB protein

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.

Characterization of Monoclonal Antibodies against HA Protein of H1N1 Swine Influenza Virus and Protective Efficacy against H1 Viruses in Mice

“…The mice spleens were collected aseptically by using euthanized anesthesia techniques. mAbs were produced using techniques similar to that described previously [ 33 , 34 , 35 ]. Splenocytes were fused with SP2/0-Ag14 myeloma cells.…”

Characterization of monoclonal antibodies against waterfowl parvoviruses VP3 protein

“…Six weeks after the initial immunization and 4 days before the mice were sacrificed for the preparation of hybridomas, a final boost was given via the same route with 30 μg of the same antigen. MAbs were produced by using techniques similar to those described previously [28]. Briefly, spleens were removed from the immunized mice, and their splenocytes were fused with NS1 myeloma cells.…”

Characterization of monoclonal antibodies against duck Tembusu virus E protein: an antigen-capture ELISA for the detection of Tembusu virus infection

“…Reactivity was visualized with the substrate 3,3 0 -diaminobenzidine (DAB; Sigma, St. Louis, MO, USA). His-E and His proteins (as a negative control) were used for dot blotting assays as described previously [26,28]. Approximately 1 lg of antigen was diluted with TNE buffer (Tiangen Biotech, Shanghai, China) and spotted onto nitrocellulose membranes (Pall Corporation, FL, USA).…”