Fluorescent and electron-microscopy immunoassays employing polyclonal and monoclonal antibodies for detection of goose parvovirus infection

Alexandrov, M.; Alexandrova, Radostina; Alexandrov, I.; Zacharieva, S.; Lasarova, S.; Doumanova, L.; Peshev, R.; Donev, Todor

doi:10.1016/s0166-0934(98)00175-x

Cited by 11 publications

(8 citation statements)

References 12 publications

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“…All uninfected cells showed no reaction to any MAbs (represented by Figure 4C). Immunofluorescence assay also indicates that the MAbs bound to the authentic viral VP3 protein, which located predominantly within the nuclei without affecting the nucleoli and rarely within the cytoplasm of infected cells, which is consistent with previous report [22,23]. In some cells GPV/MDPV appeared as granules scattered throughout the nucleus (indicated by red arrow), while in other cells GPV/MDPV were distributed homogeneously in nuclei (indicated by purple arrow or in Figure 4B).…”

Section: Resultssupporting

confidence: 91%

“…After screening and sub-cloning, three MAbs against His-VP3 were isolated and characterized. Western blotting and immunofluorescence assay indicated that the MAbs bound to the authentic viral VP3 protein of GPV and MDPV and that this protein localized mainly in the nucleus of infected cells, which is consistent with a previous report [22,23]. The MAbs bound to the His-VP3 in its native conformation, and when SDS and 2-mercaptoethanol were used to denature the His-VP3 protein, this binding was retained, indicating that the epitopes were not affected by breaking of disulfide bonds.…”

Section: Discussionsupporting

confidence: 92%

“…A similar finding was obtained when MAbs specificities were analyzed by Western blotting. Three major proteins (VP1, VP2 and VP3) MW are in consistent with previously reports of Muscovy duck (91, 78 and 58 kDa) and goose (85, 61 and 57 kDa) parvovirus virions [22-24]. …”

Section: Discussionsupporting

confidence: 91%

“…Dot blotting analysis showed that the epitopes on the VP3 recognized by three MAbs were also present on the native viral VP3 of GPV and DPV particles, which is consistent with immunofluorescence assay analysis. By the immunofluorescence assay, it could also identify the GPV/MGPV antigen with MAbs because of its distinctive nuclear localization within infected cells, which consistent with previous report [22]. A similar finding was obtained when MAbs specificities were analyzed by Western blotting.…”

Section: Discussionsupporting

confidence: 90%

“…All MAbs recognized the nature structure of His-VP3 in TNE buffer (Figure 2), but did not react with the 6.7 His proteins. Three major proteins (VP 1, VP 2 and VP 3) were identified by SDS-PAGE in a purified Muscovy duck (91, 78 and 58 kDa) and goose (85, 61 and 57 kDa) parvovirus virions [22-24]. Western blotting analysis showed that MAbs reacted with molecular weight of 90, 77, and 64 kDa of VP1, VP2, and VP3 from denatured GPV EP22 (represented by Figure 3, lane 2) and MDPV J3D6 (represented by Figure 3, lane 4) antigen, which were similar to VP1, VP2, and VP3 of goose parvovirus and Muscovy duck parvovirus.…”

Section: Resultsmentioning

confidence: 99%

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Characterization of monoclonal antibodies against waterfowl parvoviruses VP3 protein

Yin

Zhang

Gao

et al. 2012

Virol J

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BackgroundThe VP3 protein of goose parvovirus (GPV) or Muscovy duck parvovirus (MDPV), a major structural protein, can induce neutralizing antibodies in geese and ducks, but monoclonal antibodies (MAbs) against VP3 protein has never been characterized.ResultsThree hybridoma cell lines secreting anti-GPV VP3 MAbs were obtained and designated 4A8, 4E2, and 2D5. Immunoglobulin subclass tests differentiated them as IgG2b (4A8 and 4E2) and IgG2a (2D5). Dot blotting assays showed that three MAbs reacted with His-VP3 protein in a conformation-independent manner. A competitive binding assay indicated that the MAbs delineated two epitopes, A and B of VP3. Immunofluorescence assay showed that MAbs 4A8, 4E2, and 2D5 could specifically bind to goose embryo fibroblast cells (GEF) or duck fibroblast cells (DEF) infected with GPV and MDPV. Dot blotting also showed that the MAbs recognized both nature GPV and MDPV antigen. Western blotting confirmed that the MAbs recognized VP3 proteins derived from purified GPV and MDPV particles. The MAbs 4A8 and 2D5 had universal reactivity to heterologous GPV and MDPV tested in an antigen-capture enzyme-linked immunosorbent assay.ConclusionsPreparation and characterization of these the MAbs suggests that they may be useful for the development of a MAb-capture ELISA for rapid detection of both GPV and MDPV. Virus isolation and PCR are reliable for detecting GPV and MDPV infection, but these procedures are laborious, time-consuming, and requiring instruments. These diagnosis problems highlight the ongoing demand for rapid, reproducible, and automatic methods for the sensitive detection of both GPV and MDPV infection.

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Section: Resultssupporting

confidence: 91%

Section: Discussionsupporting

confidence: 92%