Polypeptide analysis with monoclonal antibodies of A type inclusion bodies induced by cowpox virus

Kitamoto, Noritoshi; Tanimoto, S.; Hiroi, Kenji; Tanaka, Tomoyuki; Miyamoto, Hiroyuki; Wakamiya, Nobutaka; Ikuta, Kazuyoshi; Ueda, Shigeharu; Kato, Shiro

doi:10.1007/bf01309876

Cited by 12 publications

(11 citation statements)

References 21 publications

(14 reference statements)

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“…First of all, we found that the 63-kDa protein appears to be essential for virus replication whereas the cowpox virus and VACV proteins are nonessential proteins. Moreover, the 63-kDa protein is a component of IMV, CEV, and EEV particles whereas the cowpox virus ATI protein was not found in purified virions (20,29). The truncated IHD-J VACV ATI protein has been shown to be present on the surface of IMV particles only and to be a specific marker of a fraction of IMVs that are destined to be included in the ATIs (48).…”

Section: Mab Isolationmentioning

confidence: 99%

Identification and Characterization of Three Immunodominant Structural Proteins of Fowlpox Virus

Boulanger

Green

Jones

et al. 2002

J Virol

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Genes encoding fowlpox virus (FWPVAvipoxviruses, which replicate only in avian cells, have been developed as safe recombinant vectors for human vaccination (23,30). In mammalian cells, avipoxvirus replication is blocked but early gene expression occurs (45,46). Late gene expression occurs in some mammalian cell types infected by fowlpox virus (FWPV), the prototypic avipoxvirus, without productive virus replication, as morphogenesis appears to be blocked (42). FWPV has been much less intensively studied than vaccinia virus (VACV). It appears to form extracellular enveloped virus (EEV) by budding rather then wrapping (6,16 (27). The complete sequence of a pathogenic strain of FWPV (1) includes genes encoding homologs of at least 21 known VACV structural proteins, but FWPV appears to lack homologs of intracellular mature virus (IMV) proteins encoded by A27L and D8L as well as an intracellular enveloped virus protein encoded by A36R and the EEV proteins encoded by B5R, A33R, and A56R (1).Although genes encoding FWPV structural proteins have been identified by sequence homology, very little is known about the proteins they encode. We produced monoclonal antibodies (MAbs) and polyclonal antibodies against FWPV proteins as markers to study FWPV morphogenesis and identified genes encoding three FWPV immunodominant structural proteins. MATERIALS AND METHODSVirus and cells. The origins, propagation, and purification of FWPV strains (FP9, HP1, and Poxine) and canarypox virus (CNPV) have been described previously (5).MAbs. BALB/c mice were immunized twice intraperitonally 3 weeks apart with a crude preparation of Poxine in Quil A, a saponin-derived adjuvant (Superfos, Vedbaek, Denmark). For the second fusion, mice were injected twice 4 weeks apart with 100 g of sucrose gradient-purified FP9 virus (5). Hybridoma supernatants were screened by enzyme-linked immunosorbent assay or by indirect immunofluorescence assay, with Poxine-infected or noninfected chicken embryo fibroblasts (CEFs) as the antigen. The 3D9 MAb directed against the 39-kDa protein was kindly provided by C. P. Czerny.Polyclonal sera. Chicken hyperimmune sera against FP9, Poxine, or CNPV were produced as described previously (5).Radioimmunoprecipitation. Infected (multiplicity of infection, 10) or uninfected CEFs were incubated in methionine-free medium containing 2% dialyzed fetal calf serum. Two hours later, the medium was replaced with fresh medium containing 1.85 MBq of [ 35 S]methionine/ml and incubated for 2 h. The medium was removed, and the cells were washed once with phosphate-buffered saline (PBS) and lysed in 2 ml of radioimmunoprecipitation assay (RIPA) buffer (2.5 mM Tris [pH 7.2], 0.15 M NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 1% sodium deoxycholate). Lysates were centrifuged for 15 min (Eppendorf centrifuge), and supernatants were stored at Ϫ70°C.Ascitic fluid (10 l) or hybridoma supernatant (500 l) was incubated for 1 h at 4°C with 50 l of 10% protein A-Sepharose CL-4B (Pharmacia) in 0.1 M sodium phosphate buffer, pH 8.1. After...

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Section: Mab Isolationmentioning

confidence: 99%

Identification and Characterization of Three Immunodominant Structural Proteins of Fowlpox Virus

Boulanger

Green

Jones

et al. 2002

J Virol

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“…Several major antigens have been shown to be induced in cells infected with CPV and VV, i.e., the nucleoprotein (NP) antigen (24,29), neutralization (NT)-associated antigen (21) and A-type inclusions (ATI) (7)(8)(9). We have identified the polypeptides of the major antigens by immunoprecipitation (IP) with polyclonal antibodies (5,6) and identified those antigens by IP and immunoblotting (western blotting, WB) with a monoclonal antibody (MAb) (11)(12)(13). The NP is a so-called common antigen that shows cross-reactivity with cells infected with either an Orthopoxvirus such as CPV, EV and VV, or a Leporipoxvirus such as the Shope fibroma virus (SFV).…”

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confidence: 99%

Preparation of Monoclonal Antibodies Cross‐Reactive with Orthopoxviruses and Their Application for Direct Immunofluorescence Test

Kitamoto

Kobayashi

Kato

et al. 2005

Microbiology and Immunology

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Variola virus (smallpox virus), vaccinia virus (VV), cowpox virus (CPV) and ectromelia virus (EV) belong to the genus Orthopoxvirus of the family Poxviridae. To establish the possible diagnosis for smallpox infection, monoclonal antibodies (MAbs) against VV and CPV were produced. The cross‐reactivity of seven MAbs with cells infected with various strains of the orthopoxviruses (CPV, VV and EV) was confirmed by an immunofluorescence (IF) test and other immunological analyses. Four and three MAbs reacted with the common antigen of all poxviruses (probably NP antigen) and the antigen involved in neutralization, respectively. We developed the IF test using these MAbs. The direct IF test required only 45 min to perform. Smallpox infection is now eradicated, but it is important to prepare for the diagnosis of smallpox in an emergency. The direct IF assay using MAbs cross‐reactive with orthopoxviruses is rapid, simple, specific, applicable for multiple samples, and will make it possible to screen for and detect orthopoxviruses that include variola virus with tissue impression smears from skin lesions in most laboratories or institutes.

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“…The dense solid ATI and the 80K polypeptide reacting with A212 are common to all the CPV and EV strains, while the surface-oriented ATI (shown in Fig. 3g; At antibody described previously by Kitamoto et al, 1986a) and the 160K polypeptide reacting with other antibodies (Kitamoto et al, 1986b) vary from strain to strain. Therefore, the former may represent a common ATI in Orthopoxvirus, whereas the latter may be virus-or strain-specific.…”

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confidence: 99%

“…The conditions for SDS-PAGE and fluorography were as described previously (Ikuta et al, 1978). The techniques for immunoblotting (Western blotting) with monoclonal antibodies by the method of Towbin et al (1979) with slight modifications have been described in detail elsewhere (Kitamoto et al, 1986a). Mock-infected and infected (24 h after infection, m.o.i.…”

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confidence: 99%

“…Hybridomas were obtained from cultures of myeloma cells fused with spleen cells from mice immunized with cells infected with the LB red strain of CPV. Ascites fluids obtained as described previously (Kitamoto et al, 1986a) were used as monoclonal antibodies. The subclasses of these antibodies were determined by immunodiffusion with a hybridoma cell lysate and class-specific immunoglobulin antisera as described by Volk et al (1982).…”

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confidence: 99%

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Monoclonal Antibodies to Cowpox Virus: Polypeptide Analysis of Several Major Antigens

Kitamoto

Tanimoto

Hiroi

et al. 1987

Journal of General Virology

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SUMMARYMonoclonal antibodies directed against major antigens induced by cowpox virus (CPV) were produced. The specificities of these antibodies were established by immunoprecipitation, immunoblotting and several serological analyses, and from the cross-reactivities of these antibodies with cells infected with various other poxviruses, ectromelia virus (EV), vaccinia virus and Shope fibroma virus. The antibodies defined included ones reacting with each of the known major antigens of poxviruses, i.e. the common antigen of all poxviruses (probably NP antigen), the Orthopoxvirus-specific antigen (probably LS antigen), the haemagglutinin, the cell surface antigen, the common A-type inclusions in CPV and EV, and the antigen involved in neutralization.

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Polypeptide analysis with monoclonal antibodies of A type inclusion bodies induced by cowpox virus

Cited by 12 publications

References 21 publications

Identification and Characterization of Three Immunodominant Structural Proteins of Fowlpox Virus

Identification and Characterization of Three Immunodominant Structural Proteins of Fowlpox Virus

Preparation of Monoclonal Antibodies Cross‐Reactive with Orthopoxviruses and Their Application for Direct Immunofluorescence Test

Monoclonal Antibodies to Cowpox Virus: Polypeptide Analysis of Several Major Antigens

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