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1.
Microbiol Immunol ; 57(1): 77-81, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23126568

RESUMO

The hemagglutinin genes (HA1 subunit) from human and animal 2009 pandemic H1N1 virus isolates were expressed with a baculovirus vector. Recombinant HA1 (rHA1) protein-based ELISA was evaluated for detection of specific influenza A(H1N1)pdm09 antibodies in serum samples from vaccinated humans. It was found that rHA1 ELISA consistently differentiated between antibodies recognizing the seasonal influenza H1N1 and pdm09 viruses, with a concordance of 94% as compared to the hemagglutination inhibition test. This study suggests the utility of rHA1 ELISA in serosurveillance.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais , Técnicas de Laboratório Clínico/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/diagnóstico , Infecções por Orthomyxoviridae/diagnóstico , Animais , Antígenos Virais/genética , Baculoviridae/genética , Ensaio de Imunoadsorção Enzimática/métodos , Expressão Gênica , Vetores Genéticos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Influenza Humana/virologia , Infecções por Orthomyxoviridae/virologia , Proteínas Recombinantes/genética , Sensibilidade e Especificidade , Soro/imunologia
2.
Hybridoma (Larchmt) ; 31(5): 340-6, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23098300

RESUMO

Monoclonal antibodies (MAbs) against the E2 protein of classical swine fever virus (CSFV) are useful for diagnosis and strain characterization. A purified, baculovirus-expressed CSFV E2 protein from the Paderborn strain was formulated with a saponin adjuvant and successfully used to induce an antigen-specific immune response in mice. After cell fusion a panel, designated F92G, of 12 mouse hybridomas (5-2, 11-1, 14-1, 25-2, 28-2, 31-1, 34-1, 35-2, 37-3, 38-2, 39-1, 41-1) producing CSFV-E2 specific MAbs were selected based on their Ig subclass and secretion level (µg IgG/mL). Nine IgG 1/k, two IgG 2b/k, and one IgG 2a/k MAbs were further characterized using immunoperoxidase reactivity, ELISA, and Western blot analysis. Immunoglobulin concentration-dependent immunoperoxidase and ELISA reactivity was observed for some of the MAbs with certain antigens. In general there were several reactivity patterns exhibited by the MAbs, with CSFV strains representing different genetic subgroups (by immunoperoxidase staining) and recombinant antigens (by ELISA). It was interesting to note that in some cases the strain-specific reactivity of a MAb was dependent on the test, thereby providing a clue regarding the nature of the binding site.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Antígenos Virais/imunologia , Vírus da Febre Suína Clássica/imunologia , Imunoglobulina G/imunologia , Proteínas do Envelope Viral/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Antígenos Virais/administração & dosagem , Antígenos Virais/genética , Baculoviridae/genética , Sítios de Ligação de Anticorpos , Western Blotting , Vírus da Febre Suína Clássica/química , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Hibridomas/imunologia , Imunização , Técnicas Imunoenzimáticas , Imunoglobulina G/biossíntese , Camundongos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Especificidade da Espécie , Suínos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética
3.
Virus Res ; 153(2): 244-9, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20713098

RESUMO

Recently, we reported the expression and glycosylation of avian metapneumovirus attachment glycoprotein (AMPV/C G protein) in eukaryotic cell lines by a transient-expression method. In the present study, we investigated the biosynthesis and O-linked glycosylation of the AMPV/C G protein in a baculovirus expression system. The results showed that the insect cell-produced G protein migrated more rapidly in SDS-PAGE as compared to LLC-MK2 cell-derived G proteins owing to glycosylation differences. The fully processed, mature form of G protein migrated between 78 and 86 kDa, which is smaller than the 110 kDa mature form of G expressed in LLC-MK2 cells. In addition, several immature G gene products migrating at 40-48 and 60-70 kDa were also detected by SDS-PAGE and represented glycosylated intermediates. The addition of the antibiotic tunicamycin, which blocks early steps of glycosylation, to insect cell culture resulted in the disappearance of two glycosylated forms of the G protein and identified a 38 kDa unglycosylated precursor. The maturation of the G protein was completely blocked by monensin, suggesting that the O-linked glycosylation of G initiated in the trans-Golgi compartment. The presence of O-linked sugars on the mature protein was further confirmed by lectin Arachis hypogaea binding assay. Furthermore, antigenic features of the G protein expressed in insect cells were evaluated by ELISA.


Assuntos
Expressão Gênica , Metapneumovirus/genética , Processamento de Proteína Pós-Traducional , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos , Glicosilação , Lectinas/metabolismo , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Spodoptera , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia
4.
Virus Res ; 147(2): 189-94, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19896993

RESUMO

Biosynthesis, glycosylation and cell surface expression of the AMPV/C G protein were examined in eukaryotic cell lines (LLC-MK2, CHO-K1, CHO-1d1D). Immature G gene products with a molecular mass of 42, 45 and 58-90 kilodaltons (kDa) were identified by SDS-PAGE and represented glycosylated intermediates. Tunicamycin treatment of transfected cells confirmed the presence of N-linked carbohydrate moieties on these intermediate species and identified a 38 kDa unglycosylated precursor. A fully processed, mature form of the protein migrated around 110 kDa. The presence of O-linked sugars on the mature G protein was confirmed by using the O-glycosylation deficient CHO-ldlD cell line supplemented with exogenous Gal and GalNAc. Binding of the lectin Arachis hypogaea (peanut agglutinin) confirmed the presence of O-linked sugars on the mature protein and its intracellular transport to the cell surface was demonstrated by indirect immunofluorescent staining.


Assuntos
Metapneumovirus/fisiologia , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Animais , Arachis/química , Linhagem Celular , Cricetinae , Cricetulus , Eletroforese em Gel de Poliacrilamida , Glicosilação , Lectinas/metabolismo , Macaca mulatta , Peso Molecular , Ligação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/isolamento & purificação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/isolamento & purificação , Proteínas Virais/química , Proteínas Virais/isolamento & purificação
6.
J Vet Diagn Invest ; 19(5): 486-91, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17823391

RESUMO

An ovine testis cell line (OA3.Ts) was evaluated and compared with primary lamb kidney (LK) cells for its utility in capripoxvirus propagation and titration. A comparison of OA3.Ts cell growth kinetics and morphology at low (<33) and high (34-36) passage levels indicated a difference in both characteristics. However, viral titers determined in low and high passage OA3.Ts cells were comparable with those obtained using LK cells. Capripoxvirus infection of OA3.Ts and LK cells resulted in a similar cytopathic effect, which allowed for the detection of discrete viral plaques following immunostaining with capripoxvirus-specific antiserum.


Assuntos
Capripoxvirus/fisiologia , Testículo/citologia , Ensaio de Placa Viral/veterinária , Cultura de Vírus/veterinária , Animais , Linhagem Celular , Rim/citologia , Masculino , Ovinos , Coloração e Rotulagem , Ensaio de Placa Viral/métodos , Cultura de Vírus/métodos
7.
Clin Diagn Lab Immunol ; 12(8): 904-9, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16085906

RESUMO

Constructs of the major core protein, designated VP7, from epizootic hemorrhagic disease virus (EHDV) type 1 were made by amino- or carboxyl-terminal fusion of a six-histidine residue tag to the VP7-1 gene. The resulting fusion proteins were produced in a baculovirus expression system and purified by a rapid, one-step procedure using nickel-nitrilotriacetic acid technology. A high level of VP7-1 protein expression was detected with the N-terminal six-histidine tag fusion construct and was comparable to the level of expression observed with an untagged VP7-1 Bam construct. In contrast, the inclusion of a six-histidine tag at the C terminus adversely affected protein expression. The antigenicity of the N-terminal six-histidine tag EHDV VP7-1 product was identical to that observed with the native virus antigen and untagged EHDV VP7-1 recombinant protein, as determined by reactivity with EHDV specific antibodies in an enzyme-linked immunosorbent assay (ELISA) and Western blot. The high production and purity levels that can be attained for the N-terminal six-histidine tag VP7-1 protein and its reactivity with EHDV-specific sera in a competitive ELISA make it a suitable assay reagent.


Assuntos
Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/isolamento & purificação , Vírus da Doença Hemorrágica Epizoótica/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Infecções por Reoviridae/diagnóstico , Animais , Baculoviridae/genética , Sequência de Bases , Proteínas do Capsídeo/genética , Linhagem Celular , Ensaio de Imunoadsorção Enzimática/normas , Insetos/citologia , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Infecções por Reoviridae/sangue , Testes Sorológicos/normas
8.
Clin Diagn Lab Immunol ; 12(1): 187-91, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15643005

RESUMO

The small hydrophobic (SH) gene of the avian pneumovirus (APV) Colorado isolate (CO), which belongs to subgroup C (APV/C), was expressed with a baculovirus vector. The recombinant SH protein was evaluated as a potential subgroup-specific diagnostic reagent in order to differentiate infections resulting from APV/C from those induced by APV/A, APV/B, and human metapneumovirus (hMPV). When the recombinant baculovirus was used to infect insect cells, a 31- to 38-kDa glycosylated form of the SH protein was produced and subsequently tested for reactivity with antibodies specific for APV/A, APV/B, APV/C, and hMPV. Western blot analysis showed that the expressed recombinant SH protein could only be recognized by APV/C-specific antibodies. This result was consistent with sequence analysis of the APV/C SH protein, which had very low (24%) amino acid identity with the corresponding protein of hMPV and no discernible identity with the SH protein of APV/A or APV/B. A recombinant SH protein-based enzyme-linked immunosorbent assay (ELISA) was developed, and it further confirmed the lack of reactivity of this protein with antisera raised to APV/A, APV/B, and hMPV and supported its designation as a subgroup-specific antigen. This finding indicated that the recombinant SH protein was a suitable antigen for ELISA-based detection of subgroup-specific antibodies in turkeys and could be used for serologically based differential diagnosis of APV and hMPV infections.


Assuntos
Especificidade de Anticorpos , Variação Genética , Metapneumovirus/imunologia , Proteínas Oncogênicas de Retroviridae/biossíntese , Proteínas Oncogênicas de Retroviridae/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Antígenos Virais/biossíntese , Antígenos Virais/genética , Antígenos Virais/imunologia , Western Blotting , Ensaio de Imunoadsorção Enzimática , Humanos , Metapneumovirus/genética , Dados de Sequência Molecular , Infecções por Paramyxoviridae/diagnóstico , Infecções por Paramyxoviridae/imunologia , Infecções por Paramyxoviridae/veterinária , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Oncogênicas de Retroviridae/genética , Homologia de Sequência de Aminoácidos , Testes Sorológicos , Perus/virologia
9.
J Virol Methods ; 120(1): 87-96, 2004 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-15234813

RESUMO

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Testes de Neutralização , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Animais , Antígenos Virais/imunologia , Western Blotting , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática , Epitopos , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Glicoproteínas de Membrana/imunologia , Camundongos , Nucleoproteínas/imunologia , Conformação Proteica , Glicoproteína da Espícula de Coronavírus , Células Vero , Proteínas do Envelope Viral/imunologia
10.
J Biochem ; 136(6): 795-804, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15671490

RESUMO

The structural glycoprotein E(rns) of classical swine fever virus (CSFV) is one of the major antibody targets upon infection of pigs with the virus. Molecular dissection of the structure of E(rns) would define the minimal immunodominant regions that induce antibody responses after infection and may thus help design an effective diagnostic reagent or vaccine. In this study, deletion analysis was made within amino acids (aa) 297 to 776 of the CSFV Alfort/187 polyprotein containing the large C-terminal portion of the E(rns) protein (aa 27 to 227), the entire E1 protein (aa 1 to 195), and the N-terminal portion of the E2 protein (aa 1 to 87). Various protein fragments with target deletions from N- or/and C-terminal ends were constructed with pET30, expressed in Escherichia coli and probed on Western blots with antisera from pigs infected with CSFV. This has resulted in the identification within E(rns) of three overlapping antigenic regions: AR1(E(rns)aa 65-145), AR2 (E(rns)aa 84-160) and AR3 (E(rns)aa 109-220). N- or C-terminal deletions as small as 3 residues introduced into these regions disrupt their reactivity with antibodies, indicating that they are the minimum requirements for recognition by pig antibodies. The three minimal antigenic regions correlated well with the hydropathy profiles and the 3D structural model of E(rns). Each individual region and a protein fragment containing AR1, AR2 and AR3 reacted equally well with pig anti-CSFV sera. Since variable and conserved sequences are present within the three overlapping antigenic regions of E(rns) of different pestiviruses, specific serological detection of CSFV infection or broad detection of pestivirus infections may be achieved with the use of a single E(rns) region or a combination of two or three E(rns) regions.


Assuntos
Antígenos Virais/imunologia , Vírus da Febre Suína Clássica/patogenicidade , Peste Suína Clássica/imunologia , Imunoglobulina G/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Peste Suína Clássica/metabolismo , Mapeamento de Epitopos , Dados de Sequência Molecular , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Suínos , Proteínas do Envelope Viral/metabolismo
11.
J Vet Diagn Invest ; 15(5): 447-53, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-14535544

RESUMO

The ability of a Japanese quail fibrosarcoma cell line (QT-35) to support the replication of avian metapneumoviruses belonging to the 3 subgroups A (14/1 virus), B (Colorado virus), and C (Hungary virus) enabled the development of assays for the detection and evaluation of virus-specific antibodies. On the basis of the results of enzyme-linked immunosorbent assay (ELISA), plaque reduction neutralization assay (PRNA), immunofluorescent assay (IFA), and Western blot analysis, some degree of antigenic cross-reactivity was observed between prototype viruses belonging to each of the 3 subgroups A, B, and C. The antigen produced in QT-35 cells was found to be superior with respect to its reactivity with virus-specific antibodies, as determined when used in ELISA and IFA. Standardization of both the input virus and the virus-specific antibodies in PRNA enabled a more detailed analysis of the antigenic relationship between these viruses. Specifically, it was observed that 14/1 virus shared more neutralizing regions with Hungary and Colorado viruses than did either of these viruses with 14/1 virus. In addition, Hungary virus shared comparatively fewer neutralizing epitopes with the Colorado virus than did 14/1 virus. Western blot analysis of the reactivity patterns of virus antigen, produced in QT-35 cells, with subgroup-specific antibodies identified a cross-reactive protein migrating at approximately 18 kD. These assays and the information from the Western blot will enable further analysis of avian metapneumovirus isolates to determine antigenic relationships.


Assuntos
Antígenos Virais/imunologia , Fibrossarcoma/imunologia , Fibrossarcoma/veterinária , Metapneumovirus/imunologia , Animais , Chlorocebus aethiops , Coturnix , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Metapneumovirus/classificação , Células Tumorais Cultivadas , Células Vero
12.
J Virol Methods ; 107(1): 9-14, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12445932

RESUMO

A plaque assay for detecting, isolating and titrating avian pneumoviruses using a Japanese quail fibrosarcoma cell line (QT-35) is described. Plaques are produced after application of either an agarose or carboxymethyl-cellulose (CMC) overlay onto cell monolayers infected with representative avian metapneumoviruses which belong to subgroup A, B or C. Virus plaques can be easily visualized by light microscopy or after staining. The parameters affecting plaque appearance include: cell seeding concentration, virus strain, overlay composition and incubation time following infection. Optimal conditions for plaquing involve seeding QT-35 cells at 40,000 cells per cm(2) when using a 1.5% CMC overlay or 100,000 cells per cm(2) when using a 1.0 or 0.8% agarose overlay. In both cases, cell monolayers are infected with virus 24 h post-seeding and clearly visible plaques develop in 6 days. Due to the robust nature of these cells, the incubation time can be extended to a maximum of 13 days after infection in order to produce larger plaques.


Assuntos
Metapneumovirus/crescimento & desenvolvimento , Ensaio de Placa Viral/métodos , Animais , Coturnix , Fibrossarcoma/virologia , Metapneumovirus/isolamento & purificação , Células Tumorais Cultivadas
13.
J Virol Methods ; 102(1-2): 73-81, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11879695

RESUMO

A Japanese quail fibrosarcoma cell line (QT-35) was evaluated and compared to Vero cells for its utility in metapneumovirus propagation, titration and serological detection by indirect immunofluorescence staining. Cell characteristics such as growth kinetics at different passage levels and seeding density in 96-well plates using various media formulations were studied in order to determine suitable assay parameters. Specifically, QT-35 cells supported the replication of a subgroup A metapneumovirus, strain 14/1, when maintained in DMEM containing a high level of glucose (4500 mg/l) and 2% gamma-irradiated fetal bovine serum (gamma-FBS). There appeared to be a decreased ability of metapneumovirus produced in chicken embryo fibroblast (CEF) cells to replicate to high titers in QT-35 cells, however, this apparent restriction was overcome after the second passage resulting in high titered stock. Metapneumovirus produced in Vero cells and propagated in QT-35 cells produced high titered stock after the first passage. Viral titers determined in Vero and QT-35 cells were comparable, when the latter cell line was used at passage levels < or = 20 and seeded between 5.0 x 10(4) and 1.0 x 10(5) cells/0.33 cm(2) in hgDMEM containing 10% gamma-FBS, with a reduction to 2% gamma-FBS when the virus was applied to the cell monolayers 24 h post-seeding. After infection with metapneumovirus, QT-35 cells exhibited syncytia, similar to those in metapneumovirus-infected Vero cells, which were readily detected by indirect immunofluorescent (IF) staining.


Assuntos
Metapneumovirus/fisiologia , Animais , Anticorpos Antivirais/análise , Divisão Celular , Chlorocebus aethiops , Coturnix , Fibrossarcoma , Técnica Indireta de Fluorescência para Anticorpo , Metapneumovirus/imunologia , Metapneumovirus/isolamento & purificação , Coloração e Rotulagem/métodos , Titulometria , Células Tumorais Cultivadas , Células Vero , Replicação Viral
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