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Registro Completo |
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Biblioteca(s): |
Embrapa Unidades Centrais. |
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Data corrente: |
15/02/2017 |
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Data da última atualização: |
29/12/2017 |
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Autoria: |
CRUZ, T. F.; KANASHIRO, T. M.; CASTRO, A. M. M. G. de; BALDIN, C. M.; RICHTZENHAIN, L. J.; ARAUJO JUNIOR, J. P. |
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Afiliação: |
TAÍS F. CRUZ, IBTEC/UNESP; TATIANA M. KANASHIRO, DMI/IB/UNESP; ALESSANDRA M. M G. DE CASTRO, FMVZ/USP; CINTIA M. BALDIN, FMVZ/USP; LEONARDO J. RICHTZENHAIN, MVZ/USP; JOÃO PAULO ARAUJO JR, UNESP/IBTEC. |
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Título: |
A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection. |
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Ano de publicação: |
2016 |
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Fonte/Imprenta: |
Pesquisa Veterinária Brasileira, Rio de Janeiro, v. 36, n. 12, p. 1171-1177, dez. 2016. |
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Idioma: |
Inglês |
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Conteúdo: |
Few studies have described enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against porcine circovirus type 2 (PCV2) based on antigens produced in cell culture. Furthermore, few articles have described viral purification techniques for members of the family Circoviridae. This occurs because circoviruses are difficult to isolate, noncytopathogenic, and produce low viral titres in cell culture. Thus, for overcoming these difficulties in the cultivation of PCV2, this study aimed to develop a double-antibody sandwich ELISA based on the cell culture antigen PCV2b for the quantification of anti-PCV2 antibodies. A 20% and 50% discontinuous sucrose cushion was used for viral purification, which enabled the separation of cell culture proteins in the 20% sucrose cushion and a greater viral concentration in the 50% sucrose cushion. Following isopycnic centrifugation, PCV2 was concentrated in the band with density values from 1.330 to 1.395g/cm3. Viral purification was assessed using SDS-PAGE, indirect ELISA and electron microscopy. The standardised ELISA revealed a strong linear correlation (r= 0.826, p<0.001) when compared with a commercial ELISA kit. The assay exhibited low variability (inter-assay coefficient of variation of 4.24% and intra-assay of 1.80%) and excellent analytical specificity conferred by the capture antibody produced in rabbit. Thus, this ELISA is a rapid, specific and convenient method for the detection of antibodies against PCV2 in studies of experimental and natural infection, and in monitoring the response to vaccination on commercial farms. MenosFew studies have described enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against porcine circovirus type 2 (PCV2) based on antigens produced in cell culture. Furthermore, few articles have described viral purification techniques for members of the family Circoviridae. This occurs because circoviruses are difficult to isolate, noncytopathogenic, and produce low viral titres in cell culture. Thus, for overcoming these difficulties in the cultivation of PCV2, this study aimed to develop a double-antibody sandwich ELISA based on the cell culture antigen PCV2b for the quantification of anti-PCV2 antibodies. A 20% and 50% discontinuous sucrose cushion was used for viral purification, which enabled the separation of cell culture proteins in the 20% sucrose cushion and a greater viral concentration in the 50% sucrose cushion. Following isopycnic centrifugation, PCV2 was concentrated in the band with density values from 1.330 to 1.395g/cm3. Viral purification was assessed using SDS-PAGE, indirect ELISA and electron microscopy. The standardised ELISA revealed a strong linear correlation (r= 0.826, p<0.001) when compared with a commercial ELISA kit. The assay exhibited low variability (inter-assay coefficient of variation of 4.24% and intra-assay of 1.80%) and excellent analytical specificity conferred by the capture antibody produced in rabbit. Thus, this ELISA is a rapid, specific and convenient method for the detection of antibodies against PCV2 in stud... Mostrar Tudo |
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Palavras-Chave: |
Isopycnic centrifugation; Sucrose cushion. |
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Thesaurus Nal: |
Antibody detection; enzyme-linked immunosorbent assay; Porcine circovirus-2. |
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Categoria do assunto: |
-- |
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URL: |
https://ainfo.cnptia.embrapa.br/digital/bitstream/item/155768/1/A-double-antibody-sandwich-ELISA.pdf
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Marc: |
LEADER 02442naa a2200241 a 4500
001 2064305
005 2017-12-29
008 2016 bl uuuu u00u1 u #d
100 1 $aCRUZ, T. F.
245 $aA double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection.$h[electronic resource]
260 $c2016
520 $aFew studies have described enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against porcine circovirus type 2 (PCV2) based on antigens produced in cell culture. Furthermore, few articles have described viral purification techniques for members of the family Circoviridae. This occurs because circoviruses are difficult to isolate, noncytopathogenic, and produce low viral titres in cell culture. Thus, for overcoming these difficulties in the cultivation of PCV2, this study aimed to develop a double-antibody sandwich ELISA based on the cell culture antigen PCV2b for the quantification of anti-PCV2 antibodies. A 20% and 50% discontinuous sucrose cushion was used for viral purification, which enabled the separation of cell culture proteins in the 20% sucrose cushion and a greater viral concentration in the 50% sucrose cushion. Following isopycnic centrifugation, PCV2 was concentrated in the band with density values from 1.330 to 1.395g/cm3. Viral purification was assessed using SDS-PAGE, indirect ELISA and electron microscopy. The standardised ELISA revealed a strong linear correlation (r= 0.826, p<0.001) when compared with a commercial ELISA kit. The assay exhibited low variability (inter-assay coefficient of variation of 4.24% and intra-assay of 1.80%) and excellent analytical specificity conferred by the capture antibody produced in rabbit. Thus, this ELISA is a rapid, specific and convenient method for the detection of antibodies against PCV2 in studies of experimental and natural infection, and in monitoring the response to vaccination on commercial farms.
650 $aAntibody detection
650 $aenzyme-linked immunosorbent assay
650 $aPorcine circovirus-2
653 $aIsopycnic centrifugation
653 $aSucrose cushion
700 1 $aKANASHIRO, T. M.
700 1 $aCASTRO, A. M. M. G. de
700 1 $aBALDIN, C. M.
700 1 $aRICHTZENHAIN, L. J.
700 1 $aARAUJO JUNIOR, J. P.
773 $tPesquisa Veterinária Brasileira, Rio de Janeiro$gv. 36, n. 12, p. 1171-1177, dez. 2016.
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Embrapa Unidades Centrais (AI-SEDE) |
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