In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203)
Hepatitis A virus (HAV) replicates relatively slowly in cell culture without a cytopathic effect, a fact that limits the use of tissue culture assays. The radioimmunofocus assay is the standard method for HAV titration, although it is labor intensive and requires the use of radioisotopes. A simple,...
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| Format: | Article |
| Language: | English |
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Associação Brasileira de Divulgação Científica
2004-07-01
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| Series: | Brazilian Journal of Medical and Biological Research |
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| Online Access: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004000700011 |
| _version_ | 1819211651734306816 |
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| author | L.M. Villar L.A. Amado A.M.C. Gaspar |
| author_facet | L.M. Villar L.A. Amado A.M.C. Gaspar |
| author_sort | L.M. Villar |
| collection | DOAJ |
| description | Hepatitis A virus (HAV) replicates relatively slowly in cell culture without a cytopathic effect, a fact that limits the use of tissue culture assays. The radioimmunofocus assay is the standard method for HAV titration, although it is labor intensive and requires the use of radioisotopes. A simple, rapid and objective infectivity assay based on an in situ enzyme immunoassay (EIA) is described here for a Brazilian cell culture-adapted HAV strain (HAF-203). The assay uses a peroxidase-labeled polyclonal antibody to fixed monolayers as an indicator of infection. EIA may be completed within 7 days using serial 5-fold dilutions of the virus, yielding a titer of 5.024 log 50% tissue culture infective dose (TCID50)/ml for HAF-203. This technique had a detection limit of 1.1 log TCID50/ml and the specificity was demonstrated by detecting no reaction on the columns of uninfected wells. The reproducibility (with intra- and inter-assay coefficients of variation ranging from 1.9 to 3.8% and from 3.5 to 9.9%, respectively) and quantitation of the assay were demonstrated by close agreement in virus infectivity titers among different assays of the same amount of virus and between assays of different amounts of virus. Furthermore, this assay does not require the use of radiolabeled antibodies. We describe here an efficient EIA that is highly reproducible and that could be used to monitor HAV growth in cell culture and to determine the quantity of HAV antigen needed for diagnostic assays. This is the first report of the infectious titer of the Brazilian cell culture-adapted HAV strain (HAF-203). |
| first_indexed | 2024-12-23T06:30:28Z |
| format | Article |
| id | doaj.art-b3d7b7df79f5439284667fe144e79d92 |
| institution | Directory Open Access Journal |
| issn | 0100-879X 1414-431X |
| language | English |
| last_indexed | 2024-12-23T06:30:28Z |
| publishDate | 2004-07-01 |
| publisher | Associação Brasileira de Divulgação Científica |
| record_format | Article |
| series | Brazilian Journal of Medical and Biological Research |
| spelling | doaj.art-b3d7b7df79f5439284667fe144e79d922022-12-21T17:56:57ZengAssociação Brasileira de Divulgação CientíficaBrazilian Journal of Medical and Biological Research0100-879X1414-431X2004-07-013771023102710.1590/S0100-879X2004000700011In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203)L.M. VillarL.A. AmadoA.M.C. GasparHepatitis A virus (HAV) replicates relatively slowly in cell culture without a cytopathic effect, a fact that limits the use of tissue culture assays. The radioimmunofocus assay is the standard method for HAV titration, although it is labor intensive and requires the use of radioisotopes. A simple, rapid and objective infectivity assay based on an in situ enzyme immunoassay (EIA) is described here for a Brazilian cell culture-adapted HAV strain (HAF-203). The assay uses a peroxidase-labeled polyclonal antibody to fixed monolayers as an indicator of infection. EIA may be completed within 7 days using serial 5-fold dilutions of the virus, yielding a titer of 5.024 log 50% tissue culture infective dose (TCID50)/ml for HAF-203. This technique had a detection limit of 1.1 log TCID50/ml and the specificity was demonstrated by detecting no reaction on the columns of uninfected wells. The reproducibility (with intra- and inter-assay coefficients of variation ranging from 1.9 to 3.8% and from 3.5 to 9.9%, respectively) and quantitation of the assay were demonstrated by close agreement in virus infectivity titers among different assays of the same amount of virus and between assays of different amounts of virus. Furthermore, this assay does not require the use of radiolabeled antibodies. We describe here an efficient EIA that is highly reproducible and that could be used to monitor HAV growth in cell culture and to determine the quantity of HAV antigen needed for diagnostic assays. This is the first report of the infectious titer of the Brazilian cell culture-adapted HAV strain (HAF-203).http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004000700011Hepatitis A virusVirus titrationEnzyme immunoassay |
| spellingShingle | L.M. Villar L.A. Amado A.M.C. Gaspar In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203) Brazilian Journal of Medical and Biological Research Hepatitis A virus Virus titration Enzyme immunoassay |
| title | In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203) |
| title_full | In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203) |
| title_fullStr | In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203) |
| title_full_unstemmed | In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203) |
| title_short | In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203) |
| title_sort | in situ enzyme immunoassay for titration of a brazilian hepatitis a virus strain haf 203 |
| topic | Hepatitis A virus Virus titration Enzyme immunoassay |
| url | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004000700011 |
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