In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203)
Braz. j. med. biol. res
;
37(7): 1023-1027, July 2004. graf
Artículo
en Inglés
| LILACS
| ID: lil-360937
RESUMO
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 percent 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 percent and from 3.5 to 9.9 percent, 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).
Texto completo:
Disponible
Índice:
LILACS (Américas)
Asunto principal:
Cultivo de Virus
/
Reproducibilidad de los Resultados
/
Técnicas para Inmunoenzimas
/
Volumetría
/
Virus de la Hepatitis A
País/Región como asunto:
America del Sur
/
Brasil
Idioma:
Inglés
Revista:
Braz. j. med. biol. res
Asunto de la revista:
Biologia
/
Medicina
Año:
2004
Tipo del documento:
Artículo
País de afiliación:
Brasil
Institución/País de afiliación:
Fiocruz/BR
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