Rapid Semi‐automated Procedures for Assaying Antiviral Activity

Miller, P. A.; Lindsay, H. L.; Cormier, Michel; Mayberry, B. R.; Trown, P. W.

doi:10.1111/j.1749-6632.1970.tb53402.x

Cited by 17 publications

(5 citation statements)

References 11 publications

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“…Since plaques are formed after multiple cycles of viral reproduction, the PRD50 method requires a longer time than the INAS50 method in which determination of viral growth is based on the synthesis of virus-specific RNA during a single-step growth. According to INAS50 methods so far proposed, cells were grown in petri dishes and therefore labeled cells had to be transferred on to Millipore filters for counting radioactivity [1,[4][5][6]. This rendered the procedure still too cumbersome to be widly used as a routine method.…”

Section: Discussionmentioning

confidence: 99%

A Rapid and Simple Method for Assaying Interferon1

Suzuki

Akaboshi

Kobayashi

1974

Japanese Journal of Microbiology

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A new procedure of assaying interferon (IF) has been developed. Cell suspension was dispensed into liquid scintillation counting vials together with IF sample. During an overnight incubation, the cells adhered sufficiently to the bottom of the vials and all the subsequent procedures were carried out without transfer of the cells from the vials. Vesicular stomatitis virus was inoculated and virusspecific RNA was labeled by adding 3H-uridine and actinomycin D to the medium. Incubation was terminated prior to completion of a single-step growth of the virus and radioactivity of the labeled cells in each vial was determined.The reciprocal of the IF dilution which reduced the radioactivity in viral RNA by 50% was taken as the titer. The present procedure consists of simple manipulations and can be completed within 24 hr. Furthermore, it is quite reproducible and gives a titer almost identical to that obtained by the conventional plaque-reduction dose method. The procedure can be applied to mouse L cells, rabbit RK-13 cells and human FL cells, without modification.The technique which is currently used most widely for assaying interferon (IF) is the plaque-reduction dose method (PRD50 method). The procedure gives reliable results but involves a longer time and laborious handlings. In order to overcome these disadvantages, several groups of workers have introduced techniques based on inhibition of nucleic acid synthesis (INAS50 method) [1,4 6], in which the IF titer is determined by measuring radioactivity of 3H-uridine incorporated into virus-specific RNA in the IFtreated cells. Although their methods have some advantages over the PRD50 method, these are still laborious and remain impractical as a routine method. In this communication we describe a new procedure of the INAS50 method which yields reliable results much faster in a simpler way. This technique will be well qualified to replace the PRD50 method. MATERIALS AND METHODSCells and media. Mouse L cells, human FL cells and rabbit RK-13 cells were employed for assaying IF preparations of the corresponding origin. Two different lines of mouse L cells (L-MS cells) and human FL cells (FL-MS cells) had been adapted to grow readily either in monolayer or in suspension in our laboratory [9]. The growth medium employed for monolayer culture was Eagle's minimum essential medium (Nissui Seiyaku Co., Japan) plus 5% calf serum (MEM + 5% CS). For suspension culture Eagle's spinner culture medium (Nissui Seiyaku Co., Japan) supplemented with 5% calf serum (SM+5%CS) was used.

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Section: Discussionmentioning

confidence: 99%

A Rapid and Simple Method for Assaying Interferon1

Suzuki

Akaboshi

Kobayashi

1974

Japanese Journal of Microbiology

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“…A semi-automated procedure has been described by MILLER et al (1970), in which both DNA and I~NA viruses can be tested. Viral nucleic acid synthesis is measured as incorporation of a radioactively labelled precursor into tissue culture cells by scintillation counting.…”

Section: Introductionmentioning

confidence: 99%

A rapid, simple and reliable method for the screening of potential antiviral compoundsin vitro

Boyd

Sommerville

1974

Archiv f Virusforschung

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“…Inhibition of viral RNA synthesis is taken as a measure of interferon titer. This method offers the advantage of being potentially applicable to both cytolytic and poorly or noncytolytic viruses; its drawbacks are that it generally requires high multiplicities of infection, large culture flasks, and mechanical removal of cells before isotope counting (1,6,10,11,13,22). Growing the cells directly in scintillation vials reduces handling to some extent (19).…”

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confidence: 99%

Microassay for interferon, using [3H]uridine, microculture plates, and a multiple automated sample harvester

Richmond¹,

Polatnick²,

Knudsen³

1980

Appl Environ Microbiol

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A microassay for interferon is described which uses target cells grown in microculture wells, [3H]uridine to measure vesicular stomatitis virus replication in target cells, and a multiple automated sample harvester to collect the radioactively labeled viral ribonucleic acid onto glass fiber filter disks. The disks were placed in minivials, and radioactivity was counted in a liquid scintillation spectrophotometer. Interferon activity was calculated as the reciprocal of the highest titer which inhibited the incorporation of [3H]uridine into viral ribonucleic acid by 50%. Interferon titers determined by the microassay were similar to the plaque reduction assay when 100 plaque-forming units of challenge vesicular stomatitis virus was used. However, it was found that the interferon titers decreased approximately 2-fold for each 10-fold increase in the concentration of challenge vesicular stomatitis virus when tested in the range of 102 to 105 plaque-forming units. Interferon titers determined by the microassay show a high degree of repeatability, and the assay can be used to measure small and large numbers of interferon samples.

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Rapid Semi‐automated Procedures for Assaying Antiviral Activity

Cited by 17 publications

References 11 publications

A Rapid and Simple Method for Assaying Interferon1

A Rapid and Simple Method for Assaying Interferon1

A rapid, simple and reliable method for the screening of potential antiviral compoundsin vitro

Microassay for interferon, using [3H]uridine, microculture plates, and a multiple automated sample harvester

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