A highly sensitive and versatile virus titration assay in the 96-well microplate format

Borisevich, Victoria; Nistler, Ryan; Hudman, Deborah; Yamshchikov, Galina V.; Seregin, Alexey; Yamshchikov, Vladimir

doi:10.1016/j.jviromet.2007.08.015

Cited by 14 publications

(14 citation statements)

References 29 publications

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“…Endotoxin quantification assays (Tamura, 1990(Tamura, , 1991(Tamura, , 1992(Tamura, , 1993, biological activity studies (Barret, 1997;Holowachuk, 2003;Lethinen, 2006), microbiological assays (Bamba, 1997;Burton, 2007;Pettit, 2005) and molecular biology studies (Borisevich, 2008;Mayo, Curnutte 1990;Zhong, Smith 1994) employ kinetic reading microplate systems, allowing analysis of a large number of samples both quickly and reliably. Bacteriostatic, bactericidal and bacteriolytic tests have been performed using microplate systems with kinetic reading of optic density fluorescence bioluminescence (Lethinen, 2006;Smith, 2008).…”

Section: Introductionmentioning

confidence: 99%

Antibiotic microbial assay using kinetic-reading microplate system

Lourenço

Pinto

2011

Braz. J. Pharm. Sci.

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The aim of this study was to determine the optimal experimental conditions to develop a methodology for microbiological assay of apramycin employing microplate and kinetic reading mode, and to validate the developed method, through evaluation of parameters of selectivity, linearity, linear range, limits of detection and quantification, accuracy and precision. The turbidimetric assay principle is simple: the test solution is added to a suspension of test microorganism in culture media, the mixture is incubated under appropriate conditions and the microbial growth is measured by photometric reading. Microplate with kinetic reading mode employed in antibiotic assay is of considerable interest since it allows reduction of material and analysis time and enables a large number of samples to be analyzed simultaneously, with automated reading and calculating. Established conditions considered the standard-curve of apramycin at concentrations from 5.0 to 35.0 mg mL -1 , and tryptic soy broth inoculated with 5% Escherichia coli (ATCC 8739) suspension. Satisfactory results were obtained with 2 hours of incubation. The developed method showed appropriate selectivity, linearity in the range from 5.0 to 35.0 mg mL , respectively, as well as satisfactory accuracy (recuperation = 98.5%) and precision (RSD = 6.0%). Microplate assay combined the characteristics of microbiological (evaluation of antibiotic activity against sensitive test microorganism) and physico-chemical (operationally straightforward and faster results) assays.Uniterms: Antibiotics/microbial assay. Apramycin/microbial assay. Apramycin sulfate. Turbidimetric assay/medicines analysis.O objetivo deste trabalho é determinar as condições experimentais ideais para o desenvolvimento de metodologia para a dosagem microbiológica de apramicina empregando microplacas e modo de leitura cinético e validar o método desenvolvido, através da avaliação dos parâmetros de especificidade e seletividade, linearidade, faixa ou intervalo linear, limite de detecção e quantificação, exatidão e precisão. O ensaio turbidimétrico é simples: a solução-teste é adicionada à suspensão de microrganismo-teste em meio de cultura, a mistura é incubada em condições apropriadas e o crescimento microbiano é medido por meio de leitura fotométrica. O emprego de método de microplacas com leitura cínética para a dosagem de antibióticos é de interesse considerável, uma vez que possibilita reduzir quantidade de material e tempo de análise necessários e permite o ensaio de grande número de amostras simultaneamente, com leitura e cálculo automatizados. As condições estabelecidas abrangem curva-padrão de apramicina com concentrações entre 5 e 35 mg/mL, e emprego de meio de cultura caldo de triptona-soja inoculado com Escherichia coli (ATCC 8739) na proporção de 5%. Foram obtidos resultados satisfatórios após 2 horas de incubação. O método desenvolvido apresentou especificidade e seletividade adequadas, linearidade na faixa de 5 a 35 mg/mL, limite de detecção e quantificação de 0,1 e 0,4 mg/mL, respectivament...

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Section: Introductionmentioning

confidence: 99%

Antibiotic microbial assay using kinetic-reading microplate system

Lourenço

Pinto

2011

Braz. J. Pharm. Sci.

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“…However, the PRNT is laborious and has limitations for screening the large numbers of serum samples needed for epidemiological investigations, because it uses 24-well plates (one and a half plates for testing one sample). Furthermore, it takes 4 or 5 days of culture to see plaques, as is the case with the TCID 50 method.A focus-reduction neutralization test (FRNT)-an alternative VN test that can be performed in 96-well plates-has been introduced for influenza virus, mumps virus, several kinds of flavivirus and some other viruses [2,12,14,15]. Although the principle of the FRNT is the same as that of the PRNT, they differ in the surface area of the culture wells, the volumes of reagents added and the visualization steps.…”

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confidence: 99%

“…Use of the Avicel overlay may not be limited to VN testing. The method that we developed here may be applicable to high-throughput focus assays for EHV-1 in drug-sensitivity testing, as previously described for influenza virus and some other viruses [2,9]. …”

mentioning

confidence: 99%

“…A focus-reduction neutralization test (FRNT)-an alternative VN test that can be performed in 96-well plates-has been introduced for influenza virus, mumps virus, several kinds of flavivirus and some other viruses [2,12,14,15]. Although the principle of the FRNT is the same as that of the PRNT, they differ in the surface area of the culture wells, the volumes of reagents added and the visualization steps.…”

mentioning

confidence: 99%

“…It can be used to visualize viral foci by immuno-staining before plaque formation, resulting in a shorter test period than with the PRNT. The sensitivity of the FRNT is sufficiently high for detecting seroconversion and comparable to that of the PRNT [2,14,15]. In this study, to overcome the problems of conventional VN tests for EHV-1, we developed an FRNT for EHV-1 that can be used to test large numbers of samples in a short period of time and with sufficient sensitivity.…”

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Development of a Focus-Reduction Neutralizing Test for Detecting Equine Herpesvirus Type-1-Neutralizing Antibodies

Bannai

Nemoto

Tsujimura

et al. 2013

The Journal of Veterinary Medical Science

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ABSTRACT. Virus-neutralizing (VN) testing is essential for evaluating virus-specific immunity in equine herpesvirus type-1 (EHV-1) infection. We developed a focus-reduction neutralization test (FRNT) for EHV-1 using 96-well plates for faster large-scale testing with sufficient sensitivity. We used an overlay medium containing Avicel (FMC Biopolymer), a microcrystalline cellulose with lower viscosity than the methylcellulose. The foci were visualized by immuno-staining with anti-EHV-1 gp14 monoclonal antibody. The FRNT successfully detected seroconversion in horses experimentally infected with EHV-1 (n=3) and in those infected naturally (n=16). The FRNT for EHV-1 was high-throughput and time saving. The FRNT can be used in large-scale seroepidemiological studies of EHV-1 and in evaluating vaccine efficacy. KEY WORDS: EHV-1, focus-reduction, virus-neutralizing test.doi: 10.1292/jvms.13-0043; J. Vet. Med. Sci. 75(9): 1209-1212, 2013 Virus-neutralization (VN) is a classical test used for the serological diagnosis of equine herpesvirus type-1 (EHV-1) infection. In addition to having diagnostic applications, the VN test is used to evaluate humoral immune responses to EHV-1 infection or EHV-1 vaccines [3][4][5]. Although the importance of cell-mediated immunity in protection against EHV-1 infection has been confirmed, induction of systemic VN antibodies is also essential for this purpose [1].There are two conventional methods of VN testing for EHV-1. One is the 50% tissue culture infective dose (TCID 50 ) method, which has been used by many researchers investigating 13]. This method has been employed as an OIE (World Organisation for Animal Health) standard protocol for the diagnosis of EHV-1 infection [8]. The method can be applied to large numbers of samples, because it uses 96-well plates (two columns of wells for testing one sample). However, it takes 4 or 5 days of culture to see clear cytopathic effects. Another method is the plaquereduction neutralizing test (PRNT) using 24-well plates [10]. The PRNT has shown greater sensitivity than the TCID 50 method in VN tests for measles virus and rubella virus [6,11], and we have confirmed the same tendency with EHV-1 (data not shown). However, the PRNT is laborious and has limitations for screening the large numbers of serum samples needed for epidemiological investigations, because it uses 24-well plates (one and a half plates for testing one sample). Furthermore, it takes 4 or 5 days of culture to see plaques, as is the case with the TCID 50 method.A focus-reduction neutralization test (FRNT)-an alternative VN test that can be performed in 96-well plates-has been introduced for influenza virus, mumps virus, several kinds of flavivirus and some other viruses [2,12,14,15]. Although the principle of the FRNT is the same as that of the PRNT, they differ in the surface area of the culture wells, the volumes of reagents added and the visualization steps. Because of such differences, the FRNT has several advantages over the PRNT. The use of 96-well plates makes it possible to...

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