Quantitation of baculovirus particles by flow cytometry

Shen, Chun Fang; Meghrous, Jamal; Kamen, Amine

doi:10.1016/s0166-0934(02)00128-3

Cited by 74 publications

(64 citation statements)

References 10 publications

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“…This method has been shown to be a rapid and robust method for not only quantifying the virus [17] but also for a qualitative assessment of the viral stock [18]. Similar to many real-time PCR methods, the protocol reported by Shen et al [17] uses the SYBR Green dye to label the viral DNA; however, unlike other methods, the virus/viral genome is not extracted from the sample to do the analysis. It is believed that this allows less loss due to any processing steps.…”

Section: Introductionmentioning

confidence: 99%

“…This may be due to the low absorbance signals associated with baculovirus when monitoring the eluent at 260 and 280 nm. SYBR Green dye is commonly used for PCR and gel electrophoresis to detect low levels of nucleic acids in biological samples, and has been used for the detection of BV using flow cytometry [17]. We therefore investigated the possibility of combining the labeling power of this dye with the separation capabilities of ion exchange chromatography.…”

Section: Introductionmentioning

confidence: 99%

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Development and validation of a HPLC method for the quantification of baculovirus particles

Transfiguracion

Mena

Aucoin

et al. 2011

Journal of Chromatography B

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A HPLC method using an anion exchange column was developed for the quantification of baculovirus particles. To properly detect the virus eluting from the column, a nucleic acid dye was used to amplify the signal projected by the virus. The viral genome was labeled by incubating the virus with SYBR Green I at 37 • C for a minimum of 1 h. The virus was specifically eluted from the contaminants in 8.9 min at a NaCl concentration of 480 mM NaCl (in 20 mM Tris-HCl, pH 7.5). The total run time of the method was 25 min. The method resulted in a linear response from 1 × 10 8 to 5.0 × 10 10 viral particles (VP/ml). The detection limit was 3.0 × 10 7 and the quantification limit was 1 × 10 8 VP/ml. The intra-assay precision was <10% for both purified and crude virus preparations whereas the inter-assay precisions were <5% and <10% for purified and crude virus preparations, respectively. The recovery/accuracy of the method ranged from 78 to 101%. This method is a robust monitoring tool to facilitate research activities with baculovirus vector and accelerate development of baculovirus-based processes for manufacturing of biologics.Crown

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Development and validation of a HPLC method for the quantification of baculovirus particles

Transfiguracion

Mena

Aucoin

et al. 2011

Journal of Chromatography B

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“…The disadvantages of the plaque assay include tedious sample preparation and the time required for sample analysis, up to a week in many cases (5). In addition, there is usually a large variance in the results of the assay, with some sources of variability including changes in media and culture conditions and interoperator, interday, and interlaboratory variations; the inherent inefficiencies of this method are well documented (2,3,6,7). Historically quantification of viruses, such as baculovirus, have relied primarily on the plaque titer assay and the limitations of the technique have hampered progress in biotechnology (8).…”

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confidence: 99%

“…Recently flow cytometric (FCM) detection and quantification of virus particles has been demonstrated (7,(9)(10)(11)(12)(13)(14)(15). The use of FCM requires staining the virus with a fluorescent dye because viruses are generally too small to efficiently scatter light (12).…”

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confidence: 99%

“…The introduction of FCM virus quantification methods has lead to decreased analysis time and decreased variability (7). Because FCM experiments to date have used various DNA/RNA stains for virus detection, the technique allows for the enumeration of only DNA/RNA events and does not permit the distinction between active and inactive virus particles (7,12,14,15).…”

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Design and characterization of a compact dual channel virus counter

2005

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Background: Although there is a growing need in the field of biotechnology to rapidly and accurately quantify viruses, time-consuming techniques such as the plaque titer method remain the ''gold standard.'' Flow cytometric methods for virus quantification offer the advantages of rapid analysis and statistical treatment. The technique presented in this work represents the first demonstration of a flow cytometric determination of a viral count that is directly related to the count obtained by plaque titer. Methods: A flow cytometric instrument for rapid quantification of virus particles was designed, constructed, and thoroughly characterized. A two-color method, which involved staining the viral genome and the protein coat for baculoviruses, was developed in addition to an algorithm to identify simultaneous events on the DNA and protein channels.

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Insect Cells and Larvae, Gene Expression Systems

Narang

Whiteley

Hussain

et al. 2010

Encyclopedia of Industrial Biotechnology

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The use of the insect cell‐baculovirus system has grown significantly over the past three decades. The present chapter elucidates the fundamentals, scientific developments, technologies, and hurdles encountered in this field. The common types of insects and insect lines used as well as their specific features and culture conditions are described. Additionally, the types of baculoviruses used and the methods for generating them are also revealed. Particular emphasis has been given to describing the construction of recombinant baculoviruses that are used in production of heterologous proteins with insect cells. A major advantage in using insect cells is their ability to perform many post‐translational processing events on these proteins including glycosylation. The types of insect cell glycosylation and the differences in glycosylation patterns between insect and mammalian systems are detailed. The parameters that affect insect cell growth and baculovirus infection including bioreactor configuration, process monitoring and control, oxygen supply, sparging, and media formulations are described. In particular, cell culture processing methods including batch, fed‐batch, perfusion, and semi‐batch processes are illustrated. Additional factors affecting baculovirus infection including multiplicity, time of infection, and the generation of mutant viruses are also considered. A final section is devoted to the use of insect larvae in combination with baculovirus as a potentially alternative to cell culture for the low‐cost generation of baculoviruses and heterologous proteins. With all the advances in molecular biology and engineering processes for the baculovirus‐insect cell system, insect cell culture and its application to recombinant protein production, vaccines, pesticides, and other areas will continue to expand in the coming decades.

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Quantitation of baculovirus particles by flow cytometry

Cited by 74 publications

References 10 publications

Development and validation of a HPLC method for the quantification of baculovirus particles

Development and validation of a HPLC method for the quantification of baculovirus particles

Design and characterization of a compact dual channel virus counter

Insect Cells and Larvae, Gene Expression Systems

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