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Quantitation of baculovirus particles by flow cytometry
Chun Fang Shen1, Jamal Meghrous, Amine Kamen
1Animal Cell Technology Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave, Montréal, Quebec, Canada H4P 2R2.
Journal of Virological Methods
|September 25, 2002
Summary
A new flow cytometry method quantitates total baculovirus particles in insect cell cultures. This rapid technique, using SYBR Green I dye, provides higher counts than traditional assays, accelerating baculovirus expression technology development.
Area of Science:
- Biotechnology
- Virology
- Cell Biology
Background:
- Baculovirus expression technology relies on accurate viral quantitation.
- Traditional methods like end-point dilution assay (EPDA) are time-consuming.
- Quantifying total baculovirus particles is crucial for process optimization.
Purpose of the Study:
- To develop and validate a rapid flow cytometry (FCM) method for quantifying total baculovirus particles.
- To optimize sample preparation for enhanced FCM analysis of baculovirus.
- To compare FCM-based particle counts with traditional EPDA titration.
Main Methods:
- Developed a direct particle count method using FCM.
- Employed SYBR Green I dye for staining baculovirus DNA.
- Optimized sample preparation including fixation, freeze-thaw, permeabilization, and heating.
- Assessed linearity, detection limits, robustness, and reproducibility.
Main Results:
- Established FCM linearity over a two-log range with a detection limit of 10(5) particles/ml.
- FCM consistently yielded higher particle counts than EPDA, with an average ratio of 3.7:1.
- Optimized sample preparation improved staining efficiency and fluorescence intensity.
- Method validation demonstrated robustness and reproducibility using large-scale bioreactor samples.
Conclusions:
- The developed FCM method offers a rapid (2-hour) and reliable way to quantitate total baculovirus particles.
- This method provides a more comprehensive measure of viral yield compared to EPDA.
- Accelerates process development in baculovirus expression technology, especially for complex product expression.