Evaluation of a Quantitative Product-enhanced Reverse Transcriptase Assay to Monitor Retrovirus in mAb Cell-culture

Brorson, Kurt; Xu, Yuan; Swann, Patrick G.; Hamilton, Elizabeth; Mustafa, Mehnaz; Wit, Christina de; Norling, Lenore A.; Stein, Kathryn E.

doi:10.1006/biol.2001.0290

Cited by 29 publications

(16 citation statements)

References 32 publications

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“…The PERT assay has been used as a method to quantify known and detect unknown retroviruses, with recognized limitations in terms of possible detection of RT-like activities of nonretroviral origin (Maudru & Peden 1997, André et al 2000, Brorson et al 2002.…”

Section: Discussionmentioning

confidence: 99%

“…At the level of the assay itself, there are some reagents that may produce RT-like activity such as Taq DNA polymerases or RNase inhibitors (Pyra et al 1994). At the level of the tested sample, there are some enzymes that may display RT-like activity such as host DNA polymerases, some other nuclear enzymes, and polymerase from mitochondria (Brorson et al 2002).…”

Section: Discussionmentioning

confidence: 99%

“…Firstly, telomerases are template specific, requiring a specific recognition sequence, TTAGGG. Thus, it is generally believed that they are unlikely to reverse transcribe MS2 phage RNA which lacks this target sequence (Brorson et al 2002). Secondly, the assay was performed on cell free haemolymph as discussed above.…”

Section: Discussionmentioning

confidence: 99%

“…Secondly, the assay was performed on cell free haemolymph as discussed above. On the other hand, TM-PERT is able to detect RT activity of endogenous retroviruses and other retroelements such as retrotransposons (Brorson et al 2002).…”

Section: Discussionmentioning

confidence: 99%

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Reverse transcriptase activity associated with haemic neoplasia in the soft-shell clam Mya arenaria

AboElkhair

Siah²,

Clark³

et al. 2009

Dis. Aquat. Org.

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Reverse transcriptase (RT) activity has been reported in bivalves affected by haemic neoplasia (HN). Since all retroviruses have RT, detection of RT activity was regarded as evidence for the retroviral etiology of HN. This study investigates the relationship between RT levels and the progress of HN as indicated by percentages of tetraploid cells in soft-shell clams Mya arenaria. The percentages of tetraploid cells were estimated by flow cytometry, and the RT levels were quantified using TaqMan product-enhanced RT (TM-PERT) assay. Results demonstrated that the amount of RT was positively correlated with the percentage of tetraploid cells circulating in clam haemolymph (R 2 = 0.974, p < 0.001). Compared to HN-negative clams (< 5% tetraploid cells), 2 stages with significantly elevated levels of RT activity were observed: the first stage at ~10 to ~20% tetraploid cells, and the second at ~30 to ~80% tetraploid cells (p < 0.01). These data support the well established fact from mammalian models that transformed cells express high levels of non-telomeric RT. The observed increase in RT levels at ~30% tetraploidy coincides with previously reported p53 gene expression. Taken together, this could indicate that using RT levels as an indicator of HN, ≥30% tetraploidy is the stage at which the disease process undergoes a change, and perhaps becomes irreversible.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Reverse transcriptase activity associated with haemic neoplasia in the soft-shell clam Mya arenaria

AboElkhair

Siah²,

Clark³

et al. 2009

Dis. Aquat. Org.

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“…However, the ES-DMA and plaque assay data did not yield the same counts (Table 7.1), which implied that not all phage particles were infectious such that a direct comparison to infectivity data led to a 10 to 40 fold disparity in the measured values, a common observation in virology when comparing the infectivity assay to methods that physically count particles [192,193]. This discrepancy between the plaque assay and the ES-DMA measured concentrations presumably involves several factors.…”

mentioning

confidence: 99%

Electrospray–differential mobility analysis of bionanoparticles

Guha

Tarlov

et al. 2012

Trends in Biotechnology

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The growth of the multibillion dollar bionanoparticle industry has spurred the development of new physical characterization methods. One such method, electrospraydifferential mobility analysis (ES-DMA) constitutes an electrospray for aerosolization of bionanoparticles (such as viruses, gold-nanoparticles, proteins, nanoparticle-protein complexes) and an ion mobility method that operates at atmospheric conditions, and separates bionanoparticles spatially. This dissertation identifies some relevant "problem" areas for ES-DMA by reviewing selected applications.Some such problems are: proteins while passing through ES capillaries are found to interact with it and thus produce time dependent size distributions. Further, it is thought that adsorbed proteins may subsequently desorb and influence size distributions with the ES-DMA which may concomitantly affect quantification of aggregates. These artifacts are studied systematically and it is demonstrated that ES-DMA can quantify adsorption-desorption of complex protein mixtures at high shear rates. Further, it is shown that desorbing proteins do not have a significant effect on size distributions. Another artifact of the ES takes place during the aersolization process. Two units (called monomers) of a bionanoparticle may get encapsulated in the same ES droplet and upon drying of the droplet create artificial dimers thus affecting quantification with ES-DMA. Assuming Poisson distribution, this thesis provides a systematic approach that can be undertaken to eliminate this artifact. A third artifact arises from the low sensitivity of the DMA to size increase. When a ligand (for e.g. protein) adsorbs to a bionanoparticle it creates an increase in the size of the later, which can be used to quantify the amount of ligand adsorbed per bionanoparticle. As ligands can change conformations upon adsorption, using ES-DMA for such applications may be flawed. This issue has been identified and a solution has been provided by integrating a mass analyzer after the ES-DMA.After correcting for these artifacts, this dissertation delves into characterization of different types of bionanoparticles and demonstrates that ES-DMA has several advantages over other traditional techniques such as transmission electron microscopy, size exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and plaque assay and thus has immense potential to become a process analytical technique in biomanufacturing environments. ELECTROSPRAY-DIFFERENTIAL MOBILITY ANALYSIS OF BIONANOPARTICLES

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Virus Retentive Filters

Miesegaes

Lute

Aranha³

et al. 2010

Encyclopedia of Industrial Biotechnology

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Ensuring the absence of detectable viruses in biopharmaceutical products is imperative from both a regulatory and patient safety standpoint. Biopharmaceutical viral safety is the result of multiple orthogonal barriers operating in concert; incorporation of virus clearance methods is an important strategy to achieving product safety. Filtration through virus retentive filters is currently a key unit operation during the production of biopharmaceuticals and plasma‐derived products. Careful process design and appropriate validation are critical for the successful implementation and performance of virus retentive filters. Here we elaborate on both process design and validation information, describe virus filters currently on the market, and discuss the historical experience with virus retentive filters as reported in the scientific literature. Detailed information for filter users can be found in a comprehensive technical report (TR 41) promulgated by the Parenteral Drug Association (PDA) (1).

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Evaluation of a Quantitative Product-enhanced Reverse Transcriptase Assay to Monitor Retrovirus in mAb Cell-culture

Cited by 29 publications

References 32 publications

Reverse transcriptase activity associated with haemic neoplasia in the soft-shell clam Mya arenaria

Reverse transcriptase activity associated with haemic neoplasia in the soft-shell clam Mya arenaria

Electrospray–differential mobility analysis of bionanoparticles

Virus Retentive Filters

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