Skip to main content
Login | Suomeksi | På svenska | In English

Browsing by Subject "assay"

Sort by: Order: Results:

  • Juntunen, Valtteri (2021)
    Adeno-Associated Viruses (AAVs) are quickly becoming one of the most applied vectors for gene therapy applications. In the recent years three new AAV-based gene therapies have been approved by U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA). The regulatory bodies require accurate and reliable characterisation of the clinical grade viral vectors during and after production. Analytic methods measuring the purity, potency and safety of the product support the up-stream and down-stream processes during the production and are used for final-drug substance characterisation. Median Tissue Culture Infectious Dose (TCID50) is a well-established method for measuring the infectious titer of a virus. Here, an assay for determining the infectious titer of AAVs, which has previously been used to characterise the existing AAV2 Reference Standard Material (AAV2RSM) was set up and optimised for research use at Kuopio Center for Gene and Cell Therapy (KCT). The assay utilizes the HeRC32-cell line, a HeLa clone, that stably expresses AAV Rep and Cap -proteins and in presence of adenovirus, enables the replication of recombinant AAV-vectors. The cells were grown in 96-well plates and infected with 10-fold dilution series of AAV vectors (AAV2 and AAV6) using human adenovirus type 5 as the co-infector. 72 hours post infection the vector genome replication of AAV was detected with quantitative PCR (qPCR). Thresholds for qPCR determined copy number and cycle threshold (Ct) were set and used for the determination of infection-positive wells. The 50-percent endpoint was observed and used to calculate the infectious titer according to the Spearman-Kärber method. The assay was set up and optimised with the AAV2 Reference standard material (AAV2RSM) using two different primer-probe sets (targeted sequences were; SV40 polyadenylation signal and AAV inverted terminal repeats (ITRs)). Plates infected with AAV2RSM were analysed separately with both primers resulting in mean infectious titers of 8.07 ± 3.13 x 108 TCID50 Infectious Units (IU) / mL (n = 9) and 1.27 ± 0.464 x 109 TCID50 IU/mL (n = 9) for SV40 and ITR, respectively. After the assay was set up with the AAV2RSM, an in-house AAV6 product was analysed with the ITR primers yielding 6.09 ± 3.94 x 109 TCID50 (IU) / mL (n = 5). The assay protocol was successfully set up for research use at the KCT laboratory. Improvements were added to the original protocol to increase assay robustness, accuracy and precision as well as to minimize the possibility of over-estimation of the infectious titer. The assay can be further optimised for a particular therapeutic AAV product in the research laboratory or technology transferred to a production facility for optimisation and validation for the analytics needs of a production pipeline.
  • Mohanraj, Ushanandini (2016)
    The rapid emergence of antibiotic resistance among many pathogenic bacteria has created a profound need to discover new alternatives to antibiotics. Bacteriophages are viruses which infect bacteria and are able to produce special proteins involved in bacterial lysis. However, for many bacteriophage-encoded gene products, the function is not known, i.e., hypothetical proteins of unknown function (HPUFs). Screening these proteins likely identifies a rich source of leads that will help in the development of novel antibacterial compounds. The current study presents two phage genomics-based screening approaches to identify phage HPUFs with antibacterial activity. Both screening assays are based on inhibition of bacterial growth when a toxic gene is expression cloned into a plasmid vector. The first approach was a luxAB/luxCDE -based luminescence screening assay. The luxCDE genes encoding the luciferase substrate producing enzymes were integrated into an Escherichia coli strain genome as a transcriptional fusion. Also, a vector carrying the luxAB genes, encoding the luciferase enzyme, and a cloning site for the phage HPUF genes, was constructed. Ligation of a toxic gene into the vector would result in few or rare transformants after electroporation while ligation of a non-toxic gene would result in large number of transformants, and the difference in number of transformants will be reflected in the amount of bioluminescence after electroporation. The proof of concept of the approach was verified using the control genes g150 (a structural, thus a non-toxic gene of phage R1-RT) and regB (a known toxic gene of phage T4). The results demonstrated a significant difference in Relative Luminescence Units (RLU) between the g150 and regB electroporation mixtures. The second screening approach was an optimized plating assay producing a significant difference in the number of transformants after ligation of the toxic and non-toxic genes into a cloning vector. This assay was tested and optimized with several known control toxic and non-toxic genes. Using the plating assay approach, in the current study, ninety-four R1-RT HPUFs were screened and ten of them showed toxicity in E. coli. In future, the identified toxic HPUFs of R1-RT could be purified and characterized to identify their bacterial targets. Further, both of these screening assays can be used to screen among HPUFs of other phages, and this should allow the discovery of a wide variety of putative inhibitors for the control of current and emerging bacterial pathogens.
  • Mohanraj, Ushanandini (2016)
    The rapid emergence of antibiotic resistance among many pathogenic bacteria has created a profound need to discover new alternatives to antibiotics. Bacteriophages are viruses which infect bacteria and are able to produce special proteins involved in bacterial lysis. However, for many bacteriophage-encoded gene products, the function is not known, i.e., hypothetical proteins of unknown function (HPUFs). Screening these proteins likely identifies a rich source of leads that will help in the development of novel antibacterial compounds. The current study presents two phage genomics-based screening approaches to identify phage HPUFs with antibacterial activity. Both screening assays are based on inhibition of bacterial growth when a toxic gene is expression cloned into a plasmid vector. The first approach was a luxAB/luxCDE -based luminescence screening assay. The luxCDE genes encoding the luciferase substrate producing enzymes were integrated into an Escherichia coli strain genome as a transcriptional fusion. Also, a vector carrying the luxAB genes, encoding the luciferase enzyme, and a cloning site for the phage HPUF genes, was constructed. Ligation of a toxic gene into the vector would result in few or rare transformants after electroporation while ligation of a non-toxic gene would result in large number of transformants, and the difference in number of transformants will be reflected in the amount of bioluminescence after electroporation. The proof of concept of the approach was verified using the control genes g150 (a structural, thus a non-toxic gene of phage R1-RT) and regB (a known toxic gene of phage T4). The results demonstrated a significant difference in Relative Luminescence Units (RLU) between the g150 and regB electroporation mixtures. The second screening approach was an optimized plating assay producing a significant difference in the number of transformants after ligation of the toxic and non-toxic genes into a cloning vector. This assay was tested and optimized with several known control toxic and non-toxic genes. Using the plating assay approach, in the current study, ninety-four R1-RT HPUFs were screened and ten of them showed toxicity in E. coli. In future, the identified toxic HPUFs of R1-RT could be purified and characterized to identify their bacterial targets. Further, both of these screening assays can be used to screen among HPUFs of other phages, and this should allow the discovery of a wide variety of putative inhibitors for the control of current and emerging bacterial pathogens.