Journal: Indian journal of medical microbiology
Article Title: A simple and rapid method for the quantitation of secreted hepatitis B virions in cell culture models.
doi: 10.4103/0255-0857.153568
Figure Lengend Snippet: Figure 1: (a) Quantitative estimation of secreted hepatitis B virions in cell culture supernatant. Secreted hepatitis B virions were estimated in undiluted supernatants and in dilutions of supernatants using immunocapture followed by quantification of virion-associated DNA using a real time PCR. Two-fold differences in quantity of secreted virions were accurately detected using our assay. No virion-associated DNA was detected from pkex 1+2 (a replication and packaging deficient mutant) and spiked DNA (growth medium spiked with 50 µL of wild type HBV constructs at a concentration of 1010 copies/mL was added to ELISA plates for immunocapture). (b) Effect of lamivudine on virion secretion. Wild type HBV constructs were transfected in Huh7 cells without lamivudine (0 µM) or with varying concentrations of lamivudine (1 µM, 5 µM and 10 µM). Cell culture supernatants (20 µL each) were used for immunocapture followed by real time PCR for quantitation of secreted virions. The lamivudine-induced reduction in hepatitis B virion secretion is shown in percentage as compared to the virions secreted in the untreated (0 µM) control. No secreted virions were detected in supernatants with 10 µM of lamivudine
Article Snippet: [11] Culture supernatants (100 μL each) from the transfected Huh7 cells (5 days post-transfection) and stably transfected HepG2.2.15 cell line were collected and subjected to DNase I (NEB, MA, USA) digestion for 30 minutes at 37°C to digest non-virion associated HBV DNA, then added to ELISA microplate wells coated with monoclonal anti-HBs antibodies (Monolisa HBsAg ULTRA ELISA kit, Bio-Rad, CA, USA) and incubated at 37°C for
Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Mutagenesis, Construct, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Quantitation Assay, Control
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