Journal: AMB Express

Article Title: Production of bioactive liver-targeting interferon Mu-IFN-CSP by soluble prokaryotic expression

doi: 10.1186/s13568-017-0493-z

Figure Lengend Snippet: Effect of IFN-CSP or mu-IFN-CSP on HBV antigens secretion and HBV-DNA replication in HepG2.2.15 cells. HepG2.2.15 cells were cultured in the presence of IFN-CSP or mu-IFN-CSP for 6 days. Hepatitis B surface antigen (HBsAg; a ) and hepatitis B e antigen (HBeAg; b ) in the culture supernatants were analyzed by enzyme-linked immunosorbant assay (ELISA). Supernatant HBV-DNA ( c ) and intracellular HBV-DNA ( d ) were measured by real-time quantitative PCR. Data represent the mean ± SEM of three experiments. ***P < 0.001 drug group vs control group; # P < 0.05, ## P < 0.01 IFN-CSP group vs mu-IFN-CSP group

Article Snippet: The treated HepG2.2.15 cells were seeded on coverslip and the primary antibody and the second antibody were goat polyclonal anti-HBsAg antibody (1:200; Novus Biologicals, CO, USA) and Alexa Fluor Cy3-conjugated donkey anti-goat IgG (1:200; Beyotime, Guangzhou, China), respectively.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Control

Journal: AMB Express

Article Title: Production of bioactive liver-targeting interferon Mu-IFN-CSP by soluble prokaryotic expression

doi: 10.1186/s13568-017-0493-z

Figure Lengend Snippet: Effect of IFN-CSP or mu-IFN-CSP on HBsAg expression in HepG2.2.15 cells. HepG2.2.15 cells stained by immunofluorescent staining with anti-HBsAg antibody and representative photographs are captured by microscope. A Controls; B IFN-CSP; C mu-IFN-CSP; 1 red stained with HBsAg; 2 blue nuclear stained with DAPI; 3 merged images of 1 and 2. Bar, 100 μm and is the same for all photomicrographs

Article Snippet: The treated HepG2.2.15 cells were seeded on coverslip and the primary antibody and the second antibody were goat polyclonal anti-HBsAg antibody (1:200; Novus Biologicals, CO, USA) and Alexa Fluor Cy3-conjugated donkey anti-goat IgG (1:200; Beyotime, Guangzhou, China), respectively.

Techniques: Expressing, Staining, Microscopy

Journal: Pathogens (Basel, Switzerland)

Article Title: Development of Low-Cost In-House Assays for Quantitative Detection of HBsAg, HBeAg, and HBV DNA to Enhance Hepatitis B Virus Diagnostics and Antiviral Screening in Resource-Limited Settings.

doi: 10.3390/pathogens14030258

Figure Lengend Snippet: Figure 1. Optimization of in-house quantitative sandwich ELISA for detection of HBsAg and HBeAg. Standard curve depicting the optimal dilution of detection antibody for the detection of (A) HBsAg and (B) HBeAg. Primary antibodies used for antigen capture were mouse monoclonal α-HBsAg (Fitzgerald, 10-H05H) and mouse monoclonal α-HBeAg (Fitzgerald, 10-H10M). Detection was achieved using HRP-conjugated α-HBsAg goat polyclonal antibody (Fitzgerald, 70-HG15S) and α-HBeAg mouse monoclonal antibody (Fitzgerald, 61-H10K). OD450 values were normalized against BSA control wells. The assay was optimized by adjusting antibody concentrations to achieve a strong signal-to-noise ratio while maintaining linearity. Data were normalized by subtracting the optical density reading at 450 nanometers (OD450nm) from bovine serum albumin (BSA) control wells. (C) Summary table of ELISA data for HBeAg and HBsAg. Assay quality was assessed based on linearity of the standard curve R2 and Z′ factor. Data from 3 biological replicates.

Article Snippet: Primary antibodies used for antigen capture were mouse monoclonal αHBsAg (Fitzgerald, 10-H05H) and mouse monoclonal α-HBeAg (Fitzgerald, 10-H10M).

Techniques: Sandwich ELISA, Control, Enzyme-linked Immunosorbent Assay, HBsAg Assay

Journal: Pathogens (Basel, Switzerland)

Article Title: Development of Low-Cost In-House Assays for Quantitative Detection of HBsAg, HBeAg, and HBV DNA to Enhance Hepatitis B Virus Diagnostics and Antiviral Screening in Resource-Limited Settings.

doi: 10.3390/pathogens14030258

Figure Lengend Snippet: Figure 2. Comparison of quantitative in-house ELISA assays for HBsAg (A) and HBeAg (B) detection in plasma samples from HBV-infected individuals (genotypes A–F), evaluated against a commercial ELISA (Abbott Architect). Dilution series (10-, 100-, 500-, 1000-fold) were conducted to determine the minimum plasma volume required for reliable quantification of HBsAg and HBeAg. Statistical analysis via one-way ANOVA with Tukey’s HSD post hoc test revealed significant differences (**) at 1000-fold dilution between groups (p < 0.05), and no significant (ns) differences between other dilution series. Data are presented as mean ± SD from three technical replicates per sample. (C) Summary of 35 samples tested, detailing HBsAg and HBeAg positivity rates across genotypes (A–F), as determined by the Abbott Architect assay. A 10-fold plasma dilution (10 µL in 100 µL of blocking buffer) reliably quantified (D) HBsAg and (E) HBeAg. Notably, HBeAg levels in samples tested from genotypes A and E were below the lower limit of detection (

Article Snippet: Primary antibodies used for antigen capture were mouse monoclonal αHBsAg (Fitzgerald, 10-H05H) and mouse monoclonal α-HBeAg (Fitzgerald, 10-H10M).

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Infection, Blocking Assay

Journal: Pathogens (Basel, Switzerland)

Article Title: Development of Low-Cost In-House Assays for Quantitative Detection of HBsAg, HBeAg, and HBV DNA to Enhance Hepatitis B Virus Diagnostics and Antiviral Screening in Resource-Limited Settings.

doi: 10.3390/pathogens14030258

Figure Lengend Snippet: Figure 4. Comparison of various DNA isolation methods for determining the EC50 of TDF in HepAD38 cells. HepAD38 cells were seeded in 24-well plates or 96-well plates and treated with TDF for 14 days with media and drug replenishment every 72 h. (A) To determine the toxicity of TDF, HepAD38 cells in the 96-well plate format were incubated with Alamar Blue for 2 h prior to reading the absorbance at 560 nm and 600 nm. Data were normalized according to the manufacturer’s directions to determine cell viability. (B) HBeAg and HBsAg could be quantified from the supernatant of 96-well HepAD38 cells treated with Alamar Blue. Extracellular (cell supernatant, top panel) and intracellular (bottom panel) HBV DNA were determined in (C) 96-well and (D) 24-well plate formats and plotted as % HBV inhibition against log [TDF] to determine the EC50. To investigate if Alamar Blue negatively impacts EC50 determination, HepAD38 cells from the 96-well plate treated with Alamar Blue were also subjected to DNA isolation and quantification. (E) Summary of TDF EC50 values obtained from the treatment of HepAD38 cells in either a 24- or 96-well format. Assay Z′

Article Snippet: Primary antibodies used for antigen capture were mouse monoclonal αHBsAg (Fitzgerald, 10-H05H) and mouse monoclonal α-HBeAg (Fitzgerald, 10-H10M).

Techniques: Comparison, DNA Extraction, Incubation, Inhibition

Journal: Cellular microbiology

Article Title: Hepatitis B virus envelope proteins can serve as therapeutic targets embedded in the host cell plasma membrane.

doi: 10.1111/cmi.13399

Figure Lengend Snippet: FIGURE 8 Targeting of membrane-localised HBs by antibodies and CAR-T cells. (a) 100 μg purified MoMab were iv injected into four AlbPsx mice that expresses HBV L protein. After 4 hr, mice were sacrificed and livers collected. HBs was stained using anti-HBs antibody 70-HG15 or an anti-human antibody to detect the MoMab that had been injected. (b) MoMab-staining of liver sections of WT-mice served as negative control. Scale bars represent 50 μm. (c-d) Huh-7 cells were transfected with plasmid DNA to express only HBV S- or L-protein or all three envelope proteins (SML). (c) Transfected cells were co-cultured with human T-cells engrafted with a scFv C8-based chimeric antigen receptor (S-CAR). Cell viability was followed by real-time cytotoxicity analysis for 4 days using an xCelligence device. Normalised cell index indicated that the engrafted T-cells specifically killed all HBs-expressing Huh-7 cells. (d) Transfected Huh-7 cells were co-cultured with human PBMCs in the presence of a bispecific T-cell engager antibody. Cell viability was followed on the xCelligence device for 4 days, indicating that the redirected T-cells specifically killed all HBs-expressing Huh-7 cells. (e) Evaluation of IFN-γ in the supernatant of PBMCs co-cultured with HBs-expressing Huh-7 cells by ELISA. HBs, hepatitis B surface proteins; IFN, interferon; PBMC, peripheral blood mononuclear cell

Article Snippet: To analyse the location of HBs, sections were stained with polyclonal anti-HBs antibodies such as 70-HG15 (goat; Fitzgerald, Acton, Massachusetts) and MoMab (human) and accordingly secondary antibodies were coupled to horseradish peroxidase (Thermo Fisher Scientific, Carlsbad, California).

Techniques: Membrane, Purification, Injection, Staining, Negative Control, Transfection, Plasmid Preparation, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay