Journal: Archives of biochemistry and biophysics

Article Title: Biochemical and biophysical characterization of maize-derived HBsAg for the development of an oral vaccine.

doi: 10.1016/j.abb.2015.10.015

Figure Lengend Snippet: Fig. 4. Electron microscopy of virus-like particles (VLPs). TEM images of VLPs formed by HBsAg present in the void volume fractions of gel filtration of proteins obtained from (A) full fat germ; (B) hexane-treated germ; (C) SFE-treated germ; (D) VLPs of SFE-treated germ shown at higher magnification. (E) Eluate obtained from peak 2 of the gel filtration chromatography (negative control). (F) Protein extract from non-transgenic maize material (negative control). (G) VLPs of yeast-derived HBsAg (Meridian) (positive control). VLPs/ proteins were negatively stained with 0.5% uranyl acetate and visualized by TEM.

Article Snippet: For immunogold labeling, sections were blocked in PBS containing 1% fish gelatin and 3% BSA for 30 min, and were labeled using a rabbit anti-HBsAg primary antibody (Genway#18-511-245179, 1:500 dilutions), and goat anti-rabbit IgG EM-grade 6 nm (Electron Microscopy Science #25104, 1:20 dilutions) as secondary antibody.

Techniques: Electron Microscopy, Virus, Chromatography, Negative Control, Transgenic Assay, Derivative Assay, Positive Control, Staining

Journal: Viruses

Article Title: Immunogenicity of Wild Type and Mutant Hepatitis B Surface Antigen Virus-like Particles (VLPs) in Mice with Pre-Existing Immunity against the Wild Type Vector

doi: 10.3390/v15020313

Figure Lengend Snippet: Expression and secretion of myc-tagged M1- and M15-HBsAgS proteins produced in HEK293T cells. Cells were transfected with plasmids expressing M1- or M15-HBsAgS. As a control, cells were mock-transfected. Detection of 35 S-labelled HBsAgS protein in the cell culture medium and cell lysate by immunoprecipitation with ( A ) polyclonal anti-HBsAg and anti-myc monoclonal antibodies. Protein (dash) and glycoproteins (asterisk) are indicated. ( B ) Anti-HBsAg (MonoLISA, Bio-Rad) and anti-myc ELISA using cell culture medium, n = 3, in duplicate, graphed mean and standard error of the mean (SEM). ( C ) The HBsAg-specific antigenicity of M15-HBsAgS was assessed relative to M1-HBsAgS using the Bio-Plex Assay. Antibodies directed against the N-terminal region, loop 1, loop 2, combination of both loops (Combo), C-terminal region, and conformational epitopes (Conform) were used to assess antigenic changes across the length of the modified HBsAg proteins. Antigenic reactivity between 0.5 and −0.5 is considered the normal variation of this assay; values greater or less than this are considered significant antigenic changes; values less than −3 are considered complete epitope losses.

Article Snippet: Iodoacetamide at a final concentration of 20 mM was added to the cell culture medium and diluted lysate, then incubated on ice with the mouse monoclonal anti-myc antibody (9E10, Invitrogen, Waltham, MA, USA) or polyclonal rabbit anti-HBsAgS antibodies (Meridian Life Science, Memphis, TN, USA) in the presence of protein A sepharose.

Techniques: Expressing, Produced, Transfection, Cell Culture, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Plex Assay, Modification

Journal: Viruses

Article Title: Immunogenicity of Wild Type and Mutant Hepatitis B Surface Antigen Virus-like Particles (VLPs) in Mice with Pre-Existing Immunity against the Wild Type Vector

doi: 10.3390/v15020313

Figure Lengend Snippet: Immuno-detection of NANP9-chimeric HBsAgS proteins by ( A ) immunoprecipitation and ( B ) ELISA. ( A ) 35 S-radiolabelled M1, M15, NANP9-M1, and NANP9-M15 proteins were immunoprecipitated from the cell culture supernatant (top) and cell lysate (bottom) using anti-HBsAg or anti-myc antibodies before separation via SDS-PAGE. The positions of the M1- and M15-HBsAgS proteins (dash) and M1 glycoprotein (asterisk) are indicated. ( B ) The antigenicity of the M1, M15, NANP9-M1, and NANP9-M15 VLPs was assessed by ELISA; anti-myc antibodies were used to capture VLPs; polyclonal anti-myc or anti-CSP were used to detect VLPs, n = 3 in duplicate, graphed mean, and SEM.

Article Snippet: Iodoacetamide at a final concentration of 20 mM was added to the cell culture medium and diluted lysate, then incubated on ice with the mouse monoclonal anti-myc antibody (9E10, Invitrogen, Waltham, MA, USA) or polyclonal rabbit anti-HBsAgS antibodies (Meridian Life Science, Memphis, TN, USA) in the presence of protein A sepharose.

Techniques: Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Cell Culture, SDS Page