Journal:

Article Title: Neutralization of Hepatitis A Virus (HAV) by an Immunoadhesin Containing the Cysteine-Rich Region of HAV Cellular Receptor-1

doi: 10.1128/JVI.75.2.717-725.2001

Figure Lengend Snippet: Expression of D1-Fc and PVR-Fc immunoadhesins in CHO cells. (A) Schematic representation of the D1-Fc and PVR-Fc immunoadhesins. To construct D1-Fc, the cysteine-rich region of havcr-1 (D1) was tagged at its N terminus with peptide DTKDDDDK (FLAG) and fused to the hinge and Fc regions of human IgG1 (IgG1 Fc). To construct PVR-Fc, the ectodomain of PVR containing the three immunoglobulin-like domains (V1, C1, and C2) was fused to the hinge and Fc regions of human IgG1. Two identical fusion proteins are linked by disulfide bonds (dashed lines), forming homodimers, which are secreted to the cell culture medium as soluble immunoadhesins. (B) D1-Fc and PVR-Fc were purified through protein A columns. The eluted immunoadhesins were analyzed by denaturing SDS-PAGE in a 4 to 20% polyacrylamide gel and stained with Coomassie blue. The arrow points to the fully glycosylated 50-kDa form of D1-Fc. The positions of prestained molecular mass markers and their sizes in kilodaltons are shown on the right.

Article Snippet: Immunoadhesins were purified from 300 ml of harvested medium by affinity chromatography using Affi-Gel protein A-agarose columns as recommended by the manufacturer (Bio-Rad Laboratories).

Techniques: Expressing, Construct, Cell Culture, Purification, SDS Page, Staining

Journal:

Article Title: Neutralization of Hepatitis A Virus (HAV) by an Immunoadhesin Containing the Cysteine-Rich Region of HAV Cellular Receptor-1

doi: 10.1128/JVI.75.2.717-725.2001

Figure Lengend Snippet: Western blot analysis of purified D1-Fc and PVR-Fc immunoadhesins. Protein A-purified D1-Fc (lanes 1 and 3) and PVR-Fc (lanes 2 and 4) were analyzed by denaturing SDS-PAGE in a 4 to 20% polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and probed with anti-FLAG MAb M2 (lanes 1 and 2) or anti-human Fc antibodies (lanes 3 and 4). The positions of prestained molecular mass markers and their sizes in kilodaltons are shown on the left.

Article Snippet: Immunoadhesins were purified from 300 ml of harvested medium by affinity chromatography using Affi-Gel protein A-agarose columns as recommended by the manufacturer (Bio-Rad Laboratories).

Techniques: Western Blot, Purification, SDS Page

Journal:

Article Title: Neutralization of Hepatitis A Virus (HAV) by an Immunoadhesin Containing the Cysteine-Rich Region of HAV Cellular Receptor-1

doi: 10.1128/JVI.75.2.717-725.2001

Figure Lengend Snippet: Binding of HAV to immunoadhesins attached to protein A-treated beads. Different amounts of purified D1-Fc or PVR-Fc were bound to 25 μl of protein A-Trisacryl beads for 2 h at 4°C. Sucrose-purified HAV (5 × 107 TCID50) was added, and the mixture was incubated with rotation overnight at 4°C. After the mixture was washed three times with PBS at 4°C, bound HAV was eluted with 100 μl of 6 M LiCl2 for 30 min at room temperature, diluted 40-fold with EMEM, and subjected to titer determination on AGMK GL37 cell monolayers. Values are the log10 of the HAV titers determined by the Reed and Muench method (32), and the standard deviations are shown as error bars.

Article Snippet: Immunoadhesins were purified from 300 ml of harvested medium by affinity chromatography using Affi-Gel protein A-agarose columns as recommended by the manufacturer (Bio-Rad Laboratories).

Techniques: Binding Assay, Purification, Incubation

Journal:

Article Title: Neutralization of Hepatitis A Virus (HAV) by an Immunoadhesin Containing the Cysteine-Rich Region of HAV Cellular Receptor-1

doi: 10.1128/JVI.75.2.717-725.2001

Figure Lengend Snippet: Inhibition of binding of HAV to D1-Fc by protective MAb 190/4. Equal amounts (3 μg) of D1-Fc and PVR-Fc were treated with 0, 0.5, 5, or 50 μg of MAb 190/4 or control MAb M2 at 4°C. Protein A-Trisacryl beads (25 μl) were added, and the mixture was incubated for 2 h at 4°C. Sucrose-purified HAV (5 × 107 TCID50) was added, and the mixture was incubated with rotation overnight at 4°C. HAV was eluted and subjected to titer determination as described in the legend to Fig. ​Fig.5.5. Values are the log10 of the HAV titers determined by the Reed and Muench method (32), and the standard deviations are shown as error bars.

Article Snippet: Immunoadhesins were purified from 300 ml of harvested medium by affinity chromatography using Affi-Gel protein A-agarose columns as recommended by the manufacturer (Bio-Rad Laboratories).

Techniques: Inhibition, Binding Assay, Incubation, Purification

Journal:

Article Title: Neutralization of Hepatitis A Virus (HAV) by an Immunoadhesin Containing the Cysteine-Rich Region of HAV Cellular Receptor-1

doi: 10.1128/JVI.75.2.717-725.2001

Figure Lengend Snippet: Neutralization of HAV by MAbs. HAV PI stock (105 TCID50) was treated with 50 μg of protein A-purified MAbs K2-4F2, K3-4C8, and VHA 813 for 1 h at 37°C. Tenfold dilutions of the neutralization reaction products were subjected to titer determination on 96-well plates containing confluent monolayers of AGMK GL37 cells. After 4 h of adsorption at 37°C, the plates were washed three times and incubated for 10 days at 35°C under CO2. HAV titers were determined by ELISA. Values are the log10 of the HAV titers calculated by the Reed and Muench method (32), and the standard deviations are shown as error bars.

Article Snippet: Immunoadhesins were purified from 300 ml of harvested medium by affinity chromatography using Affi-Gel protein A-agarose columns as recommended by the manufacturer (Bio-Rad Laboratories).

Techniques: Neutralization, Purification, Adsorption, Incubation, Enzyme-linked Immunosorbent Assay