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Results: 4

1.
FIG. 3.

FIG. 3. From: Hepatitis C Virus E2 Has Three Immunogenic Domains Containing Conformational Epitopes with Distinct Properties and Biological Functions.

Epitopes exposed on HCV glycoproteins associated with HCVpp. HEK293T cells transfected to produce HCVpp were labeled for 24 h. Supernatants (HCVpp) (A) and cell lysates (B) were immunoprecipitated with HCV HMAbs as noted.

Zhen-Yong Keck, et al. J Virol. 2004 September;78(17):9224-9232.
2.
FIG. 4.

FIG. 4. From: Hepatitis C Virus E2 Has Three Immunogenic Domains Containing Conformational Epitopes with Distinct Properties and Biological Functions.

Saturation binding of HMAbs to HCV E2. Transiently transfected 293T cells with 1a E2 were incubated with the HCV HMAbs in increasing concentrations as indicated. Staining and flow cytometry analysis were performed as described in Materials and Methods. The data points are means of two determinations and are representative of three independent experiments. Binding affinity data shown in Table 2 were analyzed using Prism software.

Zhen-Yong Keck, et al. J Virol. 2004 September;78(17):9224-9232.
3.
FIG. 1.

FIG. 1. From: Hepatitis C Virus E2 Has Three Immunogenic Domains Containing Conformational Epitopes with Distinct Properties and Biological Functions.

Competition analysis of HCV HMAbs. (A) Binding of biotinylated test antibody (as indicated on top of each panel) to HCV Q1b E2 protein captured onto GNA lectin-coated microtiter plates was competed by various concentrations of competing antibodies, as described in Materials and Methods. The bound biotinylated test antibody was detected by using alkaline phosphatase-conjugated streptavidin and p-nitrophenyl phosphate as the substrate. The y axis value (mean ± standard deviation; n = 2) is the absorbance reading obtained in the presence of competing antibody as a percentage of the reading in the absence of competing antibody. Competing antibodies were CBH-2 (▴), CBH-5 (▪), CBH-8C (♦), CBH-8E (▾), CBH-11 (•), CBH-7 (□), CBH-4G (▿), CBH-4B (▵), and R04 (*). R04 is an isotype-matched control antibody to a cytomegalovirus protein. (B) Cross-competition matrix. (C) Phylogenetic grouping of HCV HMAbs based on the competitive binding assay. Solid lines with numbers indicate the relatedness of the two adjoining antibodies. Circles are clusters of antibodies in a specific domain. Competition results used for calculation are the mean values obtained from two to five separate experiments.

Zhen-Yong Keck, et al. J Virol. 2004 September;78(17):9224-9232.
4.
FIG. 2.

FIG. 2. From: Hepatitis C Virus E2 Has Three Immunogenic Domains Containing Conformational Epitopes with Distinct Properties and Biological Functions.

Epitopes recognized by HCV HMAbs are located outside of HVR1. (A) Construct designation (on the left) showing the first amino acid sequence included in the two E2 deletion constructs employed in this study. All constructs were expressed in the pDisplay vector, which includes a heterologous signal sequence and TM domain (solid black lines) as well as epitopes recognized by MAbs to influenza virus HA (vertical bars) and c-myc (horizontal bars). HCV 1b E2 sequences are indicated in white, and strain H E2 sequences are shaded. (B) Western blot analysis showing the expression of HCV E2 proteins. Wild-type 1b and 1a (strain H) E2 proteins are shown as sf1b and sfH1a, respectively. The HVR1 1b and 1a deletions (aa 384 to 410 were deleted) are shown as pDN-411 and pDNH-411, respectively. A protein size marker is indicated in kilodaltons. (C) Reactivity of HCV HMAbs with E2 deletion constructs. HEK293 cells were mock transfected (white bars) or transfected with the HCV E2 constructs sf1b (stippled bars) or pDN-411 (black bars). Twenty-four hours posttransfection, cytoplasmic extracts were prepared and equivalent aliquots were captured onto GNA lectin-coated microtiter plates as described in Materials and Methods. The captured E2 proteins were then incubated with 10 μg of the indicated HCV HMAb/ml (x axis), and the amount of bound antibody was determined. Bars represent the mean absorbance values obtained from duplicate wells. Error bars indicate one standard deviation from the mean. (D) Same experiment as in panel C, except that HEK293 cells were transfected with sfH1a E2 (stippled bars) or pDNH-411 (black bars).

Zhen-Yong Keck, et al. J Virol. 2004 September;78(17):9224-9232.

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