pbluescript  (Addgene inc)

Journal: Journal of Virology

Article Title: Epitope Mapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients

doi: 10.1128/jvi.00453-10

Figure Lengend Snippet: FIG. 1. The epitope of ibalizumab is likely to be conformational. (A) Binding of ibalizumab to soluble hCD4, CD4-IgG2, PR14 (aa 121 to 134), or TG26 (aa 115 to 140) as measured by ELISA. (B) Com- petition for ibalizumab binding to sCD4 in solution by CD4-IgG2 or PR14 as measured by ELISA. (C) Binding titers of mouse anti-PR14 antiserum to hCD4 or PR14. (D) FACS analysis of cell surface staining by ibalizumab in control 293T cells or cells transfected with hD2- mCD4 or hCD4 plasmid DNA.

Article Snippet: Mouse CD4 (mCD4)/pVax was constructed by PCR amplification of pBS mCD4 (Addgene, Cambridge, MA) using primers 5 -CCC CCGAATTCCACCATGTGCCGAGCCATCTCTCTT-3 and 5 -CCCCCCTC GAGTCAGATGAGATTATGGCTCTT-3 , which contain the EcoRI and XhoI restriction sites.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Staining, Control, Transfection, Plasmid Preparation

Journal: Journal of Virology

Article Title: Epitope Mapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients

doi: 10.1128/jvi.00453-10

Figure Lengend Snippet: FIG. 2. Map of the ibalizumab epitope obtained by constructing and testing mouse-human chimeric CD4 molecules in binding studies using FACS. Sequence identity between mouse and human CD4 is denoted by an asterisk above the amino acid alignment. The -strands defined by Ryu et al. (24) are shown below the sequence alignment.

Article Snippet: Mouse CD4 (mCD4)/pVax was constructed by PCR amplification of pBS mCD4 (Addgene, Cambridge, MA) using primers 5 -CCC CCGAATTCCACCATGTGCCGAGCCATCTCTCTT-3 and 5 -CCCCCCTC GAGTCAGATGAGATTATGGCTCTT-3 , which contain the EcoRI and XhoI restriction sites.

Techniques: Binding Assay, Sequencing

Journal: Journal of Virology

Article Title: Epitope Mapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients

doi: 10.1128/jvi.00453-10

Figure Lengend Snippet: FIG. 3. Results of fine-mapping the ibalizumab epitope by point mutations in the chimeric mCD4-hGACFG (A) or hCD4 (B). Amino acids in regions of interest for ibalizumab binding were mutated to alanine, glycine, or the corresponding mouse amino acid as shown by single-letter amino acid symbols above the CD4 sequence alignment. Mutations with no impact on CD4 expression or ibalizumab binding are unmarked. Residues critical to ibalizumab binding are enclosed by boxes. A gray dot denotes the amino acid that partially affected ibali- zumab binding. The asterisks indicate mutations that resulted in a substantial reduction (to 25%) in CD4 expression.

Article Snippet: Mouse CD4 (mCD4)/pVax was constructed by PCR amplification of pBS mCD4 (Addgene, Cambridge, MA) using primers 5 -CCC CCGAATTCCACCATGTGCCGAGCCATCTCTCTT-3 and 5 -CCCCCCTC GAGTCAGATGAGATTATGGCTCTT-3 , which contain the EcoRI and XhoI restriction sites.

Techniques: Binding Assay, Sequencing, Expressing

Journal: Journal of Virology

Article Title: Epitope Mapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients

doi: 10.1128/jvi.00453-10

Figure Lengend Snippet: FIG. 4. Localization of ibalizumab and M-T441 epitopes on three- dimensional structures of hCD4. The epitopes of ibalizumab (pink) and M-T441 (yellow) are highlighted on CD4 (blue) complexed to the core of gp120 (brown), with two views of the structure rotated 90° around a central vertical axis (A), or space-filling model of three CD4s (blue) bound to the trimer of gp120 core (brown), before and after a conformational rearrangement (B), as derived from the cryo-electron microscopy study of Liu et al. (17). The red dot in D1 denotes F43, a critical component that binds HIV-1 Env. The parts in green highlight the V5 loop in gp120.

Article Snippet: Mouse CD4 (mCD4)/pVax was constructed by PCR amplification of pBS mCD4 (Addgene, Cambridge, MA) using primers 5 -CCC CCGAATTCCACCATGTGCCGAGCCATCTCTCTT-3 and 5 -CCCCCCTC GAGTCAGATGAGATTATGGCTCTT-3 , which contain the EcoRI and XhoI restriction sites.

Techniques: Derivative Assay, Cryo-Electron Microscopy

Journal: Journal of Virology

Article Title: Epitope Mapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients

doi: 10.1128/jvi.00453-10

Figure Lengend Snippet: FIG. 6. Results of fine-mapping of the M-T441 epitope by point muta- tions in the backbone of hD2-mCD4 (A) or hCD4 (B). Unmarked mutations have no impact on CD4 expression or M-T441 binding. Mutations enclosed by boxes substantially decreased M-T441 binding.

Article Snippet: Mouse CD4 (mCD4)/pVax was constructed by PCR amplification of pBS mCD4 (Addgene, Cambridge, MA) using primers 5 -CCC CCGAATTCCACCATGTGCCGAGCCATCTCTCTT-3 and 5 -CCCCCCTC GAGTCAGATGAGATTATGGCTCTT-3 , which contain the EcoRI and XhoI restriction sites.

Techniques: Expressing, Binding Assay