Journal: Glycovirology Protocols

Article Title: The C Type Lectins DC-SIGN and L-SIGN

doi: 10.1007/978-1-59745-393-6_4

Figure Lengend Snippet: Structure of DC-SIGN and L-SIGN proteins. The C-type lectins DC-SIGN and L-SIGN are type II transmembrane proteins. Their cytoplasmic tails contain internalization signals (di-leucine, tyrosine, and tri-acidic) which are involved in internalization of the lectin. The extracellular domain is composed of a carbohydrate recognition domain (CRD) and a neck domain (conserved in the case of DC-SIGN, variable for L-SIGN) implicated in the oligomerization of these lectins. The oligomerization is probably important for the orientation and subsequently for the function of the CRDs.

Article Snippet: Phycoerythrin (PE)-conjugated mouse monoclonal antibodies (mAbs) directed against DC-SIGN (FAb161P), L-SIGN (FAb162P), or both lectins (FAb1621P) were purchased from R&D Systems.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Internalizing antibodies to the C-type lectins, L-SIGN and DC-SIGN, inhibit viral glycoprotein binding and deliver antigen to human dendritic cells for the induction of T cell responses.

doi: 10.4049/jimmunol.176.1.426

Figure Lengend Snippet: FIGURE 1. Screening of phage-displayed Fabs for L-SIGN reactivity. A, Ninety-six Fab phage clones selected from three rounds of pan- ning on recombinant DC-SIGN (negative selec- tion) and L-SIGN (positive selection) proteins were screened for binding to K562 (negative control) and K562/L-SIGN-transfected cells by flow cytometry using goat anti-mouse IgG PE conjugate for detection. Only clones showing at least a 5-fold higher binding to K562/L-SIGN cells are shown. B, To select Fab phage clones uniquely reactive with L-SIGN, but not DC- SIGN, K562/L-SIGN cell-reactive clones were screened for reactivity with DC-SIGN-Fc and L- SIGN-Fc fusion proteins in a capture ELISA. mAb162 (L-SIGN specific), mAb1621, and mAb16211 (DC-SIGN/L-SIGN cross-reactive) were used as positive controls. Data are repre- sentative of two independent experiments.

Article Snippet: Before their use in assays, RBC were lysed and any remaining dead cells were further depleted using a dead cell removal kit (Miltenyi Biotec; catalog 130-090-101), per the manufacturer’s instructions. mAbs mAb162 (reactive only with L-SIGN), mAb1621, and mAb16211 (cross-reactive with DC-SIGN and L-SIGN) were purchased from R&D Systems.

Techniques: Clone Assay, Recombinant, Selection, Binding Assay, Negative Control, Transfection, Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Internalizing antibodies to the C-type lectins, L-SIGN and DC-SIGN, inhibit viral glycoprotein binding and deliver antigen to human dendritic cells for the induction of T cell responses.

doi: 10.4049/jimmunol.176.1.426

Figure Lengend Snippet: FIGURE 2. Receptor specificity analysis of soluble L-SIGN Fabs. A, 5 105 K562, K562/DC-SIGN, or K562/L-SIGN cells were incubated with pu- rified soluble Fabs (20 g/ml) representing six unique clones for 1 h, and the extent of their binding was assessed by flow cytometry using goat anti-mouse IgG PE conjugate. Values represent mean (bars, SD) of two independent ex- periments. B, Histograms showing similar expression of SIGN molecules on K562 cells after staining with SIGN-cross-reactive mAb16211 (20 g/ml) and detecting with goat anti-mouse IgG PE conjugate.

Article Snippet: Before their use in assays, RBC were lysed and any remaining dead cells were further depleted using a dead cell removal kit (Miltenyi Biotec; catalog 130-090-101), per the manufacturer’s instructions. mAbs mAb162 (reactive only with L-SIGN), mAb1621, and mAb16211 (cross-reactive with DC-SIGN and L-SIGN) were purchased from R&D Systems.

Techniques: Incubation, Clone Assay, Binding Assay, Cytometry, Expressing, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Internalizing antibodies to the C-type lectins, L-SIGN and DC-SIGN, inhibit viral glycoprotein binding and deliver antigen to human dendritic cells for the induction of T cell responses.

doi: 10.4049/jimmunol.176.1.426

Figure Lengend Snippet: FIGURE 5. Conversion of Fab to IgG improves receptor binding and blocking of viral protein adhesion. A, K562, K562/DC-SIGN, and K562/ L-SIGN cells were incubated with purified Abs, and the extent of their binding was assessed by flow cytometry using PE-conjugated goat anti- mouse (Fab detection) or goat anti-human (IgG detection) secondary Abs. mAb162 (L-SIGN specific) and mAb16211 (DC-SIGN/L-SIGN cross-re- active) were used as positive controls. B, K562/L-SIGN and K562/DC- SIGN cells were incubated with fluorescent beads coated with HIVgp120 in the presence of Abs and assessed by flow cytometry. C, Same as in B, except that Ebola envelope glycoprotein-coated beads were used instead of HIVgp120-coated beads. mAb162 (L-SIGN specific) and mAbAZND1 (DC-SIGN specific) were used as controls for comparison. Percentage of binding is determined as 100 times the number of cells bound to protein- coated beads with Ab divided by the number of cells bound to protein- coated beads without Ab. Values represent mean (bars, SD) of four inde- pendent experiments. , Highly significant values (p 0.00005) compared with samples not treated with Abs.

Article Snippet: Before their use in assays, RBC were lysed and any remaining dead cells were further depleted using a dead cell removal kit (Miltenyi Biotec; catalog 130-090-101), per the manufacturer’s instructions. mAbs mAb162 (reactive only with L-SIGN), mAb1621, and mAb16211 (cross-reactive with DC-SIGN and L-SIGN) were purchased from R&D Systems.

Techniques: Binding Assay, Blocking Assay, Incubation, Cytometry, Comparison