Structured Review

Affibody ad5 vector
Reported Interactions of <t>Ad5</t> with Blood Components In Vivo . 1. Ad5 binding to CAR-expressing erythrocytes (species-specific expression of CAR) can cause trapping of virus in the circulation [ 81 , 82 ]. In the presence of antibody and complement, Ad5 can bind human erythrocytes via CR-1 [ 81 ]. 2. Opsonization of Ad5 with natural IgM and/or complement promotes KC uptake via complement receptor-3 (CR-3) or Fc Receptor [ 83 ]. 3. Ad interactions with T-cells [ 84 ]. 4. FX binding to the Ad5 hexon promotes hepatocyte entry through HSPGs [ 66 ]. 5. FIX/C4BP binding to the fiber knob has been proposed to mediate hepatocyte entry via HSPGs or LRP, and has been suggested to direct KC uptake [ 65 ]. 6. Ad binding to platelets has been shown to enhance uptake by KCs [ 79 ]. Von Willebrand factor (vWF) and P-selectin have been associated with the formation of activated platelet-leukocyte aggregates which are cleared by scavenging macrophages [ 85 ].
Ad5 Vector, supplied by Affibody, used in various techniques. Bioz Stars score: 86/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors"

Article Title: Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

Journal: Viruses

doi: 10.3390/v2102290

Reported Interactions of Ad5 with Blood Components In Vivo . 1. Ad5 binding to CAR-expressing erythrocytes (species-specific expression of CAR) can cause trapping of virus in the circulation [ 81 , 82 ]. In the presence of antibody and complement, Ad5 can bind human erythrocytes via CR-1 [ 81 ]. 2. Opsonization of Ad5 with natural IgM and/or complement promotes KC uptake via complement receptor-3 (CR-3) or Fc Receptor [ 83 ]. 3. Ad interactions with T-cells [ 84 ]. 4. FX binding to the Ad5 hexon promotes hepatocyte entry through HSPGs [ 66 ]. 5. FIX/C4BP binding to the fiber knob has been proposed to mediate hepatocyte entry via HSPGs or LRP, and has been suggested to direct KC uptake [ 65 ]. 6. Ad binding to platelets has been shown to enhance uptake by KCs [ 79 ]. Von Willebrand factor (vWF) and P-selectin have been associated with the formation of activated platelet-leukocyte aggregates which are cleared by scavenging macrophages [ 85 ].
Figure Legend Snippet: Reported Interactions of Ad5 with Blood Components In Vivo . 1. Ad5 binding to CAR-expressing erythrocytes (species-specific expression of CAR) can cause trapping of virus in the circulation [ 81 , 82 ]. In the presence of antibody and complement, Ad5 can bind human erythrocytes via CR-1 [ 81 ]. 2. Opsonization of Ad5 with natural IgM and/or complement promotes KC uptake via complement receptor-3 (CR-3) or Fc Receptor [ 83 ]. 3. Ad interactions with T-cells [ 84 ]. 4. FX binding to the Ad5 hexon promotes hepatocyte entry through HSPGs [ 66 ]. 5. FIX/C4BP binding to the fiber knob has been proposed to mediate hepatocyte entry via HSPGs or LRP, and has been suggested to direct KC uptake [ 65 ]. 6. Ad binding to platelets has been shown to enhance uptake by KCs [ 79 ]. Von Willebrand factor (vWF) and P-selectin have been associated with the formation of activated platelet-leukocyte aggregates which are cleared by scavenging macrophages [ 85 ].

Techniques Used: In Vivo, Binding Assay, Expressing

In Vitro Entry Pathway of Ad5. 1. Ad5 attachment is mediated by binding of the fiber knob to the 46 kDa transmembrane receptor CAR [ 26 – 32 ]. 2. An interaction between the RGD motif with the penton base triggers internalization by clathrin-mediated endocytosis, via ανβ3/5 integrins [ 33 ]. 3. Partial disassembly of the capsid is induced upon acidification of the endosome [ 43 ]. Endosomal escape is modulated through the lytic action of protein VI [ 45 ]. 4. The nucleocapsid-hexon core is translocated to the nuclear pore complex (NPC) along the microtubule network using the microtubule-associated motor, dynein [ 46 , 47 ]. 5. The capsid undergoes its final dissociation event at the nuclear pore complex [ 47 ], allowing the core DNA to extrude into the nucleus for subsequent transcription and replication [ 48 ].
Figure Legend Snippet: In Vitro Entry Pathway of Ad5. 1. Ad5 attachment is mediated by binding of the fiber knob to the 46 kDa transmembrane receptor CAR [ 26 – 32 ]. 2. An interaction between the RGD motif with the penton base triggers internalization by clathrin-mediated endocytosis, via ανβ3/5 integrins [ 33 ]. 3. Partial disassembly of the capsid is induced upon acidification of the endosome [ 43 ]. Endosomal escape is modulated through the lytic action of protein VI [ 45 ]. 4. The nucleocapsid-hexon core is translocated to the nuclear pore complex (NPC) along the microtubule network using the microtubule-associated motor, dynein [ 46 , 47 ]. 5. The capsid undergoes its final dissociation event at the nuclear pore complex [ 47 ], allowing the core DNA to extrude into the nucleus for subsequent transcription and replication [ 48 ].

Techniques Used: In Vitro, Binding Assay

2) Product Images from "Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies"

Article Title: Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies

Journal: Molecular Biotechnology

doi: 10.1007/s12033-010-9367-1

Affibody molecule ribosome display system design . Schematic overview of the constructed ribosome display system. a An expression cassette was assembled in a vector denoted pRD, containing a T7 promoter, a Shine Dalgarno sequence and a FLAG epitope encoding sequence, followed by a cloning window and a sequence encoding a bipartite spacer element containing a 88-residue phage M13 protein 3 sequence (p3 spacer) followed by a 21-residue element derived from the E. coli SecM. The Arg residue ( in brackets ) indicates the extension of the SecM motif by this codon during PCR amplification prior to transcription; b , c for initial enrichment studies, gene fragments encoding either Z WT -ABD or Z Taq proteins were inserted in the expression cassette as indicated; d for affibody molecule library construction, two oligonucleotides (Zlib-for and Zlib-rev) encoding randomized helices one and two of the three-helix bundle scaffold were extended together utilizing mutually overlapping sequences for annealing, followed by cloning into the expression cassette where the extension products replaced a dummy fragment. A gene fragment encoding helix three was here already present in the pRD vector. Restriction sites and annealing sites for some of the oligonucleotide primers used during cloning, reverse transcription and PCR work are indicated (see text for details)
Figure Legend Snippet: Affibody molecule ribosome display system design . Schematic overview of the constructed ribosome display system. a An expression cassette was assembled in a vector denoted pRD, containing a T7 promoter, a Shine Dalgarno sequence and a FLAG epitope encoding sequence, followed by a cloning window and a sequence encoding a bipartite spacer element containing a 88-residue phage M13 protein 3 sequence (p3 spacer) followed by a 21-residue element derived from the E. coli SecM. The Arg residue ( in brackets ) indicates the extension of the SecM motif by this codon during PCR amplification prior to transcription; b , c for initial enrichment studies, gene fragments encoding either Z WT -ABD or Z Taq proteins were inserted in the expression cassette as indicated; d for affibody molecule library construction, two oligonucleotides (Zlib-for and Zlib-rev) encoding randomized helices one and two of the three-helix bundle scaffold were extended together utilizing mutually overlapping sequences for annealing, followed by cloning into the expression cassette where the extension products replaced a dummy fragment. A gene fragment encoding helix three was here already present in the pRD vector. Restriction sites and annealing sites for some of the oligonucleotide primers used during cloning, reverse transcription and PCR work are indicated (see text for details)

Techniques Used: Construct, Expressing, Plasmid Preparation, Sequencing, FLAG-tag, Clone Assay, Derivative Assay, Polymerase Chain Reaction, Amplification

Results from initial enrichment experiments . Agarose gel electrophoresis analysis of PCR products obtained using DNA templates produced after reverse transcription of mRNA recovered after different numbers of rounds of IgG affinity selection of ternary complexes containing Z WT -ABD proteins. Lane 1 : marker DNA; lane 2 : PCR product obtained after reverse transcription and amplification of an initial 1:1000 mixture of Z WT -ABD mRNA in a background of Z Taq mRNA; lane 3 : PCR product obtained after a first round of enrichment; lane 4 : PCR product obtained after a second round of enrichment. Arrows indicate marker DNA bands and the expected sizes for amplicons corresponding to amplification of Z WT -ABD and Z Taq constructs, respectively
Figure Legend Snippet: Results from initial enrichment experiments . Agarose gel electrophoresis analysis of PCR products obtained using DNA templates produced after reverse transcription of mRNA recovered after different numbers of rounds of IgG affinity selection of ternary complexes containing Z WT -ABD proteins. Lane 1 : marker DNA; lane 2 : PCR product obtained after reverse transcription and amplification of an initial 1:1000 mixture of Z WT -ABD mRNA in a background of Z Taq mRNA; lane 3 : PCR product obtained after a first round of enrichment; lane 4 : PCR product obtained after a second round of enrichment. Arrows indicate marker DNA bands and the expected sizes for amplicons corresponding to amplification of Z WT -ABD and Z Taq constructs, respectively

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Produced, Selection, Marker, Amplification, Construct

3) Product Images from "Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity"

Article Title: Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity

Journal: Scientific Reports

doi: 10.1038/srep07518

Alanine scanning of first-generation VEGFR2-specific Affibody molecules. (a) Z VEGFR2_1 (b) Z VEGFR2_2 . The 13 residues in the VEGFR2-binding Affibody molecules that were substituted with alanine are represented on the X axis, and the fold change in normalized binding signal (a ratio of FL-1 fluorescence intensity, corresponding to VEGFR2 binding, and FL-6 fluorescence intensity, corresponding to surface expression level) compared to the corresponding non-mutated binder (Z VEGFR2_1 or Z VEGFR2_2 ) is represented on the Y axis. Binding to human (green bars) or murine (purple bars) VEGFR2 is shown. The error bars show the standard deviation of two independent experiments.
Figure Legend Snippet: Alanine scanning of first-generation VEGFR2-specific Affibody molecules. (a) Z VEGFR2_1 (b) Z VEGFR2_2 . The 13 residues in the VEGFR2-binding Affibody molecules that were substituted with alanine are represented on the X axis, and the fold change in normalized binding signal (a ratio of FL-1 fluorescence intensity, corresponding to VEGFR2 binding, and FL-6 fluorescence intensity, corresponding to surface expression level) compared to the corresponding non-mutated binder (Z VEGFR2_1 or Z VEGFR2_2 ) is represented on the Y axis. Binding to human (green bars) or murine (purple bars) VEGFR2 is shown. The error bars show the standard deviation of two independent experiments.

Techniques Used: Binding Assay, Fluorescence, Expressing, Standard Deviation

Characterization of VEGFR2-binding Affibody molecules from phage display selection. (a) Sensorgrams from SPR analysis of Z VEGFR2_1 and Z VEGFR2_2 binding to immobilized human VEGFR2. Affibody molecules were injected at concentrations ranging from 50 nM to 500 nM. Data is double referenced by subtraction of simultaneous responses from reference surface and a buffer injection. The experiment was performed in duplicates. (b) Representative results from SPR-based competition assay. Sensorgrams were obtained from a double injection, where a first injection of 1 μM of Z VEGFR2_1 (I) was immediately followed by a second injection (II) of either a combination of 1 μM of Z VEGFR2_1 and 1 μM of Z VEGFR2_2 , or 2 μM of Z VEGFR2_1 , over immobilized human VEGFR2. The experiment was performed in duplicates. (c) Representative results from SPR-based analysis of human VEGF blocking. 40 nM of human VEGFR2, which had been pre-incubated for 40 min with a 25 × molar excess of Z VEGFR2_1 , Z VEGFR2_2 or PBS (control), was injected over a surface of immobilized human VEGF-A. The experiment was performed in duplicates.
Figure Legend Snippet: Characterization of VEGFR2-binding Affibody molecules from phage display selection. (a) Sensorgrams from SPR analysis of Z VEGFR2_1 and Z VEGFR2_2 binding to immobilized human VEGFR2. Affibody molecules were injected at concentrations ranging from 50 nM to 500 nM. Data is double referenced by subtraction of simultaneous responses from reference surface and a buffer injection. The experiment was performed in duplicates. (b) Representative results from SPR-based competition assay. Sensorgrams were obtained from a double injection, where a first injection of 1 μM of Z VEGFR2_1 (I) was immediately followed by a second injection (II) of either a combination of 1 μM of Z VEGFR2_1 and 1 μM of Z VEGFR2_2 , or 2 μM of Z VEGFR2_1 , over immobilized human VEGFR2. The experiment was performed in duplicates. (c) Representative results from SPR-based analysis of human VEGF blocking. 40 nM of human VEGFR2, which had been pre-incubated for 40 min with a 25 × molar excess of Z VEGFR2_1 , Z VEGFR2_2 or PBS (control), was injected over a surface of immobilized human VEGF-A. The experiment was performed in duplicates.

Techniques Used: Binding Assay, Selection, SPR Assay, Injection, Competitive Binding Assay, Blocking Assay, Incubation

Sorting of the affinity maturation libraries. (a) Z VEGFR2_1matlib (b) Z VEGFR2_2matlib displayed on staphylococcal cells. The VEGFR2 binding signal (monitored by the binding of fluorescently labeled anti-Fc antibody to human VEGFR2/Fc) is represented on the Y axis and the surface expression level (monitored by fluorescently labeled HSA binding) is represented on the X axis. The dot plots show staphylococcal cell populations from the original unsorted library as well as cell populations isolated in the 1 st , 2 nd , 3 rd and 4 th selection round, respectively. For the 4 th selection round, dot plots are shown for selections including an off-rate experiment of 0 min, 30 min or 4.5 h.
Figure Legend Snippet: Sorting of the affinity maturation libraries. (a) Z VEGFR2_1matlib (b) Z VEGFR2_2matlib displayed on staphylococcal cells. The VEGFR2 binding signal (monitored by the binding of fluorescently labeled anti-Fc antibody to human VEGFR2/Fc) is represented on the Y axis and the surface expression level (monitored by fluorescently labeled HSA binding) is represented on the X axis. The dot plots show staphylococcal cell populations from the original unsorted library as well as cell populations isolated in the 1 st , 2 nd , 3 rd and 4 th selection round, respectively. For the 4 th selection round, dot plots are shown for selections including an off-rate experiment of 0 min, 30 min or 4.5 h.

Techniques Used: Binding Assay, Labeling, Expressing, Isolation, Selection

Characterization of affinity matured VEGFR2-binding Affibody molecules selected by staphylococcal display. (a) Representative sensorgrams from SPR analysis of affinity-matured Affibody molecules (Z VEGFR2_22 and Z VEGFR2_40 ) binding to immobilized VEGFR2, showing the response signal. Affibody molecules were injected at concentrations of 5, 10 and 20 nM. Data is referenced by subtraction of simultaneous responses from reference surface. The experiment was performed in duplicates. (b) Representative results from SPR-based competition assay. Sensorgrams were obtained from a double injection, where 1 μM of Z VEGFR2_22 was injected (1), immediately followed by a second injection (2) of either a combination of 1 μM of Z VEGFR2_22 and 1 μM of Z VEGFR2_40 , or 2 μM of Z VEGFR2_22 , over immobilized human or murine VEGFR2. The experiment was performed in duplicates. (c) Representative results from SPR-based assay of VEGF blocking. 40 nM of human VEGFR2, which had been pre-incubated for 40 min with a 25 × molar excess of Z VEGFR2_22 , was injected over a surface of immobilized human VEGF-A. The experiment was performed in duplicates.
Figure Legend Snippet: Characterization of affinity matured VEGFR2-binding Affibody molecules selected by staphylococcal display. (a) Representative sensorgrams from SPR analysis of affinity-matured Affibody molecules (Z VEGFR2_22 and Z VEGFR2_40 ) binding to immobilized VEGFR2, showing the response signal. Affibody molecules were injected at concentrations of 5, 10 and 20 nM. Data is referenced by subtraction of simultaneous responses from reference surface. The experiment was performed in duplicates. (b) Representative results from SPR-based competition assay. Sensorgrams were obtained from a double injection, where 1 μM of Z VEGFR2_22 was injected (1), immediately followed by a second injection (2) of either a combination of 1 μM of Z VEGFR2_22 and 1 μM of Z VEGFR2_40 , or 2 μM of Z VEGFR2_22 , over immobilized human or murine VEGFR2. The experiment was performed in duplicates. (c) Representative results from SPR-based assay of VEGF blocking. 40 nM of human VEGFR2, which had been pre-incubated for 40 min with a 25 × molar excess of Z VEGFR2_22 , was injected over a surface of immobilized human VEGF-A. The experiment was performed in duplicates.

Techniques Used: Binding Assay, SPR Assay, Injection, Competitive Binding Assay, Blocking Assay, Incubation

Characterization of biparatopic Affibody constructs. (a) Schematic overview of the design of the dimeric constructs. (b) Schematic overview of SPR-based off rate analysis assay. HSA was immobilized on the chip surface. A first injection of dimeric Affibody constructs resulted in a negligible off rate due to the femtomolar affinity of ABD for HSA. VEGFR2 binding was analyzed by subsequent injection of monomeric VEGFR2. The experiments were performed in duplicates. (c) Representative sensorgrams obtained from the SPR-based off-rate analysis assay, showing the injection of 40 nM monomeric human VEGFR2 over each of the four dimeric Affibody molecules. Data is double referenced by subtraction of simultaneous responses from reference surface and a buffer injection. (d) Flow-cytometric analysis of binding of dimeric Affibody constructs to VEGFR2-expressing 293/KDR cells. Binding of the Affibody constructs is monitored by the binding of fluorescently labeled HSA to the ABD tag. A higher shift in mean log fluorescence intensity compared to the negative control construct Z Taq -ABD-Z Taq or cells labeled with secondary reagent only was observed for the heterodimeric constructs than for the homodimeric constructs upon binding to VEGFR2-expressing cells. The experiment was performed in duplicates. (e) Inhibition of VEGF-A induced phosphorylation of VEGFR2 on 293/KDR cells. Cells were pre-treated with the biparatopic binder Z VEGFR2_22 -(S 4 G) 4 -ABD 035 -(S 4 G) 4 -Z VEGFR2_40 , a combination of 30 nM or 3 nM of each of the monomeric binders Z VEGFR2_22 and Z VEGFR2_40 , 30 nM or 3 nM of the negative control construct Z Taq -ABD 035 -Z Taq , or PBS, followed by stimulation with VEGF-A. VEGFR2 phosphorylation was determined by ELISA. The biparatopic binder and the combination of monomers both resulted in a decrease in phosphorylation level compared to the controls, and a more potent inhibition was observed for the biparatopic binder. The data is presented as the OD450 for each sample normalized against the OD450 of untreated cells. The experiment was performed in duplicates.
Figure Legend Snippet: Characterization of biparatopic Affibody constructs. (a) Schematic overview of the design of the dimeric constructs. (b) Schematic overview of SPR-based off rate analysis assay. HSA was immobilized on the chip surface. A first injection of dimeric Affibody constructs resulted in a negligible off rate due to the femtomolar affinity of ABD for HSA. VEGFR2 binding was analyzed by subsequent injection of monomeric VEGFR2. The experiments were performed in duplicates. (c) Representative sensorgrams obtained from the SPR-based off-rate analysis assay, showing the injection of 40 nM monomeric human VEGFR2 over each of the four dimeric Affibody molecules. Data is double referenced by subtraction of simultaneous responses from reference surface and a buffer injection. (d) Flow-cytometric analysis of binding of dimeric Affibody constructs to VEGFR2-expressing 293/KDR cells. Binding of the Affibody constructs is monitored by the binding of fluorescently labeled HSA to the ABD tag. A higher shift in mean log fluorescence intensity compared to the negative control construct Z Taq -ABD-Z Taq or cells labeled with secondary reagent only was observed for the heterodimeric constructs than for the homodimeric constructs upon binding to VEGFR2-expressing cells. The experiment was performed in duplicates. (e) Inhibition of VEGF-A induced phosphorylation of VEGFR2 on 293/KDR cells. Cells were pre-treated with the biparatopic binder Z VEGFR2_22 -(S 4 G) 4 -ABD 035 -(S 4 G) 4 -Z VEGFR2_40 , a combination of 30 nM or 3 nM of each of the monomeric binders Z VEGFR2_22 and Z VEGFR2_40 , 30 nM or 3 nM of the negative control construct Z Taq -ABD 035 -Z Taq , or PBS, followed by stimulation with VEGF-A. VEGFR2 phosphorylation was determined by ELISA. The biparatopic binder and the combination of monomers both resulted in a decrease in phosphorylation level compared to the controls, and a more potent inhibition was observed for the biparatopic binder. The data is presented as the OD450 for each sample normalized against the OD450 of untreated cells. The experiment was performed in duplicates.

Techniques Used: Construct, SPR Assay, Chromatin Immunoprecipitation, Injection, Binding Assay, Flow Cytometry, Expressing, Labeling, Fluorescence, Negative Control, Inhibition, Enzyme-linked Immunosorbent Assay

4) Product Images from "Cellular Effects of HER3-Specific Affibody Molecules"

Article Title: Cellular Effects of HER3-Specific Affibody Molecules

Journal: PLoS ONE

doi: 10.1371/journal.pone.0040023

Analysis of cellular growth inhibitory effects of the HER3-specific Affibody molecules. Mean absorbance values at 450 nm ± SD, which is proportional to the number of living cells, is given on the y-axis. A. Proliferation of MCF-7 and SKBR-3 cells grown in a dilution series of HRG. B. Proliferation of MCF-7 and SKBR-3 cells grown in 40 pM HRG and a dilution series of Affibody molecules Z05416, Z05417 or ZTaq. C. Proliferation of cells grown in medium containing 40 nM Affibody molecules, 0.04 nM HRG. Results are compared to unstimulated cells (no Affibody molecules or HRG added).
Figure Legend Snippet: Analysis of cellular growth inhibitory effects of the HER3-specific Affibody molecules. Mean absorbance values at 450 nm ± SD, which is proportional to the number of living cells, is given on the y-axis. A. Proliferation of MCF-7 and SKBR-3 cells grown in a dilution series of HRG. B. Proliferation of MCF-7 and SKBR-3 cells grown in 40 pM HRG and a dilution series of Affibody molecules Z05416, Z05417 or ZTaq. C. Proliferation of cells grown in medium containing 40 nM Affibody molecules, 0.04 nM HRG. Results are compared to unstimulated cells (no Affibody molecules or HRG added).

Techniques Used:

Immunofluorescent staining of human cancer cell lines. Images showing AU565, SKBR-3, MCF-7 and SKOV-3 cells stained with HER3-specific Affibody molecules Z05416 and Z05417, respectively. The polyclonal anti-HER3 antibody A234 and ZTaq were used as positive and negative staining controls, respectively. Affibody molecules and antibodies binding to cells are shown in green while nuclear staining by DAPI is given in blue. AU565 and SKOV-3 images were acquired on the same day using the same detection gain and laser power, enabling comparison between staining intensities. Cell staining of MCF-7 and SKBR-3 was analysed on different days, using different detection gains for optimal image acquisition. Additionally, MCF-7 images were acquired using increased laser power.
Figure Legend Snippet: Immunofluorescent staining of human cancer cell lines. Images showing AU565, SKBR-3, MCF-7 and SKOV-3 cells stained with HER3-specific Affibody molecules Z05416 and Z05417, respectively. The polyclonal anti-HER3 antibody A234 and ZTaq were used as positive and negative staining controls, respectively. Affibody molecules and antibodies binding to cells are shown in green while nuclear staining by DAPI is given in blue. AU565 and SKOV-3 images were acquired on the same day using the same detection gain and laser power, enabling comparison between staining intensities. Cell staining of MCF-7 and SKBR-3 was analysed on different days, using different detection gains for optimal image acquisition. Additionally, MCF-7 images were acquired using increased laser power.

Techniques Used: Staining, Negative Staining, Binding Assay

5) Product Images from "Affibody-Mediated Sequestration of Amyloid β Demonstrates Preventive Efficacy in a Transgenic Alzheimer’s Disease Mouse Model"

Article Title: Affibody-Mediated Sequestration of Amyloid β Demonstrates Preventive Efficacy in a Transgenic Alzheimer’s Disease Mouse Model

Journal: Frontiers in Aging Neuroscience

doi: 10.3389/fnagi.2019.00064

Semi-quantitative analysis of cerebral microhemorrhages. Representative brain sections demonstrating the degree of cerebral microhemorrhages in from (ZTaq)2-ABD (A) control or ZSYM73-ABD (B) treated mice by staining with Perl’s stain for ferric iron in hemosiderin. Semi-quantitation of iron positive profiles is shown in (C) ( p = 0.29, one-tailed t -test). Scale bar (50 μm).
Figure Legend Snippet: Semi-quantitative analysis of cerebral microhemorrhages. Representative brain sections demonstrating the degree of cerebral microhemorrhages in from (ZTaq)2-ABD (A) control or ZSYM73-ABD (B) treated mice by staining with Perl’s stain for ferric iron in hemosiderin. Semi-quantitation of iron positive profiles is shown in (C) ( p = 0.29, one-tailed t -test). Scale bar (50 μm).

Techniques Used: Mouse Assay, Staining, Quantitation Assay, One-tailed Test

6) Product Images from "Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake"

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

Journal: EJNMMI Research

doi: 10.1186/s13550-016-0213-8

PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

Techniques Used: Positron Emission Tomography

PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

Techniques Used: Positron Emission Tomography

7) Product Images from "Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies"

Article Title: Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies

Journal: Molecular Biotechnology

doi: 10.1007/s12033-010-9367-1

Results from initial enrichment experiments . Agarose gel electrophoresis analysis of PCR products obtained using DNA templates produced after reverse transcription of mRNA recovered after different numbers of rounds of IgG affinity selection of ternary complexes containing Z WT -ABD proteins. Lane 1 : marker DNA; lane 2 : PCR product obtained after reverse transcription and amplification of an initial 1:1000 mixture of Z WT -ABD mRNA in a background of Z Taq mRNA; lane 3 : PCR product obtained after a first round of enrichment; lane 4 : PCR product obtained after a second round of enrichment. Arrows indicate marker DNA bands and the expected sizes for amplicons corresponding to amplification of Z WT -ABD and Z Taq constructs, respectively
Figure Legend Snippet: Results from initial enrichment experiments . Agarose gel electrophoresis analysis of PCR products obtained using DNA templates produced after reverse transcription of mRNA recovered after different numbers of rounds of IgG affinity selection of ternary complexes containing Z WT -ABD proteins. Lane 1 : marker DNA; lane 2 : PCR product obtained after reverse transcription and amplification of an initial 1:1000 mixture of Z WT -ABD mRNA in a background of Z Taq mRNA; lane 3 : PCR product obtained after a first round of enrichment; lane 4 : PCR product obtained after a second round of enrichment. Arrows indicate marker DNA bands and the expected sizes for amplicons corresponding to amplification of Z WT -ABD and Z Taq constructs, respectively

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Produced, Selection, Marker, Amplification, Construct

8) Product Images from "Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake"

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

Journal: EJNMMI Research

doi: 10.1186/s13550-016-0213-8

PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

Techniques Used: Positron Emission Tomography

PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

Techniques Used: Positron Emission Tomography

9) Product Images from "Staphylococcal Surface Display of Immunoglobulin A (IgA)- and IgE-Specific In Vitro-Selected Binding Proteins (Affibodies) Based on Staphylococcus aureus Protein A"

Article Title: Staphylococcal Surface Display of Immunoglobulin A (IgA)- and IgE-Specific In Vitro-Selected Binding Proteins (Affibodies) Based on Staphylococcus aureus Protein A

Journal: Applied and Environmental Microbiology

doi:

Expression vectors developed for surface display in S. carnosus ), suitable for surface display in S. carnosus , with the processed gene fusion product PP-ABP-XM′ illustrated as anchored to the cell surface. (B and C) Expression cassettes of the surface expression vectors encoding the chimeric surface proteins containing the monomeric and dimeric versions of the Z IgA affibody, respectively, with their gene fusion products anchored to the S. carnosus cell surface. (D and E) Expression cassettes of the surface expression vectors encoding the chimeric surface proteins containing the monomeric and dimeric versions of the Z IgE affibody, respectively, with their cell surface-anchored gene products. The names of the constructed expression vectors are given below the expression cassettes, the molecular sizes of the encoded surface proteins are indicated in kilodaltons, and the abbreviated names of the recombinant staphylococci are given at the right.
Figure Legend Snippet: Expression vectors developed for surface display in S. carnosus ), suitable for surface display in S. carnosus , with the processed gene fusion product PP-ABP-XM′ illustrated as anchored to the cell surface. (B and C) Expression cassettes of the surface expression vectors encoding the chimeric surface proteins containing the monomeric and dimeric versions of the Z IgA affibody, respectively, with their gene fusion products anchored to the S. carnosus cell surface. (D and E) Expression cassettes of the surface expression vectors encoding the chimeric surface proteins containing the monomeric and dimeric versions of the Z IgE affibody, respectively, with their cell surface-anchored gene products. The names of the constructed expression vectors are given below the expression cassettes, the molecular sizes of the encoded surface proteins are indicated in kilodaltons, and the abbreviated names of the recombinant staphylococci are given at the right.

Techniques Used: Expressing, Construct, Recombinant

10) Product Images from "HER2-Affitoxin: A Potent Therapeutic Agent for the Treatment of HER2-Overexpressing Tumors"

Article Title: HER2-Affitoxin: A Potent Therapeutic Agent for the Treatment of HER2-Overexpressing Tumors

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

doi: 10.1158/1078-0432.CCR-10-2887

Electropherogram of purified Taq-Affitoxin and HER2-Affitoxin (A) and their efficacy in eliminating NCI-N87 cells (B and C).
Figure Legend Snippet: Electropherogram of purified Taq-Affitoxin and HER2-Affitoxin (A) and their efficacy in eliminating NCI-N87 cells (B and C).

Techniques Used: Purification

Efficacy of HER2-Affitoxin and Taq-Affitoxin in subcutaneous xenografts.
Figure Legend Snippet: Efficacy of HER2-Affitoxin and Taq-Affitoxin in subcutaneous xenografts.

Techniques Used:

11) Product Images from "Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake"

Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

Journal: EJNMMI Research

doi: 10.1186/s13550-016-0213-8

PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

Techniques Used: Positron Emission Tomography

PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used
Figure Legend Snippet: PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

Techniques Used: Positron Emission Tomography

Related Articles

Amplification:

Article Title: Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
Article Snippet: Ribosome Display System Design and Evaluation A plasmid construct denoted pRD was constructed to use as assembly vector and PCR amplification template of linear expression cassettes for RD-mediated selection of binding proteins from libraries of affibody molecules (Fig. a). .. For system evaluation, test enrichment experiments were performed involving an mRNA mixture, prepared from mRNA obtained after in vitro transcription using PCR product expression cassettes encoding the model proteins ZWT -ABD (IgG and HSA binding fusion protein; Fig. b) or ZTaq (affibody molecule binding to Taq DNA polymerase [ ]; Fig. c) fused to the p3-SecM spacer.

Positive Control:

Article Title: Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity
Article Snippet: .. The positive control antibody confirmed that VEGFR2 was present at the cell surface of 293/KDR but not on HEK293 cells ( ). .. Incubation with heterodimeric constructs resulted in higher fluorescence signal intensity compared to the homodimeric constructs, confirming that the heterodimeric constructs had higher affinities for VEGFR2 also expressed on the cell surface.

Cytometry:

Article Title: Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity
Article Snippet: .. The 26 mutants were expressed and displayed on the surface of staphylococci and binding to VEGFR2 was analyzed by flow cytometry ( ). ..

Construct:

Article Title: Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity
Article Snippet: .. The slower dissociation rates for the heterodimeric constructs verified that ZVEGFR2_22 and ZVEGFR2_40 could bind simultaneously to VEGFR2 also when linked together in one fusion protein. ..

Article Title: Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
Article Snippet: Ribosome Display System Design and Evaluation A plasmid construct denoted pRD was constructed to use as assembly vector and PCR amplification template of linear expression cassettes for RD-mediated selection of binding proteins from libraries of affibody molecules (Fig. a). .. For system evaluation, test enrichment experiments were performed involving an mRNA mixture, prepared from mRNA obtained after in vitro transcription using PCR product expression cassettes encoding the model proteins ZWT -ABD (IgG and HSA binding fusion protein; Fig. b) or ZTaq (affibody molecule binding to Taq DNA polymerase [ ]; Fig. c) fused to the p3-SecM spacer.

Incubation:

Article Title: Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity
Article Snippet: .. The bacterial displayed library was incubated with 50 nM of labeled VEGFR2, followed by washing and incubation for 30 min or 4.5 h with an excess of unlabeled VEGFR2 to minimize rebinding of dissociated labeled target. .. In addition to the off-rate selection in the last round, the selection stringency was also increased throughout the four sorting rounds by using more stringent gating and sorting parameters.

Activity Assay:

Article Title: Pretargeted Imaging and Therapy
Article Snippet: Honarvar et al. demonstrated the efficacy of Affibody molecule–based pretargeting using ZHER2 -HP1 and 111 In-labeled HP2 in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts ( ) ( ). .. The pretargeting strategy produced tumoral uptake of 19 ± 2 %ID/g 1 h after injection along with a 54 ± 19 tumor-to-blood activity concentration ratio at the same time point.

Expressing:

Article Title: Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
Article Snippet: .. For system evaluation, test enrichment experiments were performed involving an mRNA mixture, prepared from mRNA obtained after in vitro transcription using PCR product expression cassettes encoding the model proteins ZWT -ABD (IgG and HSA binding fusion protein; Fig. b) or ZTaq (affibody molecule binding to Taq DNA polymerase [ ]; Fig. c) fused to the p3-SecM spacer. .. Two rounds of RD selection were performed using human IgG-coated beads for enrichment of the rare IgG-binding ZWT -ABD encoding constructs present in an initial 1000-fold molar excess of Taq DNA polymerase binder encoding control constructs, and the results monitored via analysis of PCR products obtained after post-selection RT-PCR.

Modification:

Article Title: Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors
Article Snippet: To date, the Affibody protein framework chosen for the modification of adenoviral tropism has been based on the Z-domain derived from Staphylococcal protein A [ , , , ]. .. Following these findings, Myhre and colleagues engineered an Ad5 vector with dual specificity, featuring both the HER2/neu-binding, linker-optimized Affibody and another Affibody molecule (Taq -polymerase binding) at different positions relative to each other within the HI loop of the fiber [ ].

Derivative Assay:

Article Title: Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors
Article Snippet: To date, the Affibody protein framework chosen for the modification of adenoviral tropism has been based on the Z-domain derived from Staphylococcal protein A [ , , , ]. .. Following these findings, Myhre and colleagues engineered an Ad5 vector with dual specificity, featuring both the HER2/neu-binding, linker-optimized Affibody and another Affibody molecule (Taq -polymerase binding) at different positions relative to each other within the HI loop of the fiber [ ].

Hybridization:

Article Title: Pretargeted Imaging and Therapy
Article Snippet: To this end, Westerlund et al. created an anti–human epidermal growth factor receptor 2 (HER2) Affibody molecule bearing a 15-mer PNA (ZHER2 -HP1) as well as a complementary PEGylated 15-mer PNA (HP2) that demonstrated fast hybridization, slow dissociation, rapid clearance from the blood, and low retention in healthy tissues ( ). .. Honarvar et al. demonstrated the efficacy of Affibody molecule–based pretargeting using ZHER2 -HP1 and 111 In-labeled HP2 in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts ( ) ( ).

Flow Cytometry:

Article Title: Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity
Article Snippet: .. The 26 mutants were expressed and displayed on the surface of staphylococci and binding to VEGFR2 was analyzed by flow cytometry ( ). ..

Article Title: Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity
Article Snippet: .. Data also revealed that the format allowed for simultaneous interaction with VEGFR2 and albumin, both in biosensor assays on recombinant receptor as well as in flow-cytometry experiments with VEGFR2-positive mammalian cells. .. Furthermore, one of the biparatopic binders was tested for the ability to inhibit VEGF-A induced phosphorylation of VEGFR2 expressed on mammalian cells.

Sequencing:

Article Title: Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
Article Snippet: This sequence was linked to a second element corresponding to a 21-residue stretch (aa 175 to 195) of the E. coli SecM, earlier reported to interact with the ribosome tunnel and capable of providing stable peptide–ribosome–mRNA ternary complexes even when followed by a downstream in-frame termination codon [ – ]. .. For system evaluation, test enrichment experiments were performed involving an mRNA mixture, prepared from mRNA obtained after in vitro transcription using PCR product expression cassettes encoding the model proteins ZWT -ABD (IgG and HSA binding fusion protein; Fig. b) or ZTaq (affibody molecule binding to Taq DNA polymerase [ ]; Fig. c) fused to the p3-SecM spacer.

Generated:

Article Title: Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors
Article Snippet: Affibodies with specific target-binding sites can be generated by simultaneous randomization of 13 amino acids on the Fc-binding face of the protein framework, creating libraries from which in vitro selection methods such as phage display can be used to identify candidate targeting moieties [ ]. .. Following these findings, Myhre and colleagues engineered an Ad5 vector with dual specificity, featuring both the HER2/neu-binding, linker-optimized Affibody and another Affibody molecule (Taq -polymerase binding) at different positions relative to each other within the HI loop of the fiber [ ].

other:

Article Title: Radiolabeling of HER2 specific Affibody(R) molecule with F-18
Article Snippet: Key Work Index: fluorine-18, HER2, breast cancer, Affibody® molecule

Polymerase Chain Reaction:

Article Title: Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
Article Snippet: .. For system evaluation, test enrichment experiments were performed involving an mRNA mixture, prepared from mRNA obtained after in vitro transcription using PCR product expression cassettes encoding the model proteins ZWT -ABD (IgG and HSA binding fusion protein; Fig. b) or ZTaq (affibody molecule binding to Taq DNA polymerase [ ]; Fig. c) fused to the p3-SecM spacer. .. Two rounds of RD selection were performed using human IgG-coated beads for enrichment of the rare IgG-binding ZWT -ABD encoding constructs present in an initial 1000-fold molar excess of Taq DNA polymerase binder encoding control constructs, and the results monitored via analysis of PCR products obtained after post-selection RT-PCR.

Injection:

Article Title: Pretargeted Imaging and Therapy
Article Snippet: Honarvar et al. demonstrated the efficacy of Affibody molecule–based pretargeting using ZHER2 -HP1 and 111 In-labeled HP2 in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts ( ) ( ). .. The pretargeting strategy produced tumoral uptake of 19 ± 2 %ID/g 1 h after injection along with a 54 ± 19 tumor-to-blood activity concentration ratio at the same time point.

Article Title: Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity
Article Snippet: .. As for the previous experiments, injection of the mix resulted in a substantially higher signal, confirming that the affinity-matured candidates had retained the ability to bind simultaneously to VEGFR2 ( ; ). .. The second competition assay was performed to verify that the affinity-matured Affibody molecules could block VEGF-A from binding to VEGFR2.

Binding Assay:

Article Title: Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors
Article Snippet: .. Following these findings, Myhre and colleagues engineered an Ad5 vector with dual specificity, featuring both the HER2/neu-binding, linker-optimized Affibody and another Affibody molecule (Taq -polymerase binding) at different positions relative to each other within the HI loop of the fiber [ ]. ..

Article Title: Affibody-Mediated Sequestration of Amyloid β Demonstrates Preventive Efficacy in a Transgenic Alzheimer’s Disease Mouse Model
Article Snippet: Production of ZSYM73 -ABD and (ZTaq )2 -ABD The therapeutic candidate was designed as a bispecific fusion protein, consisting of a genetic linkage between the high-affinity Aβ-capturing ZSYM73 affibody molecule and an ABD ( ; Lindberg et al., ). .. In this format, ZSYM73 -ABD (16.8 kDa) would be able to capture soluble Aβ peptides while simultaneously binding and circulating with HSA and thus extending the in vivo half-life of the protein (Andersen et al., ; Frejd, ).

Article Title: Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
Article Snippet: .. For system evaluation, test enrichment experiments were performed involving an mRNA mixture, prepared from mRNA obtained after in vitro transcription using PCR product expression cassettes encoding the model proteins ZWT -ABD (IgG and HSA binding fusion protein; Fig. b) or ZTaq (affibody molecule binding to Taq DNA polymerase [ ]; Fig. c) fused to the p3-SecM spacer. .. Two rounds of RD selection were performed using human IgG-coated beads for enrichment of the rare IgG-binding ZWT -ABD encoding constructs present in an initial 1000-fold molar excess of Taq DNA polymerase binder encoding control constructs, and the results monitored via analysis of PCR products obtained after post-selection RT-PCR.

Article Title: Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity
Article Snippet: .. In addition, non-transfected HEK293 cells were included as controls in the assay to verify that potential cell binding was specific for VEGFR2. .. An anti-human VEGFR2 monoclonal antibody (R & D Systems) was included as a positive control.

Article Title: Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity
Article Snippet: .. The 26 mutants were expressed and displayed on the surface of staphylococci and binding to VEGFR2 was analyzed by flow cytometry ( ). ..

In Vivo:

Article Title: Affibody-Mediated Sequestration of Amyloid β Demonstrates Preventive Efficacy in a Transgenic Alzheimer’s Disease Mouse Model
Article Snippet: Production of ZSYM73 -ABD and (ZTaq )2 -ABD The therapeutic candidate was designed as a bispecific fusion protein, consisting of a genetic linkage between the high-affinity Aβ-capturing ZSYM73 affibody molecule and an ABD ( ; Lindberg et al., ). .. In this format, ZSYM73 -ABD (16.8 kDa) would be able to capture soluble Aβ peptides while simultaneously binding and circulating with HSA and thus extending the in vivo half-life of the protein (Andersen et al., ; Frejd, ).

Labeling:

Article Title: Pretargeted Imaging and Therapy
Article Snippet: The high renal reabsorption of Affibody molecules, however, presents a substantial obstacle to using directly labeled variants for targeted radiotherapy. .. Honarvar et al. demonstrated the efficacy of Affibody molecule–based pretargeting using ZHER2 -HP1 and 111 In-labeled HP2 in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts ( ) ( ).

Article Title: Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity
Article Snippet: .. The bacterial displayed library was incubated with 50 nM of labeled VEGFR2, followed by washing and incubation for 30 min or 4.5 h with an excess of unlabeled VEGFR2 to minimize rebinding of dissociated labeled target. .. In addition to the off-rate selection in the last round, the selection stringency was also increased throughout the four sorting rounds by using more stringent gating and sorting parameters.

Mouse Assay:

Article Title: Pretargeted Imaging and Therapy
Article Snippet: .. Honarvar et al. demonstrated the efficacy of Affibody molecule–based pretargeting using ZHER2 -HP1 and 111 In-labeled HP2 in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts ( ) ( ). .. The pretargeting strategy produced tumoral uptake of 19 ± 2 %ID/g 1 h after injection along with a 54 ± 19 tumor-to-blood activity concentration ratio at the same time point.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
Article Snippet: For system evaluation, test enrichment experiments were performed involving an mRNA mixture, prepared from mRNA obtained after in vitro transcription using PCR product expression cassettes encoding the model proteins ZWT -ABD (IgG and HSA binding fusion protein; Fig. b) or ZTaq (affibody molecule binding to Taq DNA polymerase [ ]; Fig. c) fused to the p3-SecM spacer. .. Two rounds of RD selection were performed using human IgG-coated beads for enrichment of the rare IgG-binding ZWT -ABD encoding constructs present in an initial 1000-fold molar excess of Taq DNA polymerase binder encoding control constructs, and the results monitored via analysis of PCR products obtained after post-selection RT-PCR.

Plasmid Preparation:

Article Title: Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors
Article Snippet: .. Following these findings, Myhre and colleagues engineered an Ad5 vector with dual specificity, featuring both the HER2/neu-binding, linker-optimized Affibody and another Affibody molecule (Taq -polymerase binding) at different positions relative to each other within the HI loop of the fiber [ ]. ..

Article Title: Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
Article Snippet: Ribosome Display System Design and Evaluation A plasmid construct denoted pRD was constructed to use as assembly vector and PCR amplification template of linear expression cassettes for RD-mediated selection of binding proteins from libraries of affibody molecules (Fig. a). .. For system evaluation, test enrichment experiments were performed involving an mRNA mixture, prepared from mRNA obtained after in vitro transcription using PCR product expression cassettes encoding the model proteins ZWT -ABD (IgG and HSA binding fusion protein; Fig. b) or ZTaq (affibody molecule binding to Taq DNA polymerase [ ]; Fig. c) fused to the p3-SecM spacer.

Functional Assay:

Article Title: Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
Article Snippet: For system evaluation, test enrichment experiments were performed involving an mRNA mixture, prepared from mRNA obtained after in vitro transcription using PCR product expression cassettes encoding the model proteins ZWT -ABD (IgG and HSA binding fusion protein; Fig. b) or ZTaq (affibody molecule binding to Taq DNA polymerase [ ]; Fig. c) fused to the p3-SecM spacer. .. An efficient enrichment for ZWT -ABD encoding constructs was repeatedly seen (Fig. ), indicating that the system was functional.

Selection:

Article Title: Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors
Article Snippet: Affibodies with specific target-binding sites can be generated by simultaneous randomization of 13 amino acids on the Fc-binding face of the protein framework, creating libraries from which in vitro selection methods such as phage display can be used to identify candidate targeting moieties [ ]. .. Following these findings, Myhre and colleagues engineered an Ad5 vector with dual specificity, featuring both the HER2/neu-binding, linker-optimized Affibody and another Affibody molecule (Taq -polymerase binding) at different positions relative to each other within the HI loop of the fiber [ ].

Article Title: Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
Article Snippet: Here, the natural stop codon in the SecM gene was, however, replaced by an AGA triplet, corresponding to a rare arginine codon in E. coli to possibly further avoid disruption of ternary complexes during selection. .. For system evaluation, test enrichment experiments were performed involving an mRNA mixture, prepared from mRNA obtained after in vitro transcription using PCR product expression cassettes encoding the model proteins ZWT -ABD (IgG and HSA binding fusion protein; Fig. b) or ZTaq (affibody molecule binding to Taq DNA polymerase [ ]; Fig. c) fused to the p3-SecM spacer.

In Vitro:

Article Title: Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors
Article Snippet: A recent study has demonstrated successful in vitro targeting to human epidermal growth factor receptor-2 (HER2)/neu, an important tumor antigen which is overexpressed in ovarian and breast carcinomas [ ], through the incorporation of a HER2/neu-directed Affibody within the HI loop of a CAR-binding ablated fiber [ ]. .. Following these findings, Myhre and colleagues engineered an Ad5 vector with dual specificity, featuring both the HER2/neu-binding, linker-optimized Affibody and another Affibody molecule (Taq -polymerase binding) at different positions relative to each other within the HI loop of the fiber [ ].

Article Title: Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
Article Snippet: .. For system evaluation, test enrichment experiments were performed involving an mRNA mixture, prepared from mRNA obtained after in vitro transcription using PCR product expression cassettes encoding the model proteins ZWT -ABD (IgG and HSA binding fusion protein; Fig. b) or ZTaq (affibody molecule binding to Taq DNA polymerase [ ]; Fig. c) fused to the p3-SecM spacer. .. Two rounds of RD selection were performed using human IgG-coated beads for enrichment of the rare IgG-binding ZWT -ABD encoding constructs present in an initial 1000-fold molar excess of Taq DNA polymerase binder encoding control constructs, and the results monitored via analysis of PCR products obtained after post-selection RT-PCR.

Produced:

Article Title: Pretargeted Imaging and Therapy
Article Snippet: Honarvar et al. demonstrated the efficacy of Affibody molecule–based pretargeting using ZHER2 -HP1 and 111 In-labeled HP2 in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts ( ) ( ). .. The pretargeting strategy produced tumoral uptake of 19 ± 2 %ID/g 1 h after injection along with a 54 ± 19 tumor-to-blood activity concentration ratio at the same time point.

Concentration Assay:

Article Title: Pretargeted Imaging and Therapy
Article Snippet: Honarvar et al. demonstrated the efficacy of Affibody molecule–based pretargeting using ZHER2 -HP1 and 111 In-labeled HP2 in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts ( ) ( ). .. The pretargeting strategy produced tumoral uptake of 19 ± 2 %ID/g 1 h after injection along with a 54 ± 19 tumor-to-blood activity concentration ratio at the same time point.

Recombinant:

Article Title: Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity
Article Snippet: .. Data also revealed that the format allowed for simultaneous interaction with VEGFR2 and albumin, both in biosensor assays on recombinant receptor as well as in flow-cytometry experiments with VEGFR2-positive mammalian cells. .. Furthermore, one of the biparatopic binders was tested for the ability to inhibit VEGF-A induced phosphorylation of VEGFR2 expressed on mammalian cells.

Variant Assay:

Article Title: Affibody-Mediated Sequestration of Amyloid β Demonstrates Preventive Efficacy in a Transgenic Alzheimer’s Disease Mouse Model
Article Snippet: Production of ZSYM73 -ABD and (ZTaq )2 -ABD The therapeutic candidate was designed as a bispecific fusion protein, consisting of a genetic linkage between the high-affinity Aβ-capturing ZSYM73 affibody molecule and an ABD ( ; Lindberg et al., ). .. The ABD-moiety used in this study was a deimmunized variant with an engineered femtomolar affinity for HSA.

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    Affibody taq polymerase binding affibody molecule ztaq
    PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z <t>Taq:3638</t> -ST-CH 3 . Comparison B illustrates uptake of the targeting <t>Affibody</t> increasing as the tumors grow from time from inoculation
    Taq Polymerase Binding Affibody Molecule Ztaq, supplied by Affibody, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq polymerase binding affibody molecule ztaq/product/Affibody
    Average 92 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    taq polymerase binding affibody molecule ztaq - by Bioz Stars, 2020-04
    92/100 stars
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    86
    Affibody taq polymerase binding affibody
    PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z <t>Taq:3638</t> -ST-CH 3 . Comparison B illustrates uptake of the targeting <t>Affibody</t> increasing as the tumors grow from time from inoculation
    Taq Polymerase Binding Affibody, supplied by Affibody, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq polymerase binding affibody/product/Affibody
    Average 86 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    taq polymerase binding affibody - by Bioz Stars, 2020-04
    86/100 stars
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    PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

    Journal: EJNMMI Research

    Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

    doi: 10.1186/s13550-016-0213-8

    Figure Lengend Snippet: PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

    Article Snippet: DNA constructions and expression of Sel-tagged Affibody molecules The EGFR-binding Affibody molecule ZEGFR:2377 [ , ] and the irrelevant Taq polymerase-binding Affibody molecule ZTaq:3638 [ ] were fused with a C-terminal ST as previously described [ , ].

    Techniques: Positron Emission Tomography

    PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

    Journal: EJNMMI Research

    Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

    doi: 10.1186/s13550-016-0213-8

    Figure Lengend Snippet: PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

    Article Snippet: DNA constructions and expression of Sel-tagged Affibody molecules The EGFR-binding Affibody molecule ZEGFR:2377 [ , ] and the irrelevant Taq polymerase-binding Affibody molecule ZTaq:3638 [ ] were fused with a C-terminal ST as previously described [ , ].

    Techniques: Positron Emission Tomography