Journal: Immunity

Article Title: Vaccination Induces Maturation in a Mouse Model of Diverse Unmutated VRC01-Class Precursors to HIV-Neutralizing Antibodies with >50% Breadth

doi: 10.1016/j.immuni.2020.12.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies with less than 10% breadth were excluded from this analysis. . Bio-layer interferometry antigenicity analysis Kinetics and affinities of antibody-antigen interactions were measured on an Octet (Forte Bio) using Anti-human IgG Fc and Anti- mouse IgG Fc capture biosensors following the instructions.

Techniques: Staining, Sequencing, Expressing, Plasmid Preparation, Software

Journal: Immunity

Article Title: Vaccination Induces Maturation in a Mouse Model of Diverse Unmutated VRC01-Class Precursors to HIV-Neutralizing Antibodies with >50% Breadth

doi: 10.1016/j.immuni.2020.12.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies with less than 10% breadth were excluded from this analysis. . Bio-layer interferometry antigenicity analysis Kinetics and affinities of antibody-antigen interactions were measured on an Octet (Forte Bio) using Anti-human IgG Fc and Anti- mouse IgG Fc capture biosensors following the instructions.

Techniques: Staining, Sequencing, Expressing, Plasmid Preparation, Software

Journal: Journal of Neuroinflammation

Article Title: Engagement of TREM2 by a novel monoclonal antibody induces activation of microglia and improves cognitive function in Alzheimer’s disease models

doi: 10.1186/s12974-020-01980-5

Figure Lengend Snippet: Binding of Ab-T1 to TREM2. a Binding of the mouse IgG antibodies Ab-T1, Ab-T2, Ab-T3, Ab-T4, Ab-T5, and control anti human TREM2 Ab to human and mouse TREM2 expressed in HEK293T cells by dot blot assay. Control naïve HEK293T cells (control) used as negative control. Binding affinity sensograms of Ab-T1 to ( b ) human TREM2 and ( c ) mouse TREM2 as measured by surface plasmon resonance (BiaCore). d Ab-T1 binding affinity table; “Ka” refers to the association rate constant; “kd” refers to the dissociation rate constant; and “KD” refers to the affinity constant. e Confocal images of Ab-T1 immunostaining non-permeabilised HEK293T transfected with wild type hTREM2-GFP (upper panels) and parental HEK293T (lower panels). Nuclei stained with DAPI. f Human TREM2 expression levels on transfected HEK293T cells recognized by Ab-T1. The y -axis represents the relative mean fluorescence intensity (relative MFI) measured by flow cytometry. g Western blot showing human TREM2 detection on stable U937 cell line recognized by Ab-T1

Article Snippet: Ab-T1 clone was selected and tested for its high affinity to extracellular domain of human and mouse TREM2 in surface plasmon resonance (Biacore SPR system, GE Healthcare Life Sciences), whereby anti-TREM2 antibodies are immobilized on the chip surface through an anti-mouse capture antibody (BR100838, GE), for example, a CM5 sensor chip and the human TREM2 and mouse TREM2 (SI 50149-M02H, Sino Biological) and TREM1 antigens are then run over the chip.

Techniques: Binding Assay, Dot Blot, Negative Control, SPR Assay, Immunostaining, Transfection, Staining, Expressing, Fluorescence, Flow Cytometry, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Engagement of TREM2 by a novel monoclonal antibody induces activation of microglia and improves cognitive function in Alzheimer’s disease models

doi: 10.1186/s12974-020-01980-5

Figure Lengend Snippet: Recognition of TREM2 in AD models/human by Ab-T1. a Western blot showing Ab-T1 binding to human entorhinal cortex extracts from Alzheimer/control group patients. HEK293T protein lysate was used as negative control (NC). GAPDH as “housekeeping” protein loading control is shown in lower panels. b Immunohistochemistry staining showing Ab-T1 binding to human brain sample (entorhinal cortex sections) from Alzheimer’s disease patient (TREM2) with microglia and beta amyloid staining of same human brain tissue sample. Mouse IgG was used as negative control staining. c Confocal microscope scan images showing co-localization of TREM2 (mouse Ab-T1) with resident Iba1 positive cells (Microglia) in 5xFAD mice brain slices (white arrows). d Immunohistochemistry staining showing Ab-T1 binding to brain tissue from 5xFAD mice (TREM2) with microglia and beta amyloid staining of same mice tissue sample. Mouse IgG was used as negative control. e Western blots of supernatants (left panel) for soluble TREM2 detection in parental HEK293T vs. HEK293T-hTREM2 cells using mouse Ab-T1 and mouse IgG1 as control Ab. Transfected HEK293T cells with no insert DNA vector were used as sham. f Western blots of soluble hTREM2 detection in human CSF from Alzheimer patients using Ab-T1 and mouse IgG1 as control IgG antibody. TREM-ECD represents soluble TREM2 recombinant protein control

Article Snippet: Ab-T1 clone was selected and tested for its high affinity to extracellular domain of human and mouse TREM2 in surface plasmon resonance (Biacore SPR system, GE Healthcare Life Sciences), whereby anti-TREM2 antibodies are immobilized on the chip surface through an anti-mouse capture antibody (BR100838, GE), for example, a CM5 sensor chip and the human TREM2 and mouse TREM2 (SI 50149-M02H, Sino Biological) and TREM1 antigens are then run over the chip.

Techniques: Western Blot, Binding Assay, Negative Control, Immunohistochemistry, Staining, Microscopy, Transfection, Plasmid Preparation, Recombinant

Journal: Journal of Neuroinflammation

Article Title: Engagement of TREM2 by a novel monoclonal antibody induces activation of microglia and improves cognitive function in Alzheimer’s disease models

doi: 10.1186/s12974-020-01980-5

Figure Lengend Snippet: Ab-T1 augments uptake of beta amyloid in a Syk-dependent manner and reduces inflammation triggered by IC delivery of soluble TREM2. Ab-T1 increases uptake of labeled oligomeric beta amyloid a in human microglia derived from differentiated human PBMC’s, b murine peritoneal macrophages, and c murine microglia in a Syk dependent manner (R406). The y -axis represents the relative geometric mean (gMFI) measured by flow cytometry. Results are mean of three repeated experiments with duplicate technical sample repetition. d Intracerebral delivery of sTREM2 with Ab-T1 attenuates the neuroinflammatory response evident by delivery of sTREM2 with control IgG ( n = 7, two-tailed student’s t test). The y -axis represents the fold-change in expression relative to sham (PBS injected animals)

Article Snippet: Ab-T1 clone was selected and tested for its high affinity to extracellular domain of human and mouse TREM2 in surface plasmon resonance (Biacore SPR system, GE Healthcare Life Sciences), whereby anti-TREM2 antibodies are immobilized on the chip surface through an anti-mouse capture antibody (BR100838, GE), for example, a CM5 sensor chip and the human TREM2 and mouse TREM2 (SI 50149-M02H, Sino Biological) and TREM1 antigens are then run over the chip.

Techniques: Labeling, Derivative Assay, Flow Cytometry, Two Tailed Test, Expressing, Injection

Journal: Journal of Neuroinflammation

Article Title: Engagement of TREM2 by a novel monoclonal antibody induces activation of microglia and improves cognitive function in Alzheimer’s disease models

doi: 10.1186/s12974-020-01980-5

Figure Lengend Snippet: Effects on Ab-T1 on beta amyloid levels and target engagement in 5XFAD mice. a Ab-T1 treatment is associated with a decrease in total brain derived beta amyloid. b Beta amyloid plaques in mice brain sections were confirmed using thioflavin S fluorescent staining. c, d Images taken on microscope were analyzed using ImageJ software for mean number of plaques per mice brain section area and average plaque size ( n = 9–10, two-tailed student’s t test, ** P = 0.001, * P = 0.032). e Number of diffuse plaques in the cortex of Ab-T1 vs. control-treated 5xFAD mice (two-tailed student’s t test, ** P < 0.005). f Ab-T1 induces a reduction of soluble TREM2 in CSF and serum (two-tailed student’s t test, * P = 0.047). g Ab-T1 levels in serum and brain of 5xFAD treated mice. ( h ; upper panels) Correlation of serum and brain levels of Ab-T1 with cognition tested in MWM ( r = 0.46; P < 0.05 and r = 0.5; P < 0.05). ( h ; lower right) Association between serum and brain levels in the same treated mice ( r = 0.89; P < 0.001). ( h ; lower left) Association of free sTREM2 levels achieved by Ab-T1 treatment, and cognition ( r = 0.61; P < 0.05). Data shown in ELISA assays is the mean of triplicate sample repetition

Article Snippet: Ab-T1 clone was selected and tested for its high affinity to extracellular domain of human and mouse TREM2 in surface plasmon resonance (Biacore SPR system, GE Healthcare Life Sciences), whereby anti-TREM2 antibodies are immobilized on the chip surface through an anti-mouse capture antibody (BR100838, GE), for example, a CM5 sensor chip and the human TREM2 and mouse TREM2 (SI 50149-M02H, Sino Biological) and TREM1 antigens are then run over the chip.

Techniques: Derivative Assay, Staining, Microscopy, Software, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: A Combination of Two Human Monoclonal Antibodies Prevents Zika Virus Escape Mutations in Non-human Primates

doi: 10.1016/j.celrep.2018.10.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Tesh TVP-1140 Chemicals, Peptides, and Recombinant Proteins Streptavidin-HRP Jackson Immu no Research Cat#016-030-084 Neutravidin ThermoScientific Cat#31000 Poly-L-lysine solution Sigma-Aldrich Cat#P4707 Medium 199 Lonza Cat#12–109F TRIzol-LS Thermo Fisher Scientific Cat#10296010 Jeffamine® ED-2001 pH 7.0 Hampton Research Cat#HR2–597 Fomblin Y oil Sigma-Aldrich Cat#317942–100G Critical Commercial Assays Q5 site-directed mutagenesis kit New England Biolabs Cat#E0554S QuikChange site-directed mutagenesis Agilent Technologies Cat#200524 Nunc MaxiSorp384-well ELISA plate Sigma-Aldrich Cat#P6491 SuperSignal ELISA Femto Substrate Thermo Fisher Scientific Cat#37075 Qiaamp viral RNAmini kit QIAGEN Cat#1020953 QIAGEN One-Step RT-PCR QIAGEN Cat#210210 Superscript III RT Thermo Fisher Scientific Cat#18080093 Nucleospin gel and PCR clean-up Macherey-Nagel Cat#740609 Deposited Data Z021-ZIKV EDIII structure This paper PDB ID: 6DFI Z021-DENV1 EDIII structure This paper PDB ID: 6DFJ Experimental Models: Cell Lines HEK293–6E National Research Council of Canada NRC file 11565 Lenti-X 293T ATCC Cat#632180; RRID:CVCL_4401 Huh-7.5 Blight et al., 2002 N/A Vero WHO Laboratory of S. Whitehead MCB-P139 STAT1 −/− Laboratory of J.-L. Casanova, Chapgier et al., 2006 N/A K-562 ATCC CAT#:CCL-243; RRID:CVCL_0004 Experimental Models: Organisms/Strains Ifnar1 −/− mice: B6.129S2-lfnar 1tm1Agt/Mmiax The Jackson Laboratory JAX stock:32045 Rhesus Macaques (Macaca mulatta) California National Primate Research Center Breeding colony Oligonucleotides Primers Table S3 this paper N/A Recombinant DNA IGγ1-, IGκ or IGλ-expression vectors Tiller et al., 2008 N/A pWNVII-Rep-REN-IB Laboratory of T. Pierson N/A pZIKV/HPF/CprME Laboratory of T. Pierson N/A pZIKV/HPF/CprM*E* Robbiani et al., 2017 N/A Wild typeZIKV EDIII expression vector Robbiani et al., 2017 N/A Software and Algorithms Prism (v7) GraphPad https://www.graph pad.com Mosflm (v7.2.1, part of CCP4 package) Battye et al., 2011 http://www.ccp4.ac.uk CCP4 (v7.0.032) Winn et al., 2011 http://www.ccp4.ac.uk Ph en ix(v1.11.1–2575) Adams et al., 2010 http://www.phenix-online.org Coot (vO.8.7, part of CCP4 package) Emsley and Cowtan, 2004 http://www.ccp4.ac.uk Antibody Database (v1.0) West et al., 2013 N/A BIAcore T200 evaluation software GE Healhcare http://www.biacore.com/lifesciences/index.html Other Biacore T200 SPR system GE Healthcare CAT#:28975001 CM5 sensor chip GE Healthcare CAT#:BR100399 Open in a separate window KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for reagents should be directed to and will be fulfilled by the Lead Contact, Davide Robbiani ( ude.rellefekcor@inaibbord ).

Techniques: Recombinant, Mutagenesis, Enzyme-linked Immunosorbent Assay, Expressing, Plasmid Preparation, Software

Journal: mAbs

Article Title: Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding

doi: 10.1080/19420862.2016.1210747

Figure Lengend Snippet: Thermostability of Fab-dsFv following exposure to pH. A thermofluor assay was used to determine the midpoint melting temperature (T m ) transition states of the individual Fab ( ) and dsFv domains ( ) of the Fab-dsFv format at a pH range of pH 2.6 to pH 7.6 (0.2 increments). The corresponding Fab no hinge ( ) was used as a control. Purified protein samples in PBS pH7.4 were mixed with SYPRO® Orange dye in quadruplicate and thermocycled (peltier-based) in a 7900HT Fast Real-Time PCR System (Agilent) from 20°C to 99°C (1.1°C/min ramp rate). A charge-coupled device (CCD) was used to measure fluorescence changes. The intensity increases in fluorescence were plotted and the inflection point of the slope(s) was used to generate the T m at each pH. The T m of each domain and Fab no hinge was plotted against pH. Standard deviation was calculated at each point and plotted as error bars.

Article Snippet: The binding affinities and kinetic parameters for the interactions of antibodies were determined by SPR conducted on a Biacore T100 or a Biacore 3000 using CM5 sensor chips (GE Healthcare Bio-Sciences AB) and HBS-EP (10 mM HEPES (pH7.4), 150 mM NaCl, 3 mM EDTA, 0.05% v/v surfactant P20) running buffer.

Techniques: Purification, Real-time Polymerase Chain Reaction, Fluorescence, Standard Deviation

Journal: mAbs

Article Title: Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding

doi: 10.1080/19420862.2016.1210747

Figure Lengend Snippet: Independent and simultaneous binding SPR kinetics and affinity of Fab-dsFv for the target antigen and serum albumin. The binding affinities and kinetic parameters for the interactions of antibodies were determined by SPR with HBS-EB buffer (pH 7.4) as the running buffer at 25°C. The antibody samples were captured to the sensor chip surface via a human F(ab') 2 -specific goat Fab. A) Binding kinetics and affinity (KD) of captured Fab or Fab-dsFv to the target antigen. Values are the arithmetic mean and standard deviation (s.d) was determined from four independent titrations. B) Binding kinetics and affinity (KD) of captured Fab-dsFv to HSA (normal form at pH 7.4) MSA or CSA. Values are the arithmetic mean and s.d was determined from three independent titrations. For both A) and B) the association rate (k on ) was determined by a 3 min injection of the target antigen or albumin over captured antibody after which dissociation rate (k off ) was monitored for 30 min for the target antigen and 10 min for albumin. C) Simultaneous binding of captured Fab-dsFv to the target antigen HSA or a mixed solution of target antigen and HSA. The k on rate was determined by injecting HSA the target ligand or a mixed solution of both HSA and the target antigen over the captured antibody for 3 min. The k off rate was monitored for 30 min. Kinetic parameters were determined by simultaneous global-fitting of the resulting sensorgrams to a standard 1:1 binding model using Biacore T200 evaluation software v1. The affinity (KD) was calculated from the k off and k on rates.

Article Snippet: The binding affinities and kinetic parameters for the interactions of antibodies were determined by SPR conducted on a Biacore T100 or a Biacore 3000 using CM5 sensor chips (GE Healthcare Bio-Sciences AB) and HBS-EP (10 mM HEPES (pH7.4), 150 mM NaCl, 3 mM EDTA, 0.05% v/v surfactant P20) running buffer.

Techniques: Binding Assay, Standard Deviation, Injection, Software

Journal: Scientific Reports

Article Title: Identification of a peptide-peptide binding motif in the coating of nab-paclitaxel nanoparticles with clinical antibodies: bevacizumab, rituximab, and trastuzumab

doi: 10.1038/s41598-017-15251-6

Figure Lengend Snippet: HSA Peptide 40 Binding to Monoclonal Antibodies Bevacizumab, Rituximab, and Trastuzumab Kinetic Binding Parameters Determined by Biacore-SPR: A peptide library of human serum albumin (HSA) was screened via Biacore over immobilized antibodies bevacizumab, rituximab, and trastuzumab. Antibodies were immobilized via amine coupling and peptides were run at pM-uM concentration ranges. ( a ) HSA Peptide 40 binding curves (180 second association period, 1200 second dissociation period) against all three antibodies and the negative control antibody pembrolizumab. ( b ) Binding kinetics were determined via Biacore Evaluation Software analysis of SPR sensograms.

Article Snippet: Binding kinetics between antibody variable domain peptides and HSA Peptide 40/HSA were determined by Biacore Evaluation Software analysis of SPR sensograms. ( b ) Multiple sequence alignment of the variable region peptides in question, performed by Clustal Omega. ( c ) Rituximab digest Biacore screening.

Techniques: Binding Assay, Bioprocessing, Concentration Assay, Negative Control, Software

Journal: Scientific Reports

Article Title: Identification of a peptide-peptide binding motif in the coating of nab-paclitaxel nanoparticles with clinical antibodies: bevacizumab, rituximab, and trastuzumab

doi: 10.1038/s41598-017-15251-6

Figure Lengend Snippet: Bevacizumab Heavy Chain Peptide Library Kinetic Binding Parameters Determined by Biacore-SPR: A peptide library of bevacizumab was screened via Biacore SPR against HSA Peptide 40. The bevacizumab library was run over HSA Peptide 40 immobilized to a CM5 chip via amine coupling and vice versa. All peptides were run in the pM-uM concentration ranges. ( a ) Sensograms of HSA Peptide 40 run over immobilized Bevaciuzmab peptides 11 and 12 (120 second association period, 180 second dissociation period). Short gaps in binding curves at transition times are from removal of solvent spikes during analysis. Binding kinetics were determined via Biacore Evaluation Software analysis of SPR sensograms. ( b ) Location and structure of amino acids 90–122 on the heavy chain variable domain from a crystal structure of bevacizumab in complex with vascular endothelial growth factor (VEGF). Structure, PDB 1BJ1, was visualized using UCSF Chimera.

Article Snippet: Binding kinetics between antibody variable domain peptides and HSA Peptide 40/HSA were determined by Biacore Evaluation Software analysis of SPR sensograms. ( b ) Multiple sequence alignment of the variable region peptides in question, performed by Clustal Omega. ( c ) Rituximab digest Biacore screening.

Techniques: Binding Assay, Concentration Assay, Solvent, Software

Journal: Scientific Reports

Article Title: Identification of a peptide-peptide binding motif in the coating of nab-paclitaxel nanoparticles with clinical antibodies: bevacizumab, rituximab, and trastuzumab

doi: 10.1038/s41598-017-15251-6

Figure Lengend Snippet: Bevacizumab, Rituximab, and Trastuzuzumab Heavy Chain Variable Domain Peptide Library Screening: A peptide library of the heavy chain variable domain for Bevacizumab, Rituximab, and Trastuzumab were screened via Biacore over immobilized HSA and an HSA peptide library, as well as vice versa. Peptides were run in the pM-uM concentration range. ( a ) The positive results of the peptide screening. Binding kinetics between antibody variable domain peptides and HSA Peptide 40/HSA were determined by Biacore Evaluation Software analysis of SPR sensograms. ( b ) Multiple sequence alignment of the variable region peptides in question, performed by Clustal Omega. ( c ) Rituximab digest Biacore screening. Rituximab was trypsin digested and separated by HPLC reverse phase chromatography. The fraction (Fraction 58) that bound both HSA Peptide 40 and albumin was sequenced via mass spec. ( d ) 3D structure prediction of peptides in solution performed by PEP-FOLD 3.0, visualized in Chimera. ( e ) Biolayer Interfermoetry (BLITZ) assay kinetics assay of either biotinylated bevacizumab or Bev Variable Domain Peptide bound to strepdavidin probe against Abraxane nanoparticles. Pembrolizumab included as a negative control.

Article Snippet: Binding kinetics between antibody variable domain peptides and HSA Peptide 40/HSA were determined by Biacore Evaluation Software analysis of SPR sensograms. ( b ) Multiple sequence alignment of the variable region peptides in question, performed by Clustal Omega. ( c ) Rituximab digest Biacore screening.

Techniques: Library Screening, Concentration Assay, Binding Assay, Software, Sequencing, Reversed-phase Chromatography, Mass Spectrometry, Negative Control