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: 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: 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: 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: mBio

Article Title: Blockade of the Adenylate Cyclase Toxin Synergizes with Opsonizing Antibodies to Protect Mice against Bordetella pertussis

doi: 10.1128/mbio.01527-22

Figure Lengend Snippet: Antibodies binding different RTX epitopes have similar biophysical characteristics. (A) Schematic of the ACT domain structure showing the antibody binding sites. Mature ACT consists of a catalytically active N-terminal domain (green), a central hydrophobic domain with acylation sites at residues 860 and 983 (gray), an RTX domain comprised of five repeat blocks (RI to RV) joined by linkers (L1 to L4), and a C-terminal secretion signal (Sec). (B) SDS-PAGE gel of purified antibodies under nonreducing and reducing conditions, with molecular weight (MW) markers shown. (C) Antibody binding to wells coated with RTX 751 (1 μg/mL) (solid lines) or uncoated wells (dashed lines) was assessed by an ELISA with anti-mouse Fc–HRP detection. The binding data were fit to a four-parameter logistic curve in GraphPad Prism. Shown are the averages from duplicates, with error bars representing the data range; the experiment was repeated twice. (D) Binding of M2B10 and M1F5 to RTX 751 via biolayer interferometry. Antibodies were immobilized on anti-mouse Fc sensors and dipped into RTX 751 (six concentrations from 25 to 0.8 nM) for a 30-min equilibration. Binding affinity was determined by data fitting to a Langmuir isotherm using Octet Red96 instrument software (FortéBio); the K d values are shown as the means and ranges from two replicate experiments.

Article Snippet: To measure the binding kinetics of RTX for purified mouse α M β 2 integrin using a Biacore X100 system, the M1F5 Fab was desalted into 10 mM Na-acetate (pH 4.0) and coupled onto both flow cells to ~2,500 response units (RU) of a CM5 SPR biosensor (catalog number BR100012; Cytiva) using an EDC/NHS amine coupling kit (catalog number BR100050; Cytiva).

Techniques: Binding Assay, SDS Page, Purification, Molecular Weight, Enzyme-linked Immunosorbent Assay, Software

Journal: mBio

Article Title: Blockade of the Adenylate Cyclase Toxin Synergizes with Opsonizing Antibodies to Protect Mice against Bordetella pertussis

doi: 10.1128/mbio.01527-22

Figure Lengend Snippet: Intranasal administration of ACT-neutralizing antibody protects mice against lethal pertussis challenge. (A) BALB/c mice ( n = 6 per group; half female; 32 days old) were administered 10 μg M2B10 or an isotype control antibody intranasally (i.n.) or M2B10 intraperitoneally (i.p.) 2 h prior to challenge with a high dose (2 × 10 8 CFU) of B. pertussis TohamaI. All antibodies were produced with mouse IgG2a/kappa constant domains. (B) Five micrograms of M2B10, 10 μg of the isotype control, or the PBS control was administered intranasally to 32-day-old BALB/c mice ( n = 6; half female) before infection with 2 × 10 8 CFU B. pertussis TohamaI. (C) Antibodies M2B10 and M1F5 and their aglycosylated N297A counterparts (10 μg M2B10, 10 μg M2B10-N297A, 20 μg M1F5, and 20 μg M1F5-N297A) were administered intranasally to mice ( n = 5) before infection with 2 × 10 8 CFU B. pertussis TohamaI. For all panels, *** indicates a P value of <0.001 as determined by the Mantel-Cox test with a Bonferroni-Dunn posttest for multiple comparisons.

Article Snippet: To measure the binding kinetics of RTX for purified mouse α M β 2 integrin using a Biacore X100 system, the M1F5 Fab was desalted into 10 mM Na-acetate (pH 4.0) and coupled onto both flow cells to ~2,500 response units (RU) of a CM5 SPR biosensor (catalog number BR100012; Cytiva) using an EDC/NHS amine coupling kit (catalog number BR100050; Cytiva).

Techniques: Control, Produced, Infection

Journal: Science immunology

Article Title: A high-affinity human TCR-like antibody detects celiac disease gluten peptide-MHC complexes and inhibits T-cell activation

doi: 10.1126/sciimmunol.abg4925

Figure Lengend Snippet: ( A ) Raji B cells were in vitro loaded with gluten peptides as annotated and stained with R-PE-conjugated 206 or 3.C11 mIgG2b (n=1) ( B ) Human EBV transduced B-cell lines expressing different HLA-DQ allotypes were loaded with gluten peptides and stained with 3.C11 as before (n=2). ( C-F ) Single-cell suspensions were prepared from untreated HLA-DQ2.5 + CeD patients (n=8, UCeD) or controls with a normal intestinal histology (n=5, two of the controls were HLA-DQ2.5 + ). PCs were gated as live, large lymphocytes, CD3 - CD11c - CD14 - CD38 + CD27 +/− CD19 + CD45 + cells (C, D), and DCs/Mfs as live, CD3 - CD19 - CD27 - CD38 - CD11c + CD14 + cells (E, F). Cells were stained with mIgG2b antibodies followed by a PE-conjugated secondary antibody. Frequency of positive cells was calculated based on gates set according to the staining of an isotype control antibody (isotype). Upper panels; percentage of pMHC + cells across individuals. Boxes illustrate minimum to maximum value with the middle line at mean percentage. Dotted lines represent mean background staining of the isotype. For each CeD subject (different shades of blue), alterations in biopsy histology according to modified Marsh scores are indicated. Statistical differences between groups were analyzed using an unpaired two-tailed t -test. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001; ns, not significant. Lower panels; representative histograms showing pMHC-specific staining within patient groups as indicated.

Article Snippet: SPR binding data values were buffer subtracted and reference-cell subtracted using the Biacore T200 Evaluation Software v1.0.

Techniques: In Vitro, Staining, Expressing, Single Cell, Control, Modification, Two Tailed Test

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Niloticin inhibits osteoclastogenesis by blocking RANKL-RANK interaction and suppressing the AKT, MAPK, and NF-κB signaling pathways.

doi: 10.1016/j.biopha.2022.112902

Figure Lengend Snippet: Fig. 1. Niloticin inhibits RANKL-induced osteoclastogenesis in RAW 264.7 cells. (A) The chemical structure of niloticin. (B) Representative TRAP staining photo graphs showing that niloticin inhibited RANKL-induced osteoclast differentiation in RAW 264.7 cells (magnification: 40 ×). (C) TRAP-positive cells with over three nuclei were considered as mature osteoclasts and counted under a microscope. (D) Niloticin showed no cytotoxic effect on RAW 264.7 cells, as determined by MTT assay. Data are presented as the mean ± SD of three independent experiments. ***P < 0.001 versus the vehicle treatment group; ##P < 0.01 and ###P < 0.001 versus the RANKL alone group. RANKL, receptor activator of nuclear factor (NF)-κB ligand; TRAP, tartrate-resistant acid phosphatase; MTT, 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide; SD, standard deviation.

Article Snippet: Recombinant mouse RANKL (E. coli-expressed) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, Microscopy, MTT Assay, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Niloticin inhibits osteoclastogenesis by blocking RANKL-RANK interaction and suppressing the AKT, MAPK, and NF-κB signaling pathways.

doi: 10.1016/j.biopha.2022.112902

Figure Lengend Snippet: Fig. 2. Niloticin suppresses the RANKL-induced expression of osteoclastogenesis-related markers. (A–D) Representative western blot bands and quantifications showing that niloticin inhibited the RANKL-induced expression of NFATc1 (A), c-Fos (B), TRAP (C), and c-Src (D) in RAW 264.7 cells. Cells were pretreated with the indicated niloticin concentrations for 20 min and then with or without RANKL (50 ng/mL) for 24 h. Protein level relative to corresponding loading control was calculated using AlphaView software and normalized relative to the vehicle group. (E and F) qRT-PCR analysis showing that niloticin inhibited the RANKL-induced mRNA expression of β3-Integrin (E) and cathepsin K (F) in RAW 264.7 cells. Data are presented as the mean ± SD of three independent experiments. **P < 0.01 and ***P < 0.001 versus the vehicle treatment group; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus the RANKL alone group. RANKL, receptor activator of nuclear factor (NF)-κB ligand; NFATc1, nuclear factor of activated T cell c1; TRAP, tartrate-resistant acid phosphatase; qRT-PCR, quantitative real-time PCR; SD, stan dard deviation.

Article Snippet: Recombinant mouse RANKL (E. coli-expressed) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Control, Software, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Niloticin inhibits osteoclastogenesis by blocking RANKL-RANK interaction and suppressing the AKT, MAPK, and NF-κB signaling pathways.

doi: 10.1016/j.biopha.2022.112902

Figure Lengend Snippet: Fig. 3. Niloticin suppresses RANKL-induced AKT and MAPK activation pathways. (A) Representative bands for the effect of niloticin on RANKL-induced phos phorylation of AKT, p38, JNK, and ERK1/2. RAW 264.7 cells were pretreated with or without niloticin (7.5 μM) for 3 h in serum-free medium and then stimulated with RANKL (50 ng/mL) for the indicated times. Cell lysates were subjected to western blot analysis using the indicated primary antibodies. (B–E) The ratios of p- AKT, p-p38, p-JNK, and p-ERK1/2 relative to total AKT (B), total p38 (C), total JNK (D), and total ERK1/2 (E) quantified using AlphaView software. Data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the RANKL alone group at the same time point. RANKL, receptor activator of nuclear factor (NF)-κB ligand; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ERK1/2, extracellular signal- regulated kinase 1/2; SD, standard deviation.

Article Snippet: Recombinant mouse RANKL (E. coli-expressed) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Western Blot, Software, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Niloticin inhibits osteoclastogenesis by blocking RANKL-RANK interaction and suppressing the AKT, MAPK, and NF-κB signaling pathways.

doi: 10.1016/j.biopha.2022.112902

Figure Lengend Snippet: Fig. 4. Niloticin suppresses RANKL-induced NF-κB activation pathways. (A) Representative bands for the effect of niloticin on RANKL-induced phosphorylation of IKKα/β, IκBα, and p65. RAW 264.7 cells were treated as in Fig. 3A. Cell lysates were subjected to western blot analysis using the indicated primary antibodies. (B–D) The ratios of p-IKKα/β, p-IκBα, and p-p65 relative to total IKKα/β (B), total IκBα (C), and total p65 (D) quantified using AlphaView software. Data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the RANKL alone group at the same time point. RANKL, receptor activator of nuclear factor (NF)-κB ligand; NF-κB, nuclear factor-κB; SD, standard deviation.

Article Snippet: Recombinant mouse RANKL (E. coli-expressed) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Software, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Niloticin inhibits osteoclastogenesis by blocking RANKL-RANK interaction and suppressing the AKT, MAPK, and NF-κB signaling pathways.

doi: 10.1016/j.biopha.2022.112902

Figure Lengend Snippet: Fig. 5. Niloticin binds to RANK and blocks RANKL-RANK interaction. (A) Real-time interaction between niloticin and RANK monitored through a BLI kinetic assay using an Octet Red 96 instrument. (B) Solution competition BLI assay curves showing that niloticin disrupted the interaction between RANKL and RANK. (C) Niloticin bound to RANK with strong affinity, as validated by SPR analysis. (D) Niloticin blocked the interaction between RANKL and RANK, as validated by SPR-based “A-B- A” competition assay. The data with (±) in parentheses are the standard errors (SE) from the global fitting. RANKL, receptor activator of nuclear factor (NF)-κB ligand; RANK, RANKL receptor; BLI, biolayer interferometry; SPR, surface plasmon resonance.

Article Snippet: Recombinant mouse RANKL (E. coli-expressed) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Kinetic Assay, Competitive Binding Assay, SPR Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Niloticin inhibits osteoclastogenesis by blocking RANKL-RANK interaction and suppressing the AKT, MAPK, and NF-κB signaling pathways.

doi: 10.1016/j.biopha.2022.112902

Figure Lengend Snippet: Fig. 6. A schematic model of the molecular mechanisms underlying the inhibitory effect of niloticin on RANKL-induced osteoclastogenesis.

Article Snippet: Recombinant mouse RANKL (E. coli-expressed) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: