Journal: European Journal of Medical Research

Article Title: Membrane-bound estrogen receptor alpha initiated signaling is dynamin dependent in breast cancer cells

doi: 10.1186/s40001-018-0328-7

Figure Lengend Snippet: Expression changes of CCND1, ERBB2, KCNK5, KDM4B, and MYC after selective GPER agonist, G1 treatment. A significantly lower expression changes were observed compared to estrogen or estrogen–BSA treatments. Y -axis represents ddCT values, 0 line indicates control level. (Error bars represent standard deviation, asterisks indicate significant changes compared to control with a p value < 0.05). Numerical ddCT values are shown in Additional file : Table Sheet 5

Article Snippet: Pre-designed TaqMan ® Gene Expression Assays (Applied Biosystems) were used for real-time PCR (CCND1—Hs00765553_m1; ERBB2—Hs01001580_m1; GAPDH—Hs99999905_m1; CKNK5—Hs00186652_m1; KDM4B—Hs00943636_m1; MYC—Hs00153408_m1; RPL13a—Hs04194366_g1).

Techniques: Expressing, Control, Standard Deviation

Journal: PLoS ONE

Article Title: Acetate Supplementation Induces Growth Arrest of NG2/PDGFRα-Positive Oligodendroglioma-Derived Tumor-Initiating Cells

doi: 10.1371/journal.pone.0080714

Figure Lengend Snippet: OG33 (A) and OG35 (B-D) cells were induced to differentiate over 6 days in vitro via activation of adenylyl cyclase (1 mM dibutyryl cAMP or 10 μM forskolin) or inhibition of MEK1/2 (1 μM PD035901), ErbB2 (1 μM PD174285), PI3K (5 μM and 10 μM LY294002), or mTOR (10 nM and 20 nM rapamycin [RAP]) signaling. DMSO (0.2% DMSO) was used as a control for PD174285, PD035901 and LY294002 treatments. Despite inhibition of PI3K-Akt-mTOR and ERK signaling pathways, both cells failed to differentiate into cells with oligodendroglial morphology (A, B) or increase immunoreactivity for GPAF or CNPase (not shown). MEK and ErbB2 inhibition actually increased OG35 proliferation in DM. While inhibition of PI3K-Akt-mTOR signaling induced morphological alterations in OG35 cells (B), this was not associated with increased CNPase (C) or ASPA (D) protein levels. In fact, ASPA expression decreased in the presence of 10 μM LY294002 and 20 nM rapamycin. Differentiation of Oli-Neu cells with cAMP for 4 days or inhibition of ErbB2 signaling for 2 days served as controls for oligodendrocyte cell morphology. DM - differentiation medium, SCM - stem cell medium. n = 3 independent cultures. *p < 0.05. Scale bar = 100 μm.

Article Snippet: For pharmacological induction of differentiation, OG33 and OG35 cells were plated in SCM or DM in the absence or presence of dibutyryl cAMP (1 mM in water, Sigma), forskolin (10 μM in ethanol, Sigma), the MEK1/2 inhibitor PD035901 (1 μM in dimethylsulfoxide [DMSO], Tocris Bioscience/R & D Systems; Minneapolis, MN), the ErbB2 inhibitor PD174285 (1 μM in DMSO, Santa Cruz Biotechnology), the PI3K inhibitor LY294002 (at 5 μM and 10 μM in DMSO, Tocris), the mTOR inhibitor rapamycin (at 10 nM and 20 nM in ethanol, Sigma) or 0.2% DMSO as a control (final DMSO concentration of 0.05% for PD174285, 0.1% for PD035901 and LY294002, and 0.2% for PD035901/LY294002/rapamycin treatment).

Techniques: In Vitro, Activation Assay, Inhibition, Control, Protein-Protein interactions, Expressing

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Generation of multispecific DB-VHH constructs. Trastuzumab IgG1-K409R mAb (anti-HER2) C-termini are fused to VHHs directed against EGFR, IL6R or NKG2D via a GS-linker. CS06 IgG1-F405L mAb (anti-c-MET) is fused to the same VHH molecules in the same manner. After recombinant production and purification, parental IgG-VHHs are recombined pairwise by reduction and reoxidation. The matching K409R and F405L mutations drive the generation of heterodimeric multispecific DB-VHHs.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Construct, Recombinant, Purification

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Binding affinities measured for each paratope using recombinant antigens

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Recombinant, Construct

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Biolayer interferometry analysis of simultaneous antigen binding of tri- and tetraspecific DB-VHHs. (a), (b) and (c) show exemplary sensorgrams for trispecific molecules and (d) a tetraspecific DB-VHH. The first association step represents binding of the DB-VHH (200 nM) via its CS06 paratope to biotinylated c-MET immobilized to streptavidin biosensors. Second (and third for (D)) association step is performed using an IL6R, EGFR or NKG2D recombinant protein (200 nM). The last association step is performed using HER2 (200 nM). Kinetic buffer (KB) controls were applied as negative controls for each association step.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Recombinant

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Specific cell clustering due to simultaneous binding of the DB-VHHs to three different cancer cell lines. Flow cytometry cytograms represent the fluorescence signals of the different cell populations. (a) Cells without antibody construct. Upper left gate = HCC-1954 (HER2 +++ ) cells stained with DeepRed, lower left gate = MDA-MB-468 (EGFR +++ ) cells stained with CMRA, lower right gate = EBC-1 (c-MET ++ ) cells stained with CFSE, upper right gate = HCC-1954 + EBC-1 cell doublets. Events in all three fluorescence channels (cell triplets) were marked in red. Cells were incubated in the presence of 1 nM (b) bispecific DB, (c) and (d) trispecific DB-VHHs, (e) and (f) tetraspecific DB-VHHs.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Flow Cytometry, Fluorescence, Construct, Staining, Incubation

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Simultaneous interaction of the DB-VHHs with HCC-1954 and EBC-1 target cells and binding of recombinant IL6R. Flow cytometry cytograms represent the fluorescence signals of the two cell populations and bound recombinant IL6R, detected with an anti-His6 detection antibody. (a) Cells without antibody construct. Upper left gate = HCC-1954 (HER2+++) cells stained with DeepRed, lower right gate = EBC-1 (c-MET++) cells stained with CFSE, upper right gate = HCC-1954 + EBC-1 cell doublets. Events in all three fluorescence channels (HCC-1954 + EBC-1 + bound recombinant IL6R-His Tag) were marked in blue. Cells were incubated in the presence of 10 nM (b) bispecific DB, (c) and (d) trispecific DB-VHHs, (e) and (f) tetraspecific DB-VHHs.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Recombinant, Flow Cytometry, Fluorescence, Construct, Staining, Incubation

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: DB-VHHs elicit potent and specific NK cell-mediated target cell killing. Tumor cells were incubated with primary effector cells (NK cells) at a 1:5 ratio in the presence of antibody constructs in different concentrations. Error bars represent standard deviation of two biological replicates. Wildtype CS06 and trastuzumab with active Fc effector functioning were used as an ADCC reference (green). (a) and (b): c-MET-positive EBC-1 target cells, (c) and (d) HER2-overexpressing SK-BR-3 target cells. (a) and (c): NK cell cytotoxicity triggered by parental antibodies. (b) and (d): DB-VHHs serve as NK cell engager and mediate tumor cell killing.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Incubation, Construct, Standard Deviation

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet:

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Variant Assay, Hydrophobic Interaction Chromatography, Mutagenesis, Molecular Weight, High Performance Liquid Chromatography

Journal: Biochemical pharmacology

Article Title: BRET-based assay to monitor EGFR transactivation by the AT 1 R reveals G q/11 protein-independent activation and AT 1 R-EGFR complexes

doi: 10.1016/j.bcp.2018.10.017

Figure Lengend Snippet: (A) Schematic of AT1R-EGFR BRET-based transactivation. EGFR fused to a BRET donor (Rluc8), is co-transfected with Grb2 adaptor protein tagged with a BRET acceptor (Venus) and the AT1R. Stimulation of the AT1R promotes activation of the EGFR and recruitment of Grb2. (B) HEK293 cells expressing AT1R, EGFR-Rluc8, and Grb2-Venus were treated with 10μM AngII, 1μM EGF or vehicle. Quantification of ligand-induced BRET ratio (maximum-minimum) between EGFR-Rluc8 and Grb2-Venus following AngII- and EGF-stimulation. Insert is HEK293 cells (stably expressing AT1R) stimulated with 100nM AngII, 10nM EGF or vehicle for 5 minutes before processing for phospho-ERK1/2:total-ERK1/2 (p-ERK:T-ERK) western blots. Blots are representative of 3 independent experiments (B inset) Cells expressing AT1R, Grb2-Venus and either EGFR-Rluc8, HER2-Rluc8 or HER3-Rluc8 and stimulated with 10μM AngII. Agonist stimulation is indicated by arrow. Data represent mean ± SEM of 3 independent experiments.

Article Snippet: EGFR-Rluc8, HER2-Rluc8 and HER3-Rluc8 were generated by inserting EGFR, HER2, and HER3, obtained from Origene (Rockville, MD, USA) into pcDNA3-Rluc8.

Techniques: Transfection, Activation Assay, Expressing, Stable Transfection, Western Blot

Journal: Scientific reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Figure 1. Unique fragments of the CDRs within the heavy and light chains of the new mouse monoclonal anti-HER2 antibody. The amino acid sequence corresponds to the characteristic nucleotide sequence of (A) anti-human HER2/70.27.58 mAb and (B) anti-human HER2/70.21.73.67 mAb.

Article Snippet: The concentration of HER2 protein in the analyzed sample was calculated based on the standard curve established with the recombinant HER2 protein Fc Tag (Acro BIOSYSTEMS, Newark, DE, USA) loaded on the plate at the concentration of 1.56–100 ng/ml.

Techniques: Sequencing

Journal: Scientific reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Figure 2. Anti-HER2 monoclonal antibodies production and characterization. (A) Morphology of the anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 hybridoma cells photographed at 20 × and 40 × magnification. (B) FPLC chromatograms were recorded during the purification of the anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 antibodies using affinity chromatography on the Protein A resin. (C) SDS-PAGE analysis of the purified anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 antibodies loaded at the amount of 1 µg/well on the 12% polyacrylamide gel under reducing conditions. (D) WB analysis of HER2 in whole cell lysates of the HER2 low expressing (MDA-MB-231) and HER2 high expressing (SK-BR-3, SK-OV-3) cells, probed with the home-made anti-human HER2/70.27.58 monoclonal antibody and detected with the secondary anti-mouse IgG-HRP (upper panel). The recombinant HER2 ECD protein was used as a reference. The loading control was performed with membrane probed with antibody binding β-actin (lower panel). (E) The formaldehyde-fixed SK-OV-3 cells were photographed in the bright field (BF) at the 40 × magnification. Immunofluorescence analysis was performed on cells stained with the commercial anti- HER2 ECD antibody followed by anti-mouse IgG-AlexaFluor594 (AF594) (red channel) and co-stained with the anti-HER2/70.27.58 or anti-HER2/70.21.73.67 antibodies detected with the AlexaFluor488-labeled (AF488) secondary antibody (green channel). Nuclei were stained with DAPI (blue channel) (F) Quantitative ELISA with the anti-human HER2/70.27.58 and anti-human HER2/70.21.73.67 antibodies loaded in a range of 0–5 µg/ ml on the plate coated with the recombinant chimera of the HER2 ECD-Fc protein. The signal generated from secondary antibody anti-mouse IgG-HRP was quantified by measuring absorbance at 450 nm and expressed after background subtraction (A450-A0).

Article Snippet: The concentration of HER2 protein in the analyzed sample was calculated based on the standard curve established with the recombinant HER2 protein Fc Tag (Acro BIOSYSTEMS, Newark, DE, USA) loaded on the plate at the concentration of 1.56–100 ng/ml.

Techniques: Bioprocessing, Purification, Affinity Chromatography, SDS Page, Expressing, Recombinant, Control, Membrane, Binding Assay, Immunofluorescence, Staining, Labeling, Enzyme-linked Immunosorbent Assay, Generated

Journal: Scientific reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Figure 3. Parameters of the sandwich ELISA for HER2 detection. (A) HER2 binding kinetics in the standard curve concentration range of 0.156–10 000 ng/well (1.56–100 ng/ml). Results are expressed as absorbance at 450 nm after background subtraction (A450-A0). (B) Assay accuracy was tested by comparison of HER2 level measured by ELISA in the samples of the known antigen concentration (mock samples). Data were collected for 2, 5, 10, 30, and 50 ng/ml of HER2 (given concentration; x-axis), covering both physiological and increased concentrations. Experimentally measured concentration [ng/ml] is shown on the y-axis. Error bars indicate SD.

Article Snippet: The concentration of HER2 protein in the analyzed sample was calculated based on the standard curve established with the recombinant HER2 protein Fc Tag (Acro BIOSYSTEMS, Newark, DE, USA) loaded on the plate at the concentration of 1.56–100 ng/ml.

Techniques: Sandwich ELISA, Binding Assay, Concentration Assay, Comparison, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

doi: 10.1038/s41598-024-54590-z

Figure Lengend Snippet: Figure 4. HER2 expression in tumors from mice with xenografted human cancer cells. (A) Immunohistochemistry staining using anti-HER2/70.27.58 mAb of the mouse tumors induced with the human ovarian cancer cells (SK-OV-3) overexpressing HER2 and (B) human epithelial breast cancer cells (MDA-MB-231) with low expression of HER.

Article Snippet: The concentration of HER2 protein in the analyzed sample was calculated based on the standard curve established with the recombinant HER2 protein Fc Tag (Acro BIOSYSTEMS, Newark, DE, USA) loaded on the plate at the concentration of 1.56–100 ng/ml.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: NPJ breast cancer

Article Title: Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates.

doi: 10.1038/s41523-021-00290-0

Figure Lengend Snippet: Fig. 1 Schematic representation of the EpiView-D4 mobile pathology platform for breast cancer. The device has a smart- phone base which uses detachable modules for brightfield (top) and fluorescence (bottom) imaging using the cell phone camera. Tumor is first sampled by FNA, and then aspirates are processed for cytology and biomarker evaluation. For cytology, aspirates are smeared on a glass slide and processed with rapid (DiffQuikTM) staining kits and then imaged with the brightfield imaging attachment (top). For biomarker evaluation, aspirates are mixed with lysis buffer and then applied to a D4 immunoassay chip, which quantify an analyte of interest (in this case, HER2). D4 chips are read using the fluorescence imaging attachment. The fluorescence intensity of the cAb spots on the D4 chip correlates with the biomarker expression level.

Article Snippet: To determine analytical sensitivity, analyte binding curves were generated using recombinant HER2 protein (R&D systems, Inc.) spiked into RIPA buffer.

Techniques: Imaging, Biomarker Discovery, Staining, Lysis, Expressing

Journal: NPJ breast cancer

Article Title: Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates.

doi: 10.1038/s41523-021-00290-0

Figure Lengend Snippet: Fig. 2 D4 sandwich immunoassay for quantification of HER2 expression level and its in vitro assessment. a–c, Schematic and operation of D4 immunoassay. a Spots of immobile cAb and an excess of “soluble” fluorescently labeled dAb are printed directly onto POEGMA-coated glass. b Dispensing sample fluid onto chip surface leads to dissolution of soluble dAb spots, followed by diffusion-driven mixing and antibody “sandwich” formation if HER2 is present. HER2 binding is detected by fluorescence imaging. c Representative fluorescence image of D4 assay after exposure to HER2-spiked RIPA buffer. Scale bar, 1 mm. d Representative dose-response curve generated from D4 chips for RIPA buffer spiked with recombinant HER2. Error bars: mean ± s.d. of duplicate assays. e, Assessment of cultured breast cancer cell lines (BT474, BT20, MDA-MB-231, MDA-MB-468) by D4 assay and comparison with western blotting. Inset: Western blot against HER2 for each cell line, with representative D4 cAb spots underneath. Main: D4 signal intensity (mean ± 95% CI) of ≥6 replicates. Significant difference by one-way ANOVA (F(4, 31) = 179.4, p < 0.0001). Bars with different letters indicate different groups (Tukey post hoc test, p ≤0.05). f Concordance analysis of 8 different patient-derived tumor cell lines in culture for HER2 expression by D4 assay vs. ELISA performed by clinical lab; specimens indexed “a” through “h”. D4 results are mean ± s.d. of duplicate assays. Pearson’s r = 0.975 (p < 0.0001, 95% CI: 0.845–0.996).

Article Snippet: To determine analytical sensitivity, analyte binding curves were generated using recombinant HER2 protein (R&D systems, Inc.) spiked into RIPA buffer.

Techniques: Expressing, In Vitro, Labeling, Dissolution, Diffusion-based Assay, Binding Assay, Imaging, Generated, Recombinant, Cell Culture, Comparison, Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: NPJ breast cancer

Article Title: Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates.

doi: 10.1038/s41523-021-00290-0

Figure Lengend Snippet: Fig. 3 EpiView mobile microscope for brightfield and fluorescence imaging. a–d Optomechanical design of EpiView. Labeled 3D schematic and photographs of the EpiView scope with brightfield (a, b) and epifluorescence (c, d) assemblies. e, f Raw resolution of brightfield imaging from unprocessed images of USAF 1951 test target. Brightfield images of test target (green channel extracted) are shown in e, with red and blue dashed lines in image corresponding to vertical (red) and horizontal (blue) intensity profiles of test target features in f. Comparison of brightfield imaging of FNA cytology from human breast cancer specimen using standard benchtop microscopy (g) and EpiView device (h). Left panels: standard microscope image obtained by a 10× objective lens (top) and native view on EpiView (bottom). Scale bar, 0.2 mm. Right panels: standard microscope image obtained by a 40× objective (top), showing the same ROI outlined with red dashed line in the 10× image, and similar view obtained by digital zoom on EpiView (bottom). Scale bar, 30 µm. i, j Resolution testing with USAF 1951 in a manner similar to e and f but in epifluorescence mode based on extracting green channel image. k Left panel: Widefield view of 200 nm fluorescence beads using EpiView in epifluorescence mode. Scale bar, 0.1 mm. Right panel: Comparison of EpiView versus and benchtop microscope (40× objective, NA = 0.6) of the 200 nm beads outlined by the red and blue ROIs in the widefield image. Images were extracted from green channel. Scale bars, 40 µm. l Dose-response curve of HER2 with D4-EpiView and a conventional glass slide scanner. Limit of detection for this experiment using conventional glass scanner (LODG) is 21 pM, while that of the D4-EpiView (LODE) is 77 pM. Data represent log signal intensity ± s.d for duplicate assays for both EpiView-D4 and the slide scanner.

Article Snippet: To determine analytical sensitivity, analyte binding curves were generated using recombinant HER2 protein (R&D systems, Inc.) spiked into RIPA buffer.

Techniques: Microscopy, Imaging, Labeling, Comparison

Journal: NPJ breast cancer

Article Title: Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates.

doi: 10.1038/s41523-021-00290-0

Figure Lengend Snippet: Fig. 4 Evaluation of solid tumor xenografts from nude mice orthotopically engrafted with human breast cancer cell lines with EpiView- D4. a Schematic of workflow. Tumor aspirates are divided into two aliquots; one aliquot from an aspirate is processed as a cytology specimen on a glass slide using a Diff-QuikTM rapid staining kit and then imaged using the brightfield imaging attachment (top row), The second aliquot is lysed in RIPA buffer and applied to a D4 chip and its HER2 level is quantified using the fluorescence attachment on the EpiView-D4. b Representative western blots against HER2 for each human breast cancer cell line used for xenografting (BT20, BT474, and MDA-MB-453). Vinculin used as loading control. c Results of HER2 credentialing for 16 different solid tumor specimens from BT20, BT474, and MDA-MB-453 xenografts (N = 5, 8, 3, respectively). Each data point represents the D4 fluorescence intensity (average of duplicates) measured by the EpiView-D4 for individual mouse tumors, categorized by xenograft line. Also shown is the mean ± 95% CI fluorescence intensity for each category, which mirrors the western blots shown in panel b. There was a statistically significant difference between groups as determined by one-way ANOVA (F (2, 13) = 76.10, p < 0.0001). Multiple comparison testing showed significant differences between each group (Tukey post hoc test, p ≤0.05). d D4 fluorescence intensity by EpiView-D4 for each mouse tumor from f plotted against corresponding HER2 ELISA. Pearson r = 0.968, p < 0.0001, 95% CI: 0.910–0.989). e–g Representative LPF (main panel) and HPF (inset) images of FNA preparations for each xenograft line confirm malignant cytology (see Supplementary Fig. 4 for representative images with standard microscope). Scale bars for LPF and HPF images are 0.2 mm and 30 µm, respectively.

Article Snippet: To determine analytical sensitivity, analyte binding curves were generated using recombinant HER2 protein (R&D systems, Inc.) spiked into RIPA buffer.

Techniques: Staining, Imaging, Western Blot, Control, Comparison, Enzyme-linked Immunosorbent Assay, Microscopy