human erbb2 her2 quantikine elisa  (R&D Systems)

Journal: Frontiers in Oncology

Article Title: Polyclonal antibody-induced downregulation of HER1/EGFR and HER2 surpasses the effect of combinations of specific registered antibodies

doi: 10.3389/fonc.2022.951267

Figure Lengend Snippet: Recognition of HER1/HER2 subdomains by the PAbs and inhibition of MAbs binding. Recognition of (A) HER1 or (B) HER2 subdomains I, III, and IV expressed on phages by the PAbs (10 µg/ml) assessed by ELISA. Samples were normalized against the absorbance obtained for the recognition of 9E10MAb. PAbs from non-immunized mice (CTR PAbs) were included as control. Graphs represent the increase of the absorbance at 490 nm corresponding to the recognition of each subdomain by specific or control PAbs, normalized considering the absorbance of the 9E10 control and expressed as relative reactivity. (C) HER1-ECD-coated plates were incubated with dilutions of PAbs-containing immune sera; next, the binding capacity of MAbs was evaluated by ELISA. Graphs represent the inhibition of HER1-ECD-targeting cetuximab (cetu, black circle), panitumumab (pani, black square), nimotuzumab (nimo, gray inverted triangle), and D1 (black triangle). Inhibition was expressed as percentage considering binding of the MAbs in the absence of PAbs as maximum reactivity control, and data were adjusted to a non-linear “response vs . log (inhibitor)” regression with variable slope. (D) Likewise, it was evaluated the inhibition of the binding of HER2-ECD recognizing trastuzumab (trastuz, open circle) and pertuzumab (pertuz, open square), mediated by the PAbs. A representative experiment of three performed is shown.

Article Snippet: Quantitative ELISA systems used for detection of HER1 (DEGFR0) or HER2 (DHER20) were obtained from R&D Biosystems.

Techniques: Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Control, Incubation

Journal: Frontiers in Oncology

Article Title: Polyclonal antibody-induced downregulation of HER1/EGFR and HER2 surpasses the effect of combinations of specific registered antibodies

doi: 10.3389/fonc.2022.951267

Figure Lengend Snippet: Promotion of the endocytosis and degradation of HER1 and HER2 by the PAbs. (A) H292, H1975, and PC9ER cells were treated for 24 h with PAbs-containing immune sera or pre-immune sera as negative control (1/100). After washing with acidic buffer (glycine 100 mM, pH 3.0), cells were incubated with antibodies against HER1 and HER2 tagged to fluorophores, and surface expression of the receptors was analyzed using flow cytometry. Death cells were excluded with DAPI. Density plots show the distribution of the cell population according to recognition of HER1 ( y -axis) and HER2 ( x -axis), and the percentage of cells expressing both receptors is specified. Data are representative of two independent experiments. (B) H292 and (C) H1975 cells were seeded in coverslips and treated as in (A) for 24 h. Cells were washed in acidic buffer, fixed in paraformaldehyde (4%), and incubated with specific primary antibodies, followed by an Alexa Fluor 555‐conjugated secondary antibody. Images were captured using a confocal microscope (40× magnification) and quantification was performed using ImageJ software. In the graphs, data are means ± SD of triplicates in a representative experiment of three conducted. Means were compared using Kruskal–Wallis test followed by a Games–Howell post-test. Significant differences among control and PAbs-treated cells are represented * p < 0.05.

Article Snippet: Quantitative ELISA systems used for detection of HER1 (DEGFR0) or HER2 (DHER20) were obtained from R&D Biosystems.

Techniques: Negative Control, Incubation, Expressing, Flow Cytometry, Microscopy, Software, Control

Journal: Frontiers in Oncology

Article Title: Polyclonal antibody-induced downregulation of HER1/EGFR and HER2 surpasses the effect of combinations of specific registered antibodies

doi: 10.3389/fonc.2022.951267

Figure Lengend Snippet: Comparative evaluation of the degradation of HER1 and HER2 in H292 cells expressing wild-type HER1. For dose-curve assessment, H292 cells were treated for 24 h with increasing concentrations (0, 0.5, 1, 2.5, 5, and 10 µg/ml) of the PAbs, cetuximab (cetu), or trastuzumab (trastuz). Thereafter, cells were washed with cold saline and lysed. Next, (A) HER1 (3 µg of total lysates) and (B) HER2 (30 µg of total lysates) expression levels were analyzed by Western blot. Detection of GAPDH was included as loading control for individual membranes. (C) For point-dose evaluation, H292 cells were treated for 24 h with the PAbs (10 µg/ml), or with combinations of cetuximab (cetu, 10 µg/ml) or nimotuzumab (nimo, 10 µg/ml) with trastuzumab (trastuz, 1 µg/ml). Unspecific PAbs (CTR, 10 µg/ml), single MAbs, or TKI AG1478 (10 µM) was included as controls. As previously described, expression levels of HER1 (3 µg of total lysates) and HER2 (30 µg of total lysates) were analyzed by Western blot, including GAPDH detection as loading control. Alternatively, quantitative ELISA was performed to measure (D) HER1 and (E) HER2 in the lysates of H292 cells treated as in (C) , following the manufacturer’s instructions. In the graphs, data are means ± SD corresponding to triplicates. A representative experiment of three performed is shown. Group means were compared using Kruskal–Wallis test followed by a Games–Howell post-test. Significant differences among PAbs and control, MAbs or its combinations are represented * p < 0.05; ** p < 0.01. MWM—molecular weight marker.

Article Snippet: Quantitative ELISA systems used for detection of HER1 (DEGFR0) or HER2 (DHER20) were obtained from R&D Biosystems.

Techniques: Expressing, Saline, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Molecular Weight, Marker