Journal: mAbs

Article Title: Discovery and characterization of COVA322, a clinical-stage bispecific TNF/IL-17A inhibitor for the treatment of inflammatory diseases

doi: 10.1080/19420862.2015.1093266

Figure Lengend Snippet: Plasma concentration of COVA322 in cynomolgus monkeys. ( A ) Bifunctional ELISA was performed to detect intact COVA322. Biotinylated TNF was immobilized in the wells of neutravidin-coated microtiter 96-well plates. Plasma containing COVA322 was added to the wells. For detection, digoxigenin-labeled IL-17A was used, followed by an anti-digoxigenin antibody-HRP conjugate for substrate processing and color development. Monofunctional ELISA was performed to detect specific TNF binding of COVA322, adalimumab or golimumab. Biotinylated TNF was immobilized in the wells of neutravidin-coated microtiter 96-well plates. Plasma containing COVA322 or anti-TNF antibody was added to the wells. TNF binding compound was detected using an anti-human IgG antibody-HRP-conjugate for substrate processing and color development. ( B ) Plasma concentrations of COVA322 and the anti-TNF antibodies adalimumab and golimumab at different time-points after a single iv injection into cynomolgus monkeys. The COVA322 or anti-TNF antibody concentration in plasma was determined by Bifunctional and Monofunctional ELISA, respectively. Mean plasma concentrations of 3 cynomolgus monkeys are plotted versus time, error bars represent standard deviations (SD). ( C ) Comparison of the plasma concentrations of COVA322 in cynomolgus monkeys determined by Bifunctional and Monofunctional ELISA. The plasma concentrations obtained from both assays are comparable, indicating that COVA322 is stable in cynomolgus monkeys for at least 220 hours.

Article Snippet: The wells were washed with wash buffer (PBS, 0.1 % Tween 20) and then incubated with 100 μl/well of a solution containing 0.2 μg/ml of biotinylated TNF-α (PeproTech, biotinylated using a biotin labeling kit from ANP Technologies according to the manufacturer's instructions) diluted in assay buffer (PBS, 1 % BSA).

Techniques: Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Labeling, Binding Assay, IV Injection, Comparison

Journal: Communications Biology

Article Title: DIAPH1 mediates progression of atherosclerosis and regulates hepatic lipid metabolism in mice

doi: 10.1038/s42003-023-04643-2

Figure Lengend Snippet: Ldlr − / − and Ldlr − / − Diaph1 − / − male mice were fed Western Diet (WD) for 16 weeks. a Representative images of en face Oil Red O staining of aortas. Quantification of plaque area as percentage of Oil Red O-stained area over total aortic surface area is shown. b – e Representative images of aortic arch sections are shown and quantified for the following: b H&E; c Oil Red O; d CD68; and e , Picrosirius Red. In d , the secondary antibody-alone control is shown. Scale bar: 250 µm. The mean ± SEM is reported. The number of independent mice/group is indicated in the figure as individual data points. Statistical analyses regarding testing for the normality of data followed by appropriate statistical analyses were described in Materials and Methods. P values were determined by unpaired T-test.

Article Snippet: Sections were incubated with propylene glycol for 2 min, then incubated with pre-heated Oil Red O staining solution (American MasterTech, STOROPT) at 60 °C for an additional 8 min.

Techniques: Western Blot, Staining

Journal: Communications Biology

Article Title: DIAPH1 mediates progression of atherosclerosis and regulates hepatic lipid metabolism in mice

doi: 10.1038/s42003-023-04643-2

Figure Lengend Snippet: Ldlr − / − and Ldlr − / − Diaph1 −/− male mice were fed WD for 16 weeks. a Representative immunofluorescence staining and quantification of DIAPH1 in the liver of Ldlr −/− and Ldlr −/− Diaph1 −/− male mice. b Representative images of H&E and Oil Red O staining in liver and quantification is shown. c Quantification of free cholesterol content in liver. d Quantification of total cholesterol content in liver. e Quantification of total liver triglycerides. f Representative images of Picrosirius Red staining in liver and quantification is shown. g Quantification of whole liver weight. The mean ± SEM is reported. The number of independent mice/group is indicated in the figure as individual data points. In a the secondary antibody–alone control is shown. Scale bars: 250 µm, and inset boxes: 50 µm. Statistical analyses regarding testing for the normality of data followed by appropriate statistical analyses were described in Materials and Methods. P- values were determined by unpaired T-test or Wilcoxon rank-sum test depending on if the data passed the Shapiro-Wilk normality test.

Article Snippet: Sections were incubated with propylene glycol for 2 min, then incubated with pre-heated Oil Red O staining solution (American MasterTech, STOROPT) at 60 °C for an additional 8 min.

Techniques: Immunofluorescence, Staining