Journal: Protein Science : A Publication of the Protein Society

Article Title: Chemical synthesis and characterization of wild-type and biotinylated N-terminal domain 1-64 of ?2-glycoprotein I

doi: 10.1002/pro.387

Figure Lengend Snippet: ELISA experiments. A: Competitive inhibition experiments with plate-bound β2GpI were carried out by mixing diluted plasma from APS patients with an equal volume of a solution at increasing concentrations of competitor: β2GpI (○), N-DmI (•), and RC-DmI (▪). The resulting solution was added to β2GpI-coated wells and incubated for 1 h at 37°C. After washing, quantification of anti-β2GpI Abs still bound to the microplate-absorbed β2GpI was performed by using a secondary alkaline phosphatase-conjugated anti-human IgG antibody in the presence of pNPP. The release of p-nitrophenol was determined by measuring the absorbance of the solution at 405 nm, after incubation for 30 min at 25°C. Fitting of data points to Eq. (4) yielded the IC50 values reported in Table ​TableI.I. B: Direct binding ELISA experiments were carried out by incubating streptavidin-coated microtiter plates with a solution of Biotin-N-DmI. Thereafter, a fixed volume of plasma from selected APS patients was added, and quantification of anti-β2GpI Abs bound to Biotin-N-DmI was carried out as detailed in A. Open symbols (○, □) refer to the experimental values, whereas filled symbols (•, ▪) are the average values together with the standard deviation.

Article Snippet: These results are unprecedented and disclose novel opportunities for developing more reliable diagnostic tools based on avidin/biotin ELISA systems.

Techniques: Enzyme-linked Immunosorbent Assay, Inhibition, Clinical Proteomics, Incubation, Binding Assay, Standard Deviation

Journal: Protein Science : A Publication of the Protein Society

Article Title: Chemical synthesis and characterization of wild-type and biotinylated N-terminal domain 1-64 of ?2-glycoprotein I

doi: 10.1002/pro.387

Figure Lengend Snippet: IC 50 values (μ M ) obtained by ELISA competition binding experiments a

Article Snippet: These results are unprecedented and disclose novel opportunities for developing more reliable diagnostic tools based on avidin/biotin ELISA systems.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Cells

Article Title: Blockade of Glial Connexin 43 Hemichannels Reduces Food Intake

doi: 10.3390/cells9112387

Figure Lengend Snippet: ( A – D ): Diffusion of biotinylated TAT-GAP19 into the hypothalamus ( A , B ) and hindbrain ( C , D ) 1 h after its i.c.v. administration. White lines delimit brain tissue. Scale bars: 1 mm in ( A ) and ( C ); 500 μm in ( C ) and ( D ). ( D – F ) : High magnification of biotinylated TAT-GAP19 25 µg diffusion within the hypothalamus 30 min ( D ), 1 h ( E ) and 2 h ( F ) after its injection into the lateral ventricles. Scale bar: 500 µm. ( G ): High magnification of biotinylated TAT-GAP19-I14A 25 µg diffusion within the hypothalamus 1 h after its injection into the lateral ventricles. Scale bar: 500 µm. ( H ): High magnification of biotinylated TAT-GAP19-I14A diffusion within the hypothalamus 1 h after its injection into the lateral ventricles. White lines ( D – G ) and yellow lines ( H ) delimit diffusion area of biotinylated peptides. ARC: arcuate nucleus; 3V: third ventricle; 4V: fourth ventricle; NTS: nucleus tractus solitarius; LV: lateral ventricle.

Article Snippet: A biotinylated goat anti-rabbit IgG (1/400, #BA1000 Vector) was used as a secondary antibody (incubated for 1 h 30 at room temperature).

Techniques: Diffusion-based Assay, Injection

Journal: NPJ Vaccines

Article Title: Three immunizations with Novavax’s protein vaccines increase antibody breadth and provide durable protection from SARS-CoV-2

doi: 10.1038/s41541-024-00806-2

Figure Lengend Snippet: WA-1 Spike-binding IgG titers in plasma before and after depletion of RBD-binding antibodies ( A ), and proportion of RBD-binding antibodies at different timepoints ( B ) ( n = 6). C Correlation between proportion of RBD-specific IgG antibodies and RBD-specific MBCs in the blood ( n = 6). D Neutralization potency index, defined as the ratio between WA-1 neutralizing and RBD-binding antibody titers 2 weeks after each immunization ( n = 6). E Avidity index of RBD-binding IgG antibodies at different timepoints ( n = 6), determined by chaotropic wash ELISA with 2 M NaSCN as the chaotropic reagent. F Schematic of RBD with representative antibodies for four defined binding classes (PDB IDs 7K90 (RBD + C144 mAb), 7BZ5 (B38 mAb), 6WPS (S309 mAb), 6W41 (CR3022 mAb)). Receptor binding site (RBS) is labeled in red. G Relative serum reactivity to each of the four defined RBD-binding antibody classes (site I-site IV, n = 6), determined through ELISA competition assay using biotinylated monoclonal antibodies listed in ( F ). Total RBD-binding IgG and neutralization titers are shown on the right. Dose 2 and 3 represent timepoints 4 weeks after each immunization. H Frequency of Spike- and RBD-specific MBCs 2 weeks after each immunization ( n = 6). Binding to different Spike subdomains (RBD, NTD, or S2) at selected timepoints, measured by multiplexed Luminex bead-based assay ( n = 6) ( I ) and the fold changes in the binding titers from timepoint to timepoint (Dose 1 = week 2/week 0, Dose 2 = week 6/week 2, Dose 3 = week 37/week 6) ( J ). Data is presented as mean ± SEM ( B , D , E ) or geometric mean ± geometric SD ( H , I ). Statistical analysis was performed using Kruskal-Wallis test with Dunn’s post hoc correction ( B ), Spearman correlation ( C ) or Friedman’s test with Dunn’s post hoc correction ( D , E , H , I ). Baseline refers to week 0, dose 1, dose 2 and dose 3 refer to 2 weeks after each immunization in all panels except in ( G ). Dotted line corresponds to lower level of detection ( A ), fold change = 1 ( D , H ). or the baseline signal at week 0 ( I ).

Article Snippet: After incubation, plates were washed with PBS-T and biotinylated protein probes were added for 1.5 h (0.25 μg/mL goat anti-human IgG Fc fragment-specific antibody (Jackson ImmunoResearch, cat# 109-065-008), 1 μg/mL S-2P/RBD/ovalbumin (OVA)).

Techniques: Binding Assay, Clinical Proteomics, Neutralization, Enzyme-linked Immunosorbent Assay, Labeling, Competitive Binding Assay, Bioprocessing, Luminex, Bead-based Assay

Journal: Nature genetics

Article Title: Therapeutic modulation of eIF2α-phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models

doi: 10.1038/ng.2853

Figure Lengend Snippet: a–d, Immunostaining for PABPC1 in the cervical spinal cord of control and ALS patients. a) PABPC1 is expressed in spinal cord motor neurons, and is localized throughout the cytoplasm in a diffuse pattern. b ) Occasionally PABPC1 cytoplasmic accumulations were observed in normal motorneurons (arrow). c) In ALS patient motor neurons, PABPC1 is present in distinct cytoplasmic and dense accumulations (arrow). d ) PABPC1 cytoplasmic accumulations were also observed in the ALS motorneurons where there was a clearing in the middle. Scale bars, 30 μm. e ) The number of motor neurons containing cytoplasmic accumulation of PABPC1 in ALS patients was significantly greater ( p =0.01, one-tailed Mann-Whitney test) than in motor neurons from controls. For quantification of accumulations of PABPC1 in individual normal versus ALS cases, see . n=5 controls, 4 ALS patients, mean ± s.e.m.

Article Snippet: The tissue was cut into 7 μm sections and PABPC1 was immunolocalized with antigen retrieval (Antigen unmasking solution, citric acid based, Vector Laboratories, catalog # H-3300) and the avidin-biotin complex detection method with 3,3-diaminobenzidine as the chromagen (Vectastain ABC detection kit, Vector Labs, catalog # PK-6100).

Techniques: Immunostaining, One-tailed Test, MANN-WHITNEY