Journal: Science advances

Article Title: Fragment-based computational design of antibodies targeting structured epitopes.

doi: 10.1126/sciadv.abp9540

Figure Lengend Snippet: Fig. 1. Workflow for the combinatorial structure-based CDR design strategy. (A) The antigen structure is shown in gray, with the epitope of interest highlighted in red. At this step, the epitope is fragmented into its structural fragments, both in a linear mode and in a surface-patch mode that yields also noncontiguous fragments (see text). Some example fragments are pointed by red arrows. (B) These fragments are used as queries for a structural search against a custom database of structures of antigen-like and CDR-like interacting fragments. Hits are selected on the basis of structural and sequence similarity with the query epitope fragments, and two example hits are depicted: an epitope fragment (pink top example, purple lower example) matching antigen-like fragments (yellow) interacting with a CDR-like fragment (cyan top example, blue lower example). These two examples originate respectively from the structures of human EML1 protein and of a bacterial transferase, as antigen-like and CDR-like fragments may be found in any structure from the PDB. (C) When possible, identified CDR-like fragments are joined together. Here, the overlapping CDR-like fragments from B are merged as they meet the stated compatibility criteria. (D) The sequence of the designed CDR fragment resulting from the merging is optimized to increase the probability of favorable CDR-epitope contacts. The final fully optimized designed CDR motif can then be grafted onto suitable antibody frameworks. The example in this figure corresponds to the designed binding motif within the CDR3 of DesAb-RBD-C1 targeting the ACE2-binding site on the RBD domain.

Article Snippet: Human ACE2 (18615) recombinant protein, used in the competition assay in Fig. 4F, was also purchased from The Native Antigen Company, where it was expressed in HEK293 cells with Sheep FcTag (REC31876).

Techniques: Sequencing, Binding Assay

Journal: Science advances

Article Title: Fragment-based computational design of antibodies targeting structured epitopes.

doi: 10.1126/sciadv.abp9540

Figure Lengend Snippet: Fig. 4. The anti-RBD DesAbs bind their target and compete with human ACE2. (A) The spike protein is shown in gray, and the RBD domain is shown in purple, with glycans in red. The designed CDRs are in blue and green for DesAb-RBD-C1 and DesAb-RBD-C2, respectively. (B and C) MST of Alexa Fluor 647–labeled trimeric spike protein (8 nM) in the presence of different concentrations of DesAb-RBD-C1 (red), DesAb-RBD-C2 (yellow), and DesAb-HSA-P2 (C; gray). Apparent Kd values are obtained by fitting a 1:1 binding model (see text). (D and E) BLI association and dissociation sensorgrams obtained with APS sensors loaded with RBD and blocked with HSA. Gray traces are obtained with 4 M of DesAb-Tryp used as a negative control. (D) DesAb-RBD-C1 (4, 2, and 1 M) (from darker to lighter blue, Kd = 214 ± 4 nM). (E) DesAb-RBD-C2 (4, 2.5, and 1 M) (from darker to lighter green, Kd = 135 ± 2 nM). Data were fitted globally. (F) Binding competition experiment with sensors loaded like in (D) and (E) and dipped in wells with 5 M DesAb-RBD-C1 (blue), DesAb-RBD-C2 (green), or buffer (gray), then in wells containing ACE2 or buffer controls (see legend), and finally back to buffer. The plot reports the last two steps, showing that the binding of ACE2 is substantially reduced by the presence of either DesAb-RBD-C1 or DesAb-RBD-C2 bound to the RBD.

Article Snippet: Human ACE2 (18615) recombinant protein, used in the competition assay in Fig. 4F, was also purchased from The Native Antigen Company, where it was expressed in HEK293 cells with Sheep FcTag (REC31876).

Techniques: Labeling, Binding Assay, Negative Control

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Serum ACE2 activity is significantly correlated with SBP in stroke-alert patients and healthy young adults, but not AIS patients. Correlation graphs of ACE2 activity and SBP among stroke-alert patients (a) and healthy young adults (b) as compared to stroke patients (c). Young adult blood plasma samples in panel (b) were from a biorepository established by Wegman et al., which were obtained from research participants undergoing baseline measurements. (d) Correlation graph of ACE activity and mRS at discharge from hospital among AIS patients. ACE2: angiotensin converting enzyme 2; AIS: acute ischemic stroke; mRS: modified Rankin score; RFU: relative fluorescence unit; SBP: systolic blood pressure.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay, Clinical Proteomics, Modification, Fluorescence

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Activity of ACE2 and ACE in serum is altered following stroke. For human serum, bar graphs are means ± SEM and represent enzyme activity levels of ACE2 (a) and ACE (c) from control, stroke-alert, or AIS patients at an average of 3.6 hours and again at 3 days after stroke. Individual differences and means ± SEM in ACE2 (b) and ACE (d) are shown. * P <0.05 versus control and † P <0.05 versus stroke-alert. ‡ P <0.05 versus AIS <6 hours. ACE: angiotensin converting enzyme; ACE2: angiotensin converting enzyme 2; AIS: acute ischemic stroke; RFU: relative fluorescence unit.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay, Control, Fluorescence

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Predictors of acute ischemic stroke by multiple linear regression analysis.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay

Journal: Viruses

Article Title: Design of Three Residues Peptides against SARS-CoV-2 Infection

doi: 10.3390/v14102103

Figure Lengend Snippet: BLI interaction analysis. RBD (0.2 µM) ( A , B ) either alone in solution or mixed with VFI ( A ) and TLH ( B ) peptides, tested at different concentrations (40 µM, blue lines; 80 µM, green lines and 160 μM, orange lines). BLI interaction was performed at 25 °C in PBST (10 mM phosphate, 150 mM NaCl, 0.05% Tween 20, pH 7.4). The corresponding plots ( C , D ) of steady-state binding from the end of the association phases (nm), after the subtraction of RBD signal, against analyte concentration were used to calculate the steady-state affinity by nonlinear regression analysis using GraphPad 5 software.

Article Snippet: An Octet ® Red 96 system ® (ForteBio, Fremont, CA, USA) was used to detect interactions between the His-tagged Spike RBD protein (Elabscience Biotechnology, Biomedical Park, Wuhan, China; Cat. No.: PKSH032068) and peptides using Octet His2 biosensors according to the standard instructions with minor modifications.

Techniques: Binding Assay, Concentration Assay, Software

Journal: medRxiv

Article Title: Development of an Optical Assay to Detect SARS-CoV-2 Spike Protein Binding Interactions with ACE2 and Disruption of these Interactions Using Electric Current

doi: 10.1101/2020.11.24.20237628

Figure Lengend Snippet: Schematic illustration of SARS-CoV-2 spike protein and ACE2 receptor binding. (a) SARS-CoV-2 binding to the ACE2 receptor on the host cell surface. (b) Binding of ACE2 and spike protein along with illustration of the spike protein subunits, S1 and S2. (c) Schematic showing the distribution of the spike protein in solution. (d) Schematic showing the distribution of ACE2 in suspension. (e) Distribution of ACE2 and S protein after binding in solution.

Article Snippet: The human ACE2 recombinant protein (ProSci Cat No. 10-114) was resuspended in sterile deionized water at a stock concentration of 500 ng/µl as per the manufacturers instruction and used for the analysis.

Techniques: Binding Assay

Journal: medRxiv

Article Title: Development of an Optical Assay to Detect SARS-CoV-2 Spike Protein Binding Interactions with ACE2 and Disruption of these Interactions Using Electric Current

doi: 10.1101/2020.11.24.20237628

Figure Lengend Snippet: Optical detection of binding interactions between ACE2 and other proteins. (a) Measured light intensities over time for individual assessment of ACE2, S1X, S1Y, and BSA. (b) The measured mixed light intensities versus time for ACE2 mixed with either S1X, or S1Y, or BSA. (c) The measured ACE2-S1X interaction profile for an extended time period.

Article Snippet: The human ACE2 recombinant protein (ProSci Cat No. 10-114) was resuspended in sterile deionized water at a stock concentration of 500 ng/µl as per the manufacturers instruction and used for the analysis.

Techniques: Binding Assay