Journal: eLife
Article Title: Structural basis of Plasmodium vivax inhibition by antibodies binding to the circumsporozoite protein repeats
doi: 10.7554/eLife.72908
Figure Lengend Snippet: Binding kinetics of twofold dilutions of 2F2 IgG and Fab ( A , upper panel and lower panel, respectively) to PvCSPvk210, and 2E10.E9 IgG and Fab ( B , upper panel and lower panel, respectively) to PvCSPvk247, as measured by biolayer interferometry (BLI). Representative sensograms are shown in black and 2:1 model best fits in red. Data shown are representative of three independent measurements. Isothermal titration calorimetry (ITC) analysis of 2F2 Fab binding to PvCSPvk210 ( C ) and 2E10.E9 Fab binding to PvCSPvk247 ( D ) at 25°C. ( C , top panels): raw data of PvCSPvk210 (5 µM) in the sample cell titrated with 2F2 Fab (240 µM) in the syringe. ( D , top panels): raw data of PvCSPvk247 (5 µM) in the sample cell titrated with 2E10.E9 Fab (400 µM) in the syringe. ( C, D , bottom panel): plot and trendline of heat of injectant corresponding to the raw data. Results from size-exclusion chromatography coupled with multiangle light scattering (SEC-MALS) for the Fab 2F2-PvCSPvk210 sample ( E , left panel) and 2E10.E9 Fab-PvCSPvk247 ( F , left panel) sample, where the Fabs are in molar excess. Measurement of the molar mass of the eluting complex is shown as a red line. Mean molar mass is indicated. SDS-PAGE analysis of resulting peaks 1 and 2 for 2F2 Fab-PvCSPvk210 ( E , right panel) and 2E10.E9 Fab-PvCSPvk247 ( F , right panel) samples from SEC-MALS. Each peak was sampled in reducing and nonreducing conditions as indicated by + and –, respectively. Figure 6—source data 1. SDS-PAGE analysis of 2F2 Fab-PvCSPvk210 and 2E10.E9 Fab-PvCSPvk247 complexes.
Article Snippet: Software, algorithm , MicroCal ITC Origin7.0 Analysis Software , Malvern , https://www.malvernpanalytical.com/ , .
Techniques: Binding Assay, Isothermal Titration Calorimetry, Size-exclusion Chromatography, Multi-Angle Light Scattering, SDS Page
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![( A ) Affinities of 2F2 Fab for peptides 210-1, 210-2, 210-3, 210-4, and 210-5 as measured by <t>isothermal</t> <t>titration</t> <t>calorimetry</t> (ITC). Open circles represent independent measurements. Mean binding constant (K D ) and binding stoichiometry (N) values are shown above the corresponding bar. Error bars represent SEM. ( B ) Upper panel: sequences of peptides used in ITC experiments with variable residues underlined. Dark gray denotes the core epitope of the peptide resolved in all the X-ray crystal structures, and light gray shading indicates residues resolved in the corresponding X-ray crystal structures. Bottom panel: comparison of the conformations of PvCSP210 peptides in X-ray crystal structures. PvCSPvk210 peptides are colored from navy to light blue, with the residues adopting one turn of a 3 10 -helix depicted in pink. ( C ) Top and side views of the 210-4 peptide (light blue) in the binding groove of the 2F2 Fab shown as surface representation (heavy chain [HC] shown in green and kappa chain [KC] shown in white). ( D ) Comparison of the conformations adopted by the core epitope of peptides 210-1, 210-2, 210-3, 210-4, and 210-5 when bound to 2F2. ( E ) Detailed interactions between Fab 2F2 and peptide 210-4. H-bonds and salt bridges are shown as black dashes, peptide 210-4 is shown in light blue, HC is shown in green, and KC is shown in gray. Fab residues are annotated with H or K letters to indicate heavy and kappa light chain, respectively.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9896/pmc08809896/pmc08809896__elife-72908-fig3.jpg)
![( A ) Affinities of 2E10.E9 Fab for peptides 247-1, 247-2, 247-3, and 247-4 as measured by isothermal titration calorimetry (ITC). Open circles represent independent measurements. Mean binding constant (K D ) and binding stoichiometry (N) values are shown above the corresponding bar. Error bars represent SEM. ( B ) Upper panel: sequences of peptides used in ITC with variable residues underlined. Dark gray denotes the core epitope of the peptide resolved in all X-ray crystal structures, and light gray shading indicates residues resolved in the corresponding X-ray crystal structures. Bottom panel: comparison of the conformations of PvCSP247 peptides in X-ray crystal structures, with peptides 247-2, 247-3, and 247-4 depicted in yellow, orange, and teal, respectively. ( C ) Top and side views of the 247-3 peptide (orange) in the binding groove of the 2E10.E9 Fab shown as surface representation (heavy chain [HC] shown in blue and kappa chain [KC] shown in white). ( D ) Comparison of the conformations adopted by the core epitope of peptides 247-2, 247-3, and 247-4 when bound to 2E10.E9. ( E ) Detailed interactions between Fab 2E10.E9 and peptide 247-3. H-bonds are shown as black dashes, peptide 247-3 is shown in orange, HC is shown in green, and KC is shown in gray. The Fab residues are annotated with H or K letters to indicate heavy and kappa light chain, respectively.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9896/pmc08809896/pmc08809896__elife-72908-fig4.jpg)
