nonlinear regression to a one-site specific binding curve function  (GraphPad Software Inc)

Journal: Pharmaceutics

Article Title: Anti-Fn14 Antibody-Conjugated Nanoparticles Display Membrane TWEAK-Like Agonism †

doi: 10.3390/pharmaceutics13071072

Figure Lengend Snippet: Specific binding of GpL-TNC-TNF to anti-TNF-AuNPs and anti-IL17A-AuNPs. Equal amounts of carboxyl-modified gold nanoparticles (AuNPs; 25 mg/mL) were conjugated with a constant concentration of anti-TNF or anti-IL17A (2 mg/mL). After removal of the excess unbound antibody molecules, the antibody-conjugated AuNPs were incubated with the indicated concentrations of the GpL-TNC-TNF fusion protein for an hour at 37 °C ( A ). Alternatively, increasing concentrations of the two antibodies were used for gold nanoparticle conjugation and were then incubated with a fixed concentration (500 ng/mL) of GpL-TNF-TNC ( B ). After removal of unbound GpL-TNF-TNF molecules, the nonspecific binding values (derived of the anti-IL17A-AuNPs) were subtracted from the corresponding total binding values (derived of the anti-TNF-AuNPs) to obtain specific binding values that were fitted using non-linear regression to a single binding site interaction plot using GraphPad Prism5 software.

Article Snippet: K D values were then calculated with the “nonlinear regression to a one-site specific binding curve” function of GraphPad Prism5.

Techniques: Binding Assay, Modification, Concentration Assay, Incubation, Conjugation Assay, Derivative Assay, Software

Journal: Pharmaceutics

Article Title: Anti-Fn14 Antibody-Conjugated Nanoparticles Display Membrane TWEAK-Like Agonism †

doi: 10.3390/pharmaceutics13071072

Figure Lengend Snippet: Specific binding of Fn14ed-GpL fusion protein to 5B6-AuNPs and PDL192-AuNPs. Three equal amounts of carboxyl-modified gold nanoparticles AuNPs (25 mg/mL) were conjugated with a constant concentration (2 mg/mL) of the anti-Fn14 antibodies 5B6 ( A ) and PDL192 ( B ) or anti-TNF as a negative control. After removal of unbound antibody molecules, the antibody-AuNP conjugates were incubated with the indicated concentrations of the Fn14ed-GpL fusion protein for an hour at 37 °C. After removal of the unbound Fn14ed-GpL molecules, nonspecific binding values were obtained from the anti-TNF-AuNPs and were subtracted from the corresponding total binding values obtained from the 5B6-AuNPs and PDL192-AuNPs to calculate the specific binding values. The latter were fitted using non-linear regression to a single binding site interaction plot using GraphPad Prism5 software.

Article Snippet: K D values were then calculated with the “nonlinear regression to a one-site specific binding curve” function of GraphPad Prism5.

Techniques: Binding Assay, Modification, Concentration Assay, Negative Control, Incubation, Software