To measure the binding affinities (KD equilibrium dissociation constant) of the purified antibodies (bispecific or monoclonal) to soluble antigens, the antibodies were first captured onto streptavidin SAX biosensor tips with a biotinylated (1 biotin/molecule), polyclonal capture antibody (Jackson ImmunoResearch, catalog# 109–005-098) and then incubated with a dilution series of each soluble antigen. This assay format was chosen so the bivalent antibodies would be immobilized and on the biosensor tips and tested versus the same serial dilution of each soluble antigen. The measured quantitative KD affinities thus represent monovalent 1:1 binding interaction and can be directly compared. Experiments were run in a ForteBio Octet HTX instrument using the 96-tip mode with standard 5 Hz data acquisition rate at 27°C and 1000 RPM. Using Genedata Screener V16 software, raw Octet binding data was processed with installed SPR kinetic curve fitting package and globally fit to a 1:1 binding model to determine the association rate constant (ka) and the dissociation rate constant (kd). The equilibrium dissociation constant (KD) was then calculated as a ratio of kd/ka. The KD values are average ± standard error from at least three independent experiments. The representative sensorgrams shown in Figure 6 are in each case from one of the experiments used in the analysis.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.