Translational Medicine

QPS and GSK Scientists Publish on the Effects of Freeze-Thaw on Biotinylated Macromolecules Used in GyrolabTM

The high affinity between biotin and avidin makes biotinylation use common in ligand-binding-assays for large-molecule bioanalysis. However, the added biotin/spacer moiety might affect the labeled molecule’s activity.

The current GyrolabTM immunoassay system requires the capture reagent to be biotinylated so it will bind to the solid phase - a streptavidin-coated-bead column. During method development for a Gyrolab assay to quantify otelixizumab, the response and assay sensitivity were found to be affected by freeze-thaw. It was thought that these variations may have been related to the spacers between the biotin and the labeled antibody. Hydrophilic polyethylene glycol (PEG) spacers enhance water solubility but could be more susceptible to freeze-thaw effects than purely hydrocarbon spacers. The overall response increased 10-fold after the PEG-reagent was incubated benchtop for 8 hours after thawing. The signal-to-background increased 8-fold for the same treatment. In contrast, no significant change upon freeze-thaw was observed for reagents with hydrocarbon spacers.

Nonetheless, the authors concluded that since Gyrolab assays do not generally require prolonged incubation, they still provide an effective tool for assessing critical reagents in assay development and optimization, especially for evaluating time-dependent parameters for immunoassays.

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