Xiaodong F Liu, Roni Weaver, Charles Hottenstein, Matthew Szapacs, Lee Abberley, LingSing Chen and Xun Wang
Due to high affinity between biotin and avidin, biotinylation is widely used in ligand-binding-assay development for large-molecule bioanalysis. However, biotinylation adds biotin/spacer moiety onto the molecule and may affect the functional activity of the labeled molecule. The current Gyrolab™ immunoassay system requires the capture reagent to be biotinylated as the solid phase comprises a streptavidin-coated-bead column. During method development of a Gyrolab assay for quantification of otelixizumab, we discovered that the response and assay sensitivity was affected by freeze-thaw, which might be related to the type of spacers used between the biotin and the labeled antibody. The hydrophilic Polyethylene Glycol (PEG) spacer enhances water solubility but might be more liable to freeze-thaw compared to antibodies labeled with reagents having only hydrocarbon spacers. The overall response increased 10 fold after 8-hour incubation of the PEG-reagent at benchtop after thaw. 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. Since Gyrolab assays do not require prolonged incubation, it provides an effective tool for assessing critical reagents in assay development and optimization, especially for evaluating time-dependent parameters for immunoassays
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