Abstract
We compare three different methods to quantify the monosaccharide fucose in solutions using the displacement of a large glycoprotein, lactoferrin. Two microfluidic analysis methods, namely fluorescence detection of (labeled) lactoferrin as it is displaced by unlabeled fucose and the displacement of (unlabeled) lactoferrin in SPR, provide fast responses and continuous data during the experiment, theoretically providing significant information regarding the interaction kinetics between the saccharide groups and binding sites. For comparison, we also performed a static displacement ELISA. The stationary binding site in all cases was immobilized S2-AAL, a monovalent polypeptide based on Aleuria aurantia lectin. Although all three assays showed a similar dynamic range, the microfluidic assays with fluorescent or SPR detection show an advantage in short analysis times. Furthermore, the microfluidic displacement assays provide a possibility to develop a one-step analytical platform.
Highlights
Competitive binding of analytes in a biological sample to an immobilized receptor in flow-based systems is used in many clinical and research applications for detection and quantification of biomolecules [1]
We show that exposing a S2-aurantia lectin (AAL)-coated surface complexed with fluorescently labeled lactoferrin to a solution containing free fucose allows the fucose concentration to be quantified by monitoring displacement of labeled lactoferrin in a microfluidic system
The microchannel was saturated with fluorescently labeled lactoferrin and perfused with various concentrations of fucose [fuc]
Summary
Competitive binding of analytes in a biological sample to an immobilized receptor in flow-based systems is used in many clinical and research applications for detection and quantification of biomolecules [1]. While most traditional affinity assays depend on the association kinetics of binding between the receptor and analyte, assays using measurements of dissociation of a labeled antigen from its receptor often provide a valuable alternative [2]. Exposure to a sample with target analyte will cause dissociation of the Applied Biochemistry and Biotechnology (2019) 188:868–877 antibody-antigen complex and a signal that is dependent on the concentration of target analyte can be registered by measuring either the amount of label that is released from the surface or the decrease of label on the surface [3]. A problem with displacement immunoassays is that the interaction between the antibody and the labeled antigen is often of high affinity, which makes the dissociation of the complex unfavorable. Unspecific interactions between the labeled ligand and other parts of the sensor surface, apart from the antibody, may affect dissociation negatively [4]
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