Abstract
Coupling of surface plasmon resonance (SPR) detection to asymmetric flow field-flow fractionation (AF4) offers the possibility to study active fractions of bio-separations on real samples, such as serum and saliva, including the assessment of activity of possibly aggregated species. The coupling of SPR with AF4 requires the possibility to select fractions from a fractogram and redirect them to the SPR. The combination of SPR with chromatography-like methods also requires a mechanism for regeneration of the receptor immobilised onto the SPR sensor surface. In recent work, the combination of size exclusion chromatography (SEC) with SPR was pioneered as a successful methodology for identification, characterisation and quantification of active biocomponents in biological samples. In this study, the approach using AF4 is evaluated for the antibody trastuzumab in buffer and serum. The particular object of this study was to test the feasibility of using AF4 in combination with SPR to detect and quantify proteins and aggregates in complex samples such as blood serum. Also, in the investigation, three different immobilisation methods for the receptor HER-2 were compared, which involved (1) direct binding via EDC/NHS, the standard approach; (2) immobilisation via NTA-Ni-Histag complexation; and (3) biotin/avidin-linked chemistry using a regenerable form of avidin. The highest specific activity was obtained for the biotin-avidin method, while the lowest specific activity was observed for the NTA-Ni-Histag linkage. The data show that AF4 can separate trastuzumab monomers and aggregates in blood serum and that SPR has the ability to selectively monitor the elution. This is an encouraging result for automated analysis of complex biological samples using AF4-SPR.
Highlights
As previously reported, the size separation methodology of asymmetrical flow field-flow fractionation (AF4) [1, 2] can separate the macromolecular components in whole blood, serum and plasma samples by hydrodynamic size without can detect and quantify the compound of interest in a complex matrix
With the carboxymethyldextran matrices (CMD)-type sensors (CMD and CMD-nitrilotriacetic acid (NTA)), total binding of the ligand is expectedly higher than sensors based on monolayers due to a thick hydrogel layer
The responses registered with the biotinylated Au sensors are generally around 350 mdeg for avidin binding, and 100– 200 mdeg for ligand capture depending on the protein MW
Summary
The size separation methodology of asymmetrical flow field-flow fractionation (AF4) [1, 2] can separate the macromolecular components in whole blood, serum and plasma samples by hydrodynamic size without can detect and quantify the compound of interest in a complex matrix. It would be more desirable to selectively measure directly on the target component without any labelling. One such methodology that has been used to great effect to study a wide range of components is surface plasmon resonance (SPR). The excitation of the “plasmon” requires a p-polarised laser beam impinging on the gold surface under a specific angle, and is in practice observed as a drop in reflected light when scanning the angle over the range in which the resonance occurs. When compounds adsorb to the outer surface of the metal film, the refractive index close to the metal changes and the position of the SPR minimum notably changes, if they are big molecules like proteins. If the protein monomer and different aggregated forms first could be physically separated and measured by SPR, the data interpretation becomes more detailed
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