Biomedical Engineering Reference
In-Depth Information
assays and surface plasmon resonance (Appukuttan, 2002; Blixt et al., 2004; Bohorov et al.,
2006; Ideo et al., 2003; Munoz et al., 2010; Song et al., 2009b; Stowell et al., 2008a). For glycan
arrays it can be important which linker is used to bind the glycan epitopes to the surface
(length, chemical structure). Moreover it is possible to use chemically and/or enzymatically
produced ligands as well as glycans from natural compounds like glycopeptides and
glycolipids (Blixt et al., 2004; Bohorov et al., 2006). The latter allows the analysis of complex
and even unknown glycan structures of different cells (Song et al., 2009a; Song et al., 2009b;
Song et al., 2010). Immobilised galectins are for example used in frontal affinity approaches
and ELISA assays (Hirabayashi et al., 2002; Sörme et al., 2002).
Variations of binding assays with immobilised partners are assays in which the surface
binding is inhibited by a soluble ligand. Such inhibition studies of surface interactions allow
a direct read-out of IC50 values and thereby the direct comparison of relative affinities
(Sörme et al., 2002). For the calculation of affinity constants assumptions have to be made to
simplify calculations which might not be correct for each single interaction measurement.
Additional the problems mentioned before still persist (Sörme et al., 2004).
Most assays with one immobilised component as well as some direct interaction assays are
based on the read-out of a fluorescence signal or other labels. Therefore either the galectin or
the glycan structures have to be labelled. This leads to some additional problems: If the
glycan is chemically labelled the linker or label itself can alter the binding affinity with
specific effects for different galectins (Sörme et al., 2004). Therefore the affinity constants
measured do not exactly fit to the unmodified glycan structures. Moreover the labelling of
glycans is time-consuming. The labelling of galectins can also alter the binding specificities.
It is in most cases done by random chemical modification of specific functional groups such
as amino or thiol functionalities (Carlsson et al., 2007; Patnaik et al., 2006; Rapoport et al.,
2008; Salomonsson et al., 2010; Song et al., 2009b; Stowell et al., 2008a; Stowell et al., 2008b).
Although this labelling is assumed not to influence binding specificity or inactive galectins
are removed after the labelling reaction, binding and oligomerisation still might be slightly
affected. Moreover lot-specific aberrations between different labelling reactions occur.
Labelled galectins are for example used for glycan arrays and ELISA-type set-ups (Blixt et
al., 2004; Carlsson et al., 2007; Rapoport et al., 2008; Salomonsson et al., 2010; Song et al.,
2009b; Stowell et al., 2008a) while fluorescence labelled glycans are used in frontal affinity
chromatography or fluorescence polarisation (Carlsson et al., 2007; Hirabayashi et al., 2002;
Salomonsson et al., 2010; Sörme et al., 2004).
Assays using both binding partners in its soluble form overcome most of the mentioned
problems. But although those assays have different advantages the results cannot directly be
compared with the natural set-up in which the glycans are immobilised on glycoproteins or
glycolipids and thereby multivalently presented. Fluorescence polarisation is one of these
methods measuring direct interactions of ligands in solution, but facing negative side effects
of glycan labelling. Similarly, titration calorimetry also measures the interaction of two
soluble binding partners. As for titration calorimetry no labelling reaction has to be
performed this assay set-up might be considered as the one with fewest problems. But
needed galectin concentrations for this test are usually (but not always) in high micromolar
ranges and therefore above the physiological range. In this concentration range galectins
tend to oligomerise, aggregate or precipitate (Ahmad et al., 2004b; Bachhawat-Sikder et al.,
2001; Cho & Cummings, 1995; Dam et al., 2005; Sörme et al., 2004). Moreover titration
