Biomedical Engineering Reference
In-Depth Information
dynamic rate constants correlated better with mitogenic activity than did the equilibrium
constants (42). A more pertinent example, however, is that of McDonnell et al. (46), who
identified a role for the C
2 domain for IgE (see Figure 20.2) in allergic responses. Removal
of this domain from IgE has little effect on the overall affinity of IgE for its receptor F c
RI,
but has a clear effect on the off rate. It was thus demonstrated that C
2 contributes to the
slow off rate of IgE-mediated mast cell sensitization during the allergic response.
Insights into binding-reaction mechanisms can be gained by analyzing binding kinetics
and thermodynamics over a range of conditions, such as temperature, ionic strength, and
pH. Measurements over a range of ionic strengths, for example, can help describe the role
of electrostatic interactions for a particular binding reaction. Thus, one can, for example,
discriminate between specific and nonspecific protein-DNA binding, due to their differ-
ence in ionic strength dependence. Indeed, nonspecific protein-DNA binding interactions
are highly dependent on ionic strength due to the significant electrostatic contribution of
the negatively charged phosphates of the DNA backbone (42).
SPR has also been used for investigating biological membrane events. This has been
made possible by the recent introduction of sensor surfaces specifically designed for this
purpose, such as the HPA (hydrophobic) or L1 (lipophilic) surfaces from Biacore (Uppsala,
Sweden) (43). These surfaces offer the ability to specifically orient immobilized ligands
and have been shown to be a successful membrane mimic (42). Danelian et al. (47), for
example, used a liposome-covered sensor surface for an assay for lipid absorption for a
panel of 27 drugs and showed a strong correlation with passive intestinal absorption.
Future advances in this field might include inserting ion channels, transmembrane recep-
tors, and cell-signaling molecules within the immobilized lipid surface in an attempt to
closer mimic in vivo systems (43).
Lastly, the general versatility, ease of automation, lack of labeling requirements and low
sample consumption of SPR analysis make it a promising means for large-scale screening
for binding events, both for small molecules in drug discovery and for macromolecules in
large-scale ligand fishing experiments (42). It is able to perform functional characterization
of “hits” from primary screens, providing information not obtainable from traditional
screening methods and making it possible to rank ligands based on affinity as well as asso-
ciation and dissociation kinetics. Additionally, some of the more advanced instruments
(BIAcore 2000 and 3000) have a throughput of 100-300 samples per day, depending on
assay conditions (43), thus making this method capable of high-throughput analysis. SPR
has in fact been used to monitor binding of thyroxin analogues to an immobilized anti-
body (48), as well as by the La Jolla Pharmaceutical Company for the development of a
new drug for the treatment of systemic lupus erythromatosis (42).
20.2.4
Future Developments
Despite the many advantages offered by SPR, there are a number of advances that will
need to be made in order for this analytical technique to become as widespread as, for
example, immunoassays, which are the major competitor of SPR within the field of analy-
sis and detection of biochemical substances, and which offer low cost and high sensitivity
and specificity testing. These advances are likely to include (32):
Improvement of detection limits. Current detection limits stand at about 1 pg/mm 2 ,
which is not sufficient for detecting low concentrations of low-molecular weight
analytes. Although optimization of SPR optical instruments and refining of data-
processing methods may lower the current detection limits, no approach cur-
rently exists that will lower this limit by the necessary orders of magnitude.
Progress will therefore need to be made in this area.
Search WWH ::




Custom Search