Biomedical Engineering Reference
In-Depth Information
used at this stage, in which the aptamers are passed over a cellulose filter in the absence
of the target. This is to eliminate aptamers that bind the filter in a target-independent
manner (54). Counter selection is also sometimes used, where aptamers that bind struc-
tures similar to that of the target are removed (51). Affinity chromatography is generally
used to isolate aptamers for small molecular targets (54).
The high-affinity aptamers are then amplified by reverse transcription-PCR (RT-PCR)
(for RNA aptamers) (Step 5) or by PCR for DNA aptamers (Step 6), to create a new
aptamer library enriched with the aptamers of high affinity. The entire process is then
repeated (Step 7), resulting in fewer and fewer unique sequences, with higher and higher
affinity to the target, being retained. Note that during each round of selection, the binding
conditions for the aptamer and the target are generally made more stringent to increase
the selective pressure on the remaining aptamers. Generally, a complete SELEX process
(between 8 and 15 cycles) will yield a final mixture of no more than ten aptamers (53; 54).
The aptamers can then be cloned and sequenced, allowing further identical aptamers to be
generated by chemical synthesis.
The SELEX procedure is clearly involved and can take weeks to months to produce a
suitable aptamer when performed manually. Cox et al. (59) first reported on the automa-
tion of the procedure. A more recent report (60) however details the automation of the pro-
cedure where approximately 12 rounds of selection can be carried out in 2 days. Based on
these figures, it has been estimated that one robot could produce aptamers against 120 tar-
gets in 1 month, exceeding manual throughput by 10-100 fold. It has also been suggested
that further development of the automated procedure could lead to the production of
aptamers to upwards of 1000 targets per month. Several robots working in parallel could
therefore generate aptamers to an entire proteome within a relatively short period of time,
further highlighting the immense potential of aptamers. An additional advantage of an
automated procedure is the consistency of the repetitive tasks that it offers, creating a
bench mark to allow comparisons between different laboratories and eliminating varia-
tions due to manual selection techniques (59).
20.3.3
Aptamers and Antibodies
Given the characteristics of aptamers described above, it is clear that they could be of great
use for therapeutic, analytical, and diagnostic procedures. Currently, antibodies are most
frequently used in procedures where high affinity and specificity for a particular target are
required, such as ELISAs. The following is a discussion of the advantages and disadvan-
tages of using aptamers or antibodies for these techniques.
The use of antibodies to detect analytes became widespread in the 1970s, when poly-
clonal sera from immunized animals were the most popular choice (52). It was not long
thereafter till the discovery of monoclonal antibody technology which allowed the produc-
tion of a unique antibody in large quantities (52). This technology allowed affinity-based
assays to be further refined and optimized and was embraced throughout the scientific
community. The many advantages of antibodies include their high affinity and specificity
for their particular antigen, typically with very low dissociation constants. Furthermore,
selected clones producing the antibody of choice can be cultured continuously, hence pro-
viding a limitless supply of a particular antibody (theoretically). Lastly, the immunogen
used for the identification of a monoclonal antibody does not have to be pure (52).
There are, however, a number of disadvantages and limitations associated with the use
of antibodies. These are listed below (52):
The production of antibodies requires animals. The generation of antibodies
against molecules that are not well tolerated by the animal (e.g., toxins) or
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