Low avidity and affinity issues are not normally things we encounter as we always start our selection process ‘with the end in mind’. We always raise the aptamer in the buffer or matrix that it is intended that the diagnostic test is required for and therefore know it will work without any matrix effects or non-specific binding.
We’ve fully automated our selection process on our robotic systems and check affinity of the aptamer pool after each selection round. This ensures ongoing quality at each step and monitors the increasing aptamer affinity. The whole process takes around 8 weeks to obtain a ‘monoclonal’ aptamer ready to be functionalised for a test kit .
Yes certainly, we have some really good examples in the food & feed sector for Food Safety applications.
Our fully automated selection process is similar to SELEX, but it is our own proprietory high throughput system that we term HARP ( High-throughput Antibody Replacement Process).
Because aptamers are physically smaller than antibodies it means that they can actually get closer to the target molecule. Commonly our aptamers are measured as nano to pico level molar affinity. We usually measure affinity using a technique called BLI (Bio Layer Interferometry).
Yes we do. The sequence of each aptamer is defined by NGS. This can be provided along with the aptamers if requested.
The physical size measurement of an aptamer is 15-25 Å, which contrasts to an antibody, which on average are 130 Å. This correlates to a molecular mass of 8-20kDa.
Whilst the aptamer sequence can be around 30-mers long, it is possible to shorten the sequence once selected so that their bulk production is more efficient, producing higher yields.
It is possible to raise aptamers against targets in suspension, although this process currently takes longer than targets capable for immobilisation.
Aptamers are vulnerable to attack by nucleases, but there are a number of different approaches to prevent degradation. Use of modified nucleotides and end capping has proven to be very successful at preventing significant loss of aptamer function in multiple tests.
Aptamers can be reverse engineered like most current affinity reagent technologies. For example, antibodies can be sequenced by peptide sequencing via mass spectroscopy if desired.
Blocking groups or structures can be added to the end of the aptamer to make this more difficult. Sequencing the aptamer would not tell the individual whether the aptamer was DNA or RNA, nor provide any information regarding modifications.
Aptamers are in various stages of clinical trial at the moment and have passed strict pre-clinical testing in order to do so. However, any potency of an aptamer is likely to be due to the aptamer target, rather than intrinsic immunogenicity or toxicity.