1. How reproducible is the system between-run, e.g. including different occasions/days?
The sensors are extremely reproducible between runs giving CVs of less than 6% at all levels of detection. This is highly dependent on the quality of the bioreagents used. The sensors can make a good assay better but cannot improve the performance of a bad assay.
2. Depending on the assay type, is it correct to assume that the stored sensor has either a labeled antibody (for example enzyme labeled) – as in the competitive case or a monoclonal antibody, as in the non-competitive case, as part of the base sensor? Is this covalently linked into the polypyrrole as part of the polymerisation reaction, and if so does this tend to alter the binding characteristics of the Ab? Are the binding characteristics of the Ab reproducible from sensor to sensor?
The sensor is stable with and without bioreagents for at least 4 months at 37°C (this was evaluated with and without a streptavidin coating, using sucrose as a stabiliser). Specific stabilising agents for some particular bioreceptors may be required, e.g. Troponin I. Commercially developed stabilisers give better results. All the solutions used are the same as those regularly used in commercial ELISA, so regular stabilisers or sterilisation protocols should apply.
We have done little work on assessing the stability of the bioreagents on our system. We are assuming that all the work previously carried out on ELISA plates and other types of solid phase can be transferred without much difficulty. The Ab can be either physi-adsorbed or immobilised via a streptavidin-biotin or other linkage methods.
It is also possible to store the base sensors. The main precaution is to seal against oxygen and moisture. Any protein stabilizers, forming a "sealing" film on the sensor surface at the stabilizing step, give additional protection and extend storage.
3. How was the sensor stored to obtain the 4 month @ 37°C? Are they stored dry or wet, and if wet is a special stabilizing buffer required to stabilize the Ab present on the sensor?
Sensors were stored dry.
4. Regarding the 4 month stability question, is this the same for the other analytes (Troponin I, TNF, Digoxin)?
The stability tests were performed on bare sensors and streptavidin coated sensors only. They were tested using our QC assay. We are not developing assays and stabilization procedures, but a platform detection technology and therefore have to show that the technology itself is stable.
Stabilizing reagents can be different for different analytes. Normally any commercial stabilizers can be used in the UTS.
5. In the sandwich assay, how is the second labeled antibody stored. If stored as a solution in the system, are the Ab’s and enzymes used as labels stable for a significant period of time? Is this shelf-life similar to the base sensor itself?
The answer to the questions about reagent storage is the same as for most rapid diagnostics already commercially available. i.e it has all been done many times before. The only new thing we have is the detection technology.
