1. Are the sample calibration curves generated with whole blood? If not, what would the blood calibration look like compared to those published?
All the calibration curves are performed in complex fluids, serum, plasma, whole blood, urine, milk or homogenates. We do not see much of a matrix effect on our sensors even between serum and whole blood or between skimmed and whole milk.
It is generally expected that there are matrix effecys for most assays for example measuring from whole blood may involve the heamatocrit effect but this can be controlled if a separation membrane is used, such as Hemasep. The matrix effect is a common occurrence for ELISA’s and each matrix generally requires its own calibration curve.
2. Is there concern of Blood/Serum proteins nonspecifically binding to the membrane and altering the electron transfer properties or potential measurement?
Nonspecific binding can and does occur and this is kept to a minimum by blocking. There is no real problem with noise unless the ELISA itself is non-specific. We only detect the enzyme linked antibody turning over, not bound proteins. In addition the measurement is done after all sample and unbound reagents have been removed and replaced by a controlled measuring solution. Potentiometry is a passive technique, i.e. no current flow, therefore unwanted electrochemical interference’s cannot occur.
3. What is the purpose of polypyrrole besides that of an immobilisation-matrix ?
The polymer film is not only the immobilisation-matrix, but also the sensing element. The properties of this sensing element can be tailored to requirements during the polymerisation step. The sensing element must possess quasi-metallic conductivity, but at the same time metals (e.g. gold and platinum) are not entirely suitable. Minimum variations in their surface morphology in particular attachment of biomolecules changes the potentiometric response significantly even for the same set of parameters and especially in aqua solutions. Polymer film is more tolerant to all these factors.
4. What is the reproducibility of the assay from day-to-day?
This depends on the type of the assay. For the QC assay, which is a one-step assay it is 10-15% for low concentrations (0.1ng/ml) and 2-3% for higher concentrations (1-10ng/ml) (this is for day-to-day and for different batches of sensors! It is less for one batch or for one day!). For multi-step assays it is a bit higher as a consequence of more non-automated steps.
Formation of the immunocomplex is still performed manually. In multi-step assay sensors are placed in the tubes for incubation with a sample, washed, transferred to tubes with conjugate, washed and then the measuring step is performed. The measuring step was manual for NLBTC, it was semi-automated in 2001.
We expect 2-3% CV’s at 0.1ng/ml with automation or semi-automation of all steps.
