Standard sandwich immunoassay.

6 spots or replicates per marker.

We use 6 spots to ensure statistical power. This also minimizes the effect of imperfections in the final image. The software can detect abnormalities in the array area; if the spot to spot CV's are>20% the software throws out the furthest outlier. If the CV is still >20% a second data point is removed. No more than 2 data points (spots) will be removed from any row regardless of %CV.

Our master curve is generated in the lab prior to shipment of every lot of biochips. Standards are run in quadruplicate on multiple instruments. The master curves are then generated from the means of these quadruplicates. We use 9 point master curves depending upon the biomarker. The master curve(s) are electronically stored in the instrument for each particular lot of biochips.

Spot to spot and biochip to biochip CV's are expected to be <10% at a low level of analyte concentration

Serum or Plasma as of 2QTR2006. Additional matrices are being tested including (saliva, urine, cell supernatant and lysate)

As of 3QT2007 we are reading both sample types off a plasma curve. Running cell culture supernatants would be slightly different because the matrix is different. Either have to prepare standards in that new matrix and generate new standard curves or throw in a fudge factor and multiply all cell culture results by that number to get actual concentrations.

No, our instrument is not set up to evaluate DNA

Yes, we are able to develop custom arrays

The assay progress can be monitored by the instrument clock timer, which indicates the time remaining in the assay

If antibodies are available or can be generated, the system is capable of measuring these protein variants, provided they are at high enough concentrations in their respective matrices for detection.

Currently up to 20 different markers but theoretically up to 40.

The glass slide is treated with a patented coating of an ultra- thin film (<0.1µ thick) of nitrocellulose Temperature of the assay?

A heater element in the door of the instrument along with two thermistors used to measure actual temperature maintains constant temperature of the biochips

Fluorescence excited by a laser and read by a CCD camera

Positive and negative assay control spots are included on each array. Additionally control samples are provided for each lot of biochips.

Today all assays run in ~1 hour. We are evaluating ways to shorten this time while maintaining performance.

Today all assays run in ~1 hour. We are evaluating ways to shorten this time while maintaining performance.

Today all assays require a 2 fold dilution of sample: 100 µl of sample and 100µl of sample diluent. Further dilution may be required if biomarker concentrations exceed the assay dynamic range.

On a CD or over the internet in the future

200ml total volume is required for each assay. The actual sample volume is dependent upon the dilution requirements for each specific assay ( 100ml of sample and 100 ml of buffer.1:1 dilution)

Two wash steps; one between antibody and label and then a final prior to imaging

Scientific literature was evaluated for the given indication and biomarkers relevant to that indication were selected. The list of biomarkers was circulated among investigators in the field for feedback. A comparison of competitive products was also done.

The instrument is capable of evaluating biomarkers that contains any anticoagulant. A specific anticoagulant however, may be recommended for a particular biomarker.

The last day.

20 minutes

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