Karen Walker, Maggie Maramba, and Rebecca Reynolds
Human ID Department, Roche Molecular Systems, Alameda, CA

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The ProfiBlot IIT is an automated strip processing instrument made by SLT lab instruments and distributed by Tecan US. The ProfiBlot IIT has a heated tray basin which can hold up to 24 AmpliType PM or HLA DQA1 strips. The temperature of the tray can be controlled from room temperature to 65^°C. As many as six solutions can be dispensed from the machine into the tray; solutions are also automatically aspirated from the tray into a waste container. All steps after sample addition can be run by the instrument without further input from the user.

The ProfiBlot IIT has been tested extensively by us for use in the AmpliType assay system. Initial studies examined the instrument’s performance, reproducibility, and performance compared to manually-performed assay. Validation studies include tests for product carry-over, detection of mixtures, temperature and buffer (SSPE and SDS concentrations) windows, and sensitivity with a wide range of input DNA amounts. We have also compared heat and base denaturation of PCR product and found that not only does base denaturation perform as well as heat, but using base also eliminated the need for having a thermal cycler next to the assay area, and prevents re-annealing of the product (such as occurs with heat denaturation).

We have developed protocols for the ProfiBlot IIT that provide acceptable performance for the AmpliType PM and PM+DQA1 assays, comparable to that of manually-performed assay. Two protocols have been developed: one is standard assay with a fixed 20 minute color development time; the second incorporates a color development step which the user can stop once the desired amount of signal is present on the strips. A typical 24 strip assay requires approximately 1.5 hours to perform, which is comparable to the length of time necessary to type only 16 strips using the manual assay. One benefit of the instrument is that it is completely "walk away" once samples have been loaded, thereby leaving the user free to pursue other activities. Another benefit of the ProfiBlot IIT is that only 3 mL of the hybridization or wash solution are used for each step instead of the 5mL used in the manual assay. Consequently, the stock solutions need to be made less frequently.