Background: The study objective was to develop a prototype for multi-antiepileptic drugs (AEDs) ImmunoChip for the purpose of therapeutic drug monitoring at the point-of-care (POC). To achieve this goal, a one-step homogeneous immunoassay was used and optimized to measure three AEDs in serum samples. The procedure utilizes the chemistry of the Cloned Enzyme Donor Immunoassay (CEDIA) and is modified for use with the Beta-Glo assay system, generating a bioluminescent signal. This method allows for immunochemical detection and quantitation of analytes in a homogeneous manner.

Methods: A novel microfabrication method was used to prototype microstructures on an ImmunoChip. The ImmunoChip allows lyophilized assay reagents to be stored in a multi-layered pattern. Using the one-step CEDIA procedure as the assay platform, antibodies specific to different AEDs were dispensed and lyophilized in different wells in an ImmunoChip. Serum sample containing AEDs in this study was dispensed on the center of the nitrocellulose filter paper, placed above the wells in the center of the array. It wicked along the membrane and into each well, where the reagents were reconstituted and the CEDIA and bioluminescent reactions took place.

Results: Luminescent signals yielded from each well in an ImmunoChip were collected using a CCD camera. The luminescent signal for each assay was recorded after a 5-minute exposure. CCD images are shown in Fig. 1A. Images were analyzed using ImageJ and MatLab to integrate the CCD counts across the area of each well for each exposure. Calibration curves (Fig. 1B) for the three AEDs, carbamazepine (CBZ), phenytoin (PHT), and valproic acid (VPA) were created by time integrating the CCD counts in each well on different chips.

Conclusion: The speed and convenience of this ImmunoChip make it well suited for the therapeutic monitoring of AEDs in serum at the POC and eventually in the home environment.