Evaluating sandwich immunoassays in microarray format in terms of the ambient analyte regime

Background: Conceptionally, antibody microarrays are simply multiplexed sandwich immunoassays in a miniaturized format. However, from the amounts of capture antibodies used, it is not apparent whether such assays are ambient analyte (Ekins. Clin Chem 1998;44:2015-30) or mass-sensing devices (Silzel et al. Clin Chem 1998;44: 2036-43). We evaluated multiplexed microarray sandwich assays for 24 mouse serum proteins in these terms within the boundaries of our experimental setup and based on theoretical considerations of the law of mass action. Methods: Capture antibodies for 24 mouse serum proteins were printed on planar microarray substrates. After incubation with mixtures of purified antigens for 1 or 18 h, mixtures of biotinylated detection antibodies were used. High assay sensitivity was achieved by use of resonance-light-scattering particles for signal generation. Titration curves were generated for assay volumes of 20, 40, and 80 μL, and detection limits were calculated and compared. The assays were modeled theoretically based on the amounts of capture antibodies and the assay volumes used. Results: As predicted, experimental variations of the assay volume by up to fourfold did not appreciably affect detection. Even for the most sensitive assay, <2% of the analyte molecules present in the sample were captured and generated signal at the detection limit. However, increasing the sample incubation time from 1 to 18 h on average lowered the detection limit threefold. Conclusions: In our experimental setup, all 24 sandwich microarray assays fulfill the criteria of the "ambient analyte" regime because depletion of analyte molecules from the assay volume is insignificant.